U.S. patent application number 12/626011 was filed with the patent office on 2010-08-05 for modified polynucleotides for use in rna interference.
This patent application is currently assigned to DHARMACON INC.. Invention is credited to Yuriy Fedorov, Anastasia Khvorova, Devin Leake, William Marshall, Kimberly Nichols, Angela Reynolds.
Application Number | 20100197023 12/626011 |
Document ID | / |
Family ID | 42470686 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100197023 |
Kind Code |
A1 |
Leake; Devin ; et
al. |
August 5, 2010 |
MODIFIED POLYNUCLEOTIDES FOR USE IN RNA INTERFERENCE
Abstract
Methods and compositions for performing RNA interference
comprising a wide variety of stabilized siRNAs suitable for use in
serum-containing media and for in vivo applications, such as
therapeutic applications, are provided. These siRNAs permit
effective and efficient applications of RNA interference to
applications such as diagnostics and therapeutics through the use
of one or more modifications including orthoesters, terminal
conjugates, modified linkages and 2' modified nucleotides. Uniquely
modified siRNAs have been developed that reduces off-target effects
incurred in gene-silencing. The modifications include
phosphorylation of the first 5' terminal antisense nucleotide; 2'
carbon modifications of the first and second or first, second, and
third 5' terminal antisense nucleotides; and optionally 2' carbon
modifications of the first and second or first, second, and third
5' terminal sense nucleotide. Control and exaequo molecules are
also provided. siRNA molecules and related control, trackability
and exaequo agents with specific stability modifications were
developed.
Inventors: |
Leake; Devin; (Denver,
CO) ; Reynolds; Angela; (Conifer, CO) ;
Khvorova; Anastasia; (Boulder, CO) ; Marshall;
William; (Boulder, CO) ; Fedorov; Yuriy;
(Superior, CO) ; Nichols; Kimberly; (Louisville,
CO) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
DHARMACON INC.
Lafayette
CO
|
Family ID: |
42470686 |
Appl. No.: |
12/626011 |
Filed: |
November 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10551350 |
Oct 19, 2006 |
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PCT/US04/10343 |
Apr 1, 2004 |
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12626011 |
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10406908 |
Apr 2, 2003 |
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10551350 |
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10613077 |
Jul 1, 2003 |
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10406908 |
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60542668 |
Feb 6, 2004 |
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60542646 |
Feb 6, 2004 |
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60543640 |
Feb 10, 2004 |
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60543661 |
Feb 10, 2004 |
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Current U.S.
Class: |
435/463 ;
536/24.5 |
Current CPC
Class: |
C12N 15/111 20130101;
C12Y 207/11024 20130101; C12N 2320/11 20130101; C12N 2310/3521
20130101; C12N 15/1137 20130101; C12N 2310/14 20130101; C12N
2310/321 20130101; C12N 2310/322 20130101; C12N 2310/315 20130101;
C12Y 301/03001 20130101; C12N 15/1138 20130101; C12N 2330/30
20130101; C12N 2310/321 20130101 |
Class at
Publication: |
435/463 ;
536/24.5 |
International
Class: |
C12N 15/87 20060101
C12N015/87; C07H 21/04 20060101 C07H021/04 |
Claims
1. A double stranded polyribonucleotide comprising: (a) a sense
strand comprising: a first nucleotide closest to the 5' end of the
sense strand, said first nucleotide having a first 2'-O-alkyl
modification; and a second nucleotide next closest to the 5' end of
the sense strand, said second nucleotide having a second 2'-O-alkyl
modification; (b) an antisense strand comprising a phosphate group
on the 5' end, wherein all pyrimidine nucleotides on said antisense
stand comprise a 2' halogen modification; wherein the sense strand
and the antisense strand are capable of forming a duplex of between
18 and 30 base pairs; wherein said antisense strand is at least 80%
complementary to said sense strand; and wherein said antisense
strand is at least 80% complementary to a target nucleic acid.
2. The double stranded polyribonucleotide of claim 1, wherein the
sense strand and antisense strand are capable of forming a duplex
of between 19 and 25 base pairs.
3. The double stranded polyribonucleotide of claim 1, wherein said
target nucleic acid is a mRNA.
4. The double stranded polyribonucleotide of claim 1 wherein said
first 2'-O-alkyl modification and said second 2'-O-alkyl
modification are each independently selected from the group
consisting of a 2'-O-methyl modification, a 2'-O-ethyl
modification, a 2'-O-propyl modification, a 2'-O-isopropyl
modification, a 2'-O-butyl modification, and a 2'-O-isobutyl
modification.
5. The double stranded polyribonucleotide of claim 1 wherein said
first 2'-O-alkyl modification and said second 2'-O-alkyl
modification are both 2'-O-methyl modifications.
6. The double stranded polyribonucleotide of claim 1 wherein said
2' halogen modification is a 2'-F modification.
7. The double stranded polyribonucleotide of claim 1, further
comprising a conjugate.
8. The double stranded polyribonucleotide of claim 7, wherein said
conjugate is a detectable label.
9. The double stranded polyribonucleotide of claim 8, wherein said
detectable label is attached to said first 5' terminal sense
nucleotide.
10. The double stranded polyribonucleotide of claim 8, wherein said
detectable label is a fluorescent dye.
11. The double stranded polyribonucleotide of claim 7 wherein said
conjugate is cholesterol.
12. The double stranded polyribonucleotide of claim 11 wherein said
cholesterol is attached to said sense strand.
13. The double stranded polyribonucleotide of claim 12 wherein said
cholesterol is attached to the 3' end of said sense strand.
14. The double stranded polyribonucleotide of claim 1, further
comprising at least one phosphorothioate internucleotide
linkage.
15. The double stranded polyribonucleotide of claim 1, further
comprising: a 3' overhang on the 3' end of the antisense strand
comprising a first overhang nucleotide and a second overhang
nucleotide; and a blunt end at the 5' terminus of the antisense
strand and the 3' terminus of the sense strand.
16. The double stranded polyribonucleotide of claim 15 wherein the
first overhang nucleotide is attached to the double stranded
polyribonucleotide by a phosphorothioate internucleotide linkage,
and the first overhang nucleotide and the second overhang
nucleotide are attached to each other by a phosphorothioate
internucleotide linkage.
17. A method of gene silencing comprising introducing a double
stranded polyribonucleotide into a cell that is expressing or is
capable of expressing a target nucleic acid, wherein said double
stranded polyribonucleotide comprises: (a) a sense strand
comprising: a first nucleotide closest to the 5' end of the sense
strand, said first nucleotide having a first 2'-O-alkyl
modification; and a second nucleotide next closest to the 5' end of
the sense strand, said second nucleotide having a second 2'-O-alkyl
modification; (b) an antisense strand comprising a phosphate group
on the 5' end, wherein all pyrimidine nucleotides on said antisense
stand comprise a 2' halogen modification; wherein the sense strand
and the antisense strand are capable of forming a duplex of between
18 and 30 base pairs; wherein said antisense strand is at least 80%
complementary to said sense strand; and wherein said antisense
strand is at least 80% complementary to said target nucleic acid.
Description
RELATED APPLICATION INFORMATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/551,350, filed Oct. 19, 2006 which is the
35 U.S.C. .sctn.371 National Stage of PCT Application Serial No.
PCT/US2004/10343, filed Apr. 1, 2004, which claims the benefit of
the filing date of U.S. Provisional Patent Application Nos.
60/542,668, filed Feb. 6, 2004; 60/542,646, filed Feb. 6, 2004;
60/543,640, filed Feb. 10, 2004; and 60/543,661, filed Feb. 10,
2004. PCT Application Serial No. PCT/US2004/10343, filed Apr. 1,
2004, is also a continuation-in-part of U.S. patent application
Ser. No. 10/406,908, filed Apr. 2, 2003, and a continuation-in-part
of U.S. patent application Ser. No. 10/613,077, filed Jul. 1, 2003.
The disclosure of each of the aforementioned applications is
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of modified
polynucleotides.
BACKGROUND
[0003] RNA-induced gene silencing in mammalian cells is presently
believed to implicate a minimum of three different levels of
control: (i) transcription inactivation (siRNA-guided DNA and
histone methylation); (ii) small interfering RNA (siRNA)-induced
mRNA degradation; and (iii) mRNA-induced transcriptional
attenuation. The RNA interference (RNAi) generated by siRNA can be
long lasting and effective over multiple cell divisions.
Consequently, the ability to assess gene function via siRNA
mediated methods, as well as to develop therapies for
over-expressed genes, represents an exciting and valuable tool that
will accelerate gene function analysis, drug target validation, and
genome-wide investigations. Moreover, RNAi has broad potential as a
therapeutic tool.
[0004] Relatively recent discoveries in the field of RNA metabolism
have revealed that the uptake of duplex RNA (dsRNA) can induce
RNAi.
[0005] In these circumstances, a Type III RNase called Dicer
processes the long ds RNA into siRNA that subsequently partner with
the RNA Interfering Silencing Complex (RISC) to mediate the
degradation of target transcripts in a sequence specific manner.
This phenomenon has been observed in a diverse group of organisms.
Unfortunately, in mammalian cells, the use of long dsRNA to induce
RNAi has been met with only limited success. In large part, this
ineffectiveness is due to induction of the interferon response,
which results in a general, as opposed to targeted, inhibition of
protein synthesis.
[0006] Recently, it has been shown that when synthetic siRNAs are
introduced into mammalian cells in culture, sequence-specific
inhibition of target mRNA can be realized without inducing an
interferon response. These short duplexes, can act catalytically at
sub-molar concentrations to cleave greater than 95% of the target
mRNA in a cell. A description of the mechanisms for siRNA activity,
as well as some of its applications is provided in Provost et al.,
Ribonuclease Activity and RNA Binding of Recombinant Human Dicer,
E.M.B.O.J., 2002 Nov. 1, 21(21): 5864-5874; Tabara et al., The
dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a
DexH-box Helicase to Direct RNAi in C. elegans, Cell. 2002, Jun.
28, 109(7):861-71; Ketting et al., Dicer Functions in RNA
Interference and in Synthesis of Small RNA Involved in
Developmental Timing in C. elegans; and Martinez et al.,
Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi,
Cell 2002, Sep. 6, 110(5):563.
[0007] There are four main issues that must be addressed when
working with siRNA: (i) functionality; (ii) specificity; (iii)
delivery methods; and (iv) stability. Functionality refers to the
ability of a particular siRNA to silence the desired target.
Methods for improving functionality are, for example, the subject
of U.S. patent application Ser. No. 10/714,333. Specificity refers
to the ability of a particular siRNA to silence a desired target
and only the desired target. Thus, specificity refers to minimizing
off-target effects. Delivery methods are the means by which a user
introduces a particular siRNA into a cell and may, for example,
include using vectors or modifications of the siRNA itself.
Stabilization refers to the ability of an siRNA to resist
degradation by enzymes and other harmful substances that exist in
intra-cellular and extra-cellular environments. For example, when
naked siRNA molecules are introduced into blood, serum, or
serum-containing media, they are not stable and are almost
immediately degraded, which reduces or eliminates their
effectiveness.
[0008] The present invention addresses the second and fourth
issues; specificity and stability, by providing modifications to
siRNA that can either increase or decrease specificity and/or
increase stability.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to compositions and
methods for performing RNA interference. In general the siRNA
chemical modifications described herein affect two critical
properties of the molecules to which they are associated: stability
and specificity. Those that affect stability are particularly
advantageous for use in applications that require exposure to
blood, serum, serum-containing media, and other biological material
that contain nucleases or other factors that tend to degrade
nucleic acids. Modifications that reduce the level of off-target
effects induced by a siRNA directed against a specific target are
particularly valuable in research and therapeutic settings.
Multiple distinct combinations of modifications that substantially
improve RNAi applications are disclosed and are applicable in the
design of optimum silencing reagents, transfection controls, and
exaequo reagents.
[0010] According to a first embodiment, the present invention
provides an siRNA having a sense strand comprising a polynucleotide
comprised of at least one orthoester modified nucleotide, and an
antisense strand comprising a polynucleotide comprised of at least
one 2' modified nucleotide unit.
[0011] According to a second embodiment, the present invention
provides an siRNA having: a sense strand comprising a
polynucleotide comprised of at least one orthoester modified
nucleotide; an antisense strand comprising a polynucleotide
comprised of at least one 2' modified nucleotide; and a
conjugate.
[0012] According to a third embodiment, the present invention
provides an siRNA having: a sense strand comprising at least one
orthoester modified nucleotide; an antisense strand; and a
conjugate.
[0013] According to a fourth embodiment, the present invention
provides an siRNA having: a sense strand; an antisense strand; and
a conjugate, wherein the sense strand and/or the antisense strand
have at least one 2' modified nucleotide.
[0014] According to a fifth embodiment, the present invention
provides an siRNA having a sense strand comprising at least one
orthoester modified nucleotide, an antisense strand comprising at
least one 2' modified nucleotide selected from the group consisting
of a 2' halogen modified nucleotide, a 2' amine modified
nucleotide, a 2'-O-alkyl modified nucleotide, and a 2' alkyl
modified nucleotide, and a conjugate selected from the group
consisting of amino acids, peptides, polypeptides, proteins,
sugars, carbohydrates, lipids, polymers, nucleotides,
polynucleotides, and combinations thereof, wherein the
polyribonucleotide comprises between 18 and 30 nucleotide base
pairs.
[0015] According to a sixth embodiment, the present invention
provides a composition comprising one of the structures below:
##STR00001##
wherein each of B.sub.1 and B.sub.2 is a nitrogenous base,
carbocycle, or heterocycle; X is selected from the group consisting
of O, S, C, and N; W is selected from the group consisting of an
OH, a phosphate, a phosphate ester, a phosphodiester, a
phosphotriester, a modified internucleotide linkage, a conjugate, a
nucleotide, and a polynucleotide; R1 is an orthoester; R2 is
selected from the group consisting of a 2'-O-alkyl group, an alkyl
group, an amine and a halogen; and Y is a nucleotide or
polynucleotide. The dashed lines between B.sub.1 and B.sub.2
indicate interaction by hydrogen bonding between nitrogenous
bases.
[0016] According to a seventh embodiment, the present invention
provides a method of performing RNA interference. This method is
comprised of exposing an siRNA to a target nucleic acid. The siRNA
is comprised of a sense strand and an antisense strand, and at
least one of said sense strand and said antisense strand comprises
at least one orthoester modified nucleotide.
[0017] According to an eighth embodiment, the present invention
provides another method of performing RNA interference. This method
is comprised of exposing an siRNA to a target nucleic acid, wherein
the siRNA is comprised of a sense strand, an antisense strand, and
a conjugate. According to this embodiment, either the sense strand
or the antisense strand comprises a 2' modified nucleotide.
[0018] The compositions of the first through eighth embodiments of
the present invention can render siRNAs resistant to nuclease
degradation, while maintaining biological functionality. By, for
example, using siRNAs with at least one orthoester modified
nucleotide, such as on the sense strand, and at least one other
modification, such as at an appropriate position on the antisense
strand, one can enhance stability while retaining functionality in
RNA interference applications. Additionally, using siRNAs with one
or more 2' modifications, and/or modified internucleotide linkages,
in conjunction with conjugates, in RNA interference applications,
can also provide enhanced stability while retaining functionality,
even in the absence of an orthoester modification on either
strand.
[0019] According to a ninth embodiment, the present invention
provides a method of performing RNA interference, said method
comprising exposing an siRNA to a target nucleic acid, wherein said
siRNA is comprised of a sense strand and an antisense strand, and
wherein said sense strand is substantially nonfunctional.
[0020] According to a tenth embodiment, the present invention
provides a method of performing RNA interference, said method
comprising exposing an siRNA to a target nucleic acid, wherein said
siRNA comprises: (a) a conjugate; (b) a sense strand comprising at
least one 2'-O-alkyl modification, wherein said sense strand is
substantially nonfunctional; and (c) an antisense strand comprising
at least one 2'-fluorine modification, wherein said sense and
antisense strands form a duplex of 18-30 base pairs.
[0021] According to an eleventh embodiment, the present invention
provides an siRNA comprised of: [0022] (a) a sense strand, wherein
said sense strand is comprised of [0023] i. a first 5' terminal
sense nucleotide and a second 5' terminal sense nucleotide, wherein
said first 5' terminal sense nucleotide comprises a first
2'-O-alkyl sense modification and said second 5' terminal sense
nucleotide comprises a second 2'-O-alkyl sense modification; and
[0024] ii. at least one 2'-O-alkyl pyrimidine modified sense
nucleotide, wherein said at least one 2'-O-alkyl pyrimidine
modified sense nucleotide is a nucleotide other than said first 5'
terminal sense nucleotide or said second 5' terminal sense
nucleotide; and [0025] (b) an antisense strand, wherein said
antisense strand is comprised of [0026] i. at least one 2' halogen
modified pyrimidine nucleotide; and [0027] ii. a first 5' terminal
antisense nucleotide, wherein said first 5' terminal antisense
nucleotide is phosphorylated at its 5' carbon position, [0028]
wherein the sense strand and the antisense strand are capable of
forming a duplex of between 18 and 30 base pairs.
[0029] The molecules of the eleventh embodiment may be used to
silence a target and/or as a control. These molecules may further
comprise a label, such as a fluorescent label and/or a third 5'
terminal sense nucleotide that comprises a third 2'-O-alkyl sense
modification.
[0030] According to a twelfth embodiment, the present invention
provides an siRNA comprised of: [0031] (a) a sense strand, wherein
said sense strand is comprised of [0032] i. a first 5' terminal
sense nucleotide and a second 5' terminal sense nucleotide, wherein
said first 5' terminal sense nucleotide comprises a first
2'-O-alkyl sense modification and said second 5' terminal sense
nucleotide comprises a second 2'-O-alkyl sense modification; and
[0033] ii. at least one 2'-O-alkyl pyrimidine modified sense
nucleotide, wherein said at least one 2'-O-alkyl pyrimidine
modified sense nucleotide is a nucleotide other than said first 5'
terminal sense nucleotide or said second 5' terminal sense
nucleotide; and [0034] (b) an antisense strand, wherein said
antisense strand is comprised of [0035] i. a first 5' terminal
antisense nucleotide and a second 5' terminal antisense nucleotide,
wherein said first 5' terminal antisense nucleotide comprises a
first 2'-O-alkyl antisense modification and said second 5' terminal
sense nucleotide comprises a second 2'-O-alkyl antisense
modification; and [0036] ii. at least one 2'-O-alkyl pyrimidine
modified antisense nucleotide, wherein said at least one 2'-O-alkyl
pyrimidine modified antisense nucleotide is a nucleotide other than
said first 5' terminal antisense nucleotide or said second 5'
terminal antisense nucleotide. [0037] wherein the sense strand and
antisense strand are capable of forming a duplex of between 16 and
28 base pairs.
[0038] Preferably, the molecules of the twelfth embodiment also
comprise a label that may for example be a fluorescent dye, and/or
comprise a third 5' terminal sense nucleotide that comprises a
third 2'-O-alkyl sense modification, and/or a third 5' terminal
antisense nucleotide that comprises a third 2'-O-alkyl antisense
modification. The molecules of the twelfth embodiment can form
duplexes of, for example, 19-25 base pairs or, for example, 19-28
base pairs.
[0039] According a thirteenth embodiment, the present invention
provides an siRNA, comprising: [0040] (a) an antisense strand,
wherein said antisense strand is comprised of a first 5' terminal
antisense nucleotide and said first 5' terminal antisense
nucleotide is phosphorylated at said first 5' terminal antisense
nucleotide's 5' carbon position; and [0041] (b) a sense strand,
wherein said sense strand is comprised of a first 5' terminal sense
nucleotide and a second 5' terminal sense nucleotide, wherein said
first 5' terminal sense nucleotide comprises a first 2' carbon
sense modification and said second 5' terminal sense nucleotide
comprises a second 2' carbon sense modification.
[0042] Preferably, the 2'-modifications are 2'-O-alkyl
modifications. According to this embodiment, the sense strand and
the antisense strand preferably contain from 18-30 base pairs that
are at least substantially complementary.
[0043] According to a fourteenth embodiment, the present invention
is directed to a unimolecular siRNA capable of forming a hairpin
siRNA. The unimolecular siRNA comprises: [0044] (a) an antisense
region, wherein said antisense region is comprised of a first 5'
terminal antisense nucleotide and wherein said first 5' terminal
antisense nucleotide is phosphorylated at said first 5' terminal
antisense nucleotide's 5' carbon position; [0045] (b) a sense
region, wherein said sense region is comprised of a first 5'
terminal sense nucleotide and a second 5' terminal sense
nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification; and [0046] (c) a loop region, wherein said loop
region is located between said sense region and said antisense
region.
[0047] According to this fourteenth embodiment, the sense region
and antisense region are at least substantially complementary and
may form a stable duplex. Similar to the thirteenth embodiment,
according to this fourteenth embodiment, the sense region and the
antisense region form a duplex that preferably contains from 18-30
base pairs. Preferably the loop region is downstream of the
antisense region.
[0048] According to a fifteenth embodiment, the present invention
is directed to a method for minimizing off-target effects, said
method comprising exposing an siRNA to a target nucleic acid or to
a cell that is expressing or is capable of expressing the target
nucleic acid, wherein said siRNA comprises an antisense strand and
a sense strand, wherein: [0049] (a) said sense strand is comprised
of a first 5' terminal sense nucleotide and a second 5' terminal
sense nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification; and [0050] (b) said antisense strand is comprised of
a first 5' terminal antisense nucleotide and said first 5' terminal
antisense nucleotide is phosphorylated at said first 5' terminal
antisense nucleotide's 5' position.
[0051] According to a sixteenth embodiment, the present invention
is directed to a method for minimizing off-target effects, said
method comprising exposing a modified unimolecular siRNA capable of
forming a hairpin to a target nucleic acid or to a cell that is
expressing or is capable of expressing the target nucleic acid,
wherein said unimolecular siRNA comprises an antisense region and a
sense region, wherein: [0052] (a) said sense region is comprised of
a first 5' terminal sense nucleotide and a second 5' terminal sense
nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification; [0053] (b) said antisense region is comprised of a
first 5' terminal antisense nucleotide and said first 5' terminal
antisense nucleotide is phosphorylated at said first 5' terminal
antisense nucleotide's 5' carbon position; and [0054] (c) a loop
region, wherein said loop region is located between said sense
region and said antisense region.
[0055] Preferably the loop region is located downstream of the
antisense region.
[0056] According to a seventeenth embodiment, the present invention
is directed to an siRNA, comprising: [0057] (a) an antisense
strand, wherein said antisense strand is comprised of a first 5'
terminal antisense nucleotide and a second 5' terminal antisense
nucleotide, wherein said first 5' terminal antisense nucleotide
comprises a first 2' carbon antisense modification and said second
5' terminal antisense nucleotide comprises a second 2' carbon
antisense modification and said first 5' terminal antisense
nucleotide is phosphorylated at said first 5' terminal antisense
nucleotide's 5' carbon position; and [0058] (b) a sense strand,
wherein said sense strand is comprised of a first 5' terminal sense
nucleotide and a second 5' terminal sense nucleotide, wherein said
first 5' terminal sense nucleotide comprises a first 2' carbon
sense modification and said second 5' terminal sense nucleotide
comprises a second 2' carbon sense modification.
[0059] Preferably, the 2'-modifications are 2'-O-alkyl
modifications. According to this seventeenth embodiment, the sense
strand and the antisense strand preferably contain from 18-30 base
pairs that are at least substantially complementary.
[0060] According to an eighteenth embodiment, the present invention
is directed to a unimolecular siRNA capable of forming a hairpin
siRNA, said unimolecular siRNA comprising: [0061] (a) an antisense
region, wherein said antisense region is comprised of a first 5'
terminal antisense nucleotide and a second 5' terminal antisense
nucleotide, wherein said first 5' terminal antisense nucleotide
comprises a first 2'carbon antisense modification and said second
5' terminal antisense nucleotide comprises a second 2' carbon
antisense modification and said first 5' terminal antisense
nucleotide is phosphorylated at said first 5' terminal antisense
nucleotide's 5' carbon position; [0062] (b) a sense region, wherein
said sense region is comprised of a first 5' terminal sense
nucleotide and a second 5' terminal sense nucleotide, wherein said
first 5' terminal sense nucleotide comprises a first 2' carbon
sense modification and said second 5' terminal sense nucleotide
comprises a second 2' carbon sense modification; and [0063] (c) a
loop region, wherein said loop region is located between said sense
region and said antisense region.
[0064] According to this eighteenth embodiment, the sense region
and antisense region are substantially complementary and may form a
stable duplex. Similar to the fourteenth embodiment, according to
this eighteenth embodiment, the sense region and the antisense
region form a duplex that preferably contains from 18-30 base pairs
that are at least substantially complementary. Preferably the loop
region is located downstream of the antisense region.
[0065] According to a nineteenth embodiment, the present invention
is directed to a method for minimizing off-target effects, said
method comprising exposing an siRNA to a target nucleic acid or to
a cell that is expressing or is capable of expressing said target
nucleic acid, wherein said siRNA comprises an antisense strand and
a sense strand, wherein: [0066] (a) said sense strand is comprised
of a first 5' terminal sense nucleotide and a second 5' terminal
sense nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification; and [0067] (b) said antisense strand is comprised of
a first 5' terminal antisense nucleotide and a second 5' terminal
antisense nucleotide, wherein said first 5' terminal antisense
nucleotide comprises a first 2' carbon antisense modification and
said second 5' terminal antisense nucleotide comprises a second 2'
carbon antisense modification, and said first 5' terminal antisense
nucleotide is phosphorylated at said first 5' terminal antisense
nucleotide's 5' position.
[0068] According to a twentieth embodiment, the present invention
is directed to a method for minimizing off-target effects, said
method comprising exposing a unimolecular siRNA that is capable of
forming a hairpin siRNA to a target nucleic acid or to a cell that
is expressing or is capable of expressing said target nucleic acid,
wherein said unimolecular siRNA comprises an antisense region and a
sense region, wherein: [0069] (a) said sense region is comprised of
a first 5' terminal sense nucleotide and a second 5' terminal sense
nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification; [0070] (b) said antisense region is comprised of a
first 5' terminal antisense nucleotide and a second 5' terminal
antisense nucleotide, wherein said first 5' terminal antisense
nucleotide comprises a first 2' carbon antisense modification and
said second 5' terminal antisense nucleotide comprises a second
2'-carbon antisense modification, and said first 5' terminal
antisense nucleotide is phosphorylated at said first 5' terminal
antisense nucleotide's 5' carbon position; and [0071] (c) a loop
region, wherein said loop region is located between said sense
region and said antisense region.
[0072] Preferably the loop region is located downstream of the
antisense region.
[0073] The present invention also provides controls, tracking
agents and exaequo agents for siRNA applications. These agents
comprise the molecules of the seventeenth and eighteenth
embodiments, except that they do not contain a phosphorylated 5'
terminal antisense nucleotide. Thus, they comprise: (i) first and
second (or first, second, and third) 5' terminal antisense
nucleotides that each contain 2' carbon antisense modifications,
preferably, 2'-O-alkyl modifications, more preferably 2'-O-methyl
modifications; and (ii) first and second (or first, second, and
third) 5' terminal sense nucleotides that each contain 2' carbon
sense modifications, preferably, 2'-O-alkyl modifications, more
preferably 2'-O-methyl modifications. Under certain embodiments,
there are no modifications of any other nucleotides other than a
label, which may for example, be a fluorescent label and located on
the first 5' terminal sense nucleotide. There also may be
modifications of one or more of the sense pyrimidines with
2'-O-alkyl groups, and/or modifications of one or more of the
antisense pyrimidines with 2'-halogen groups, and/or a blocking
group on the 5' carbon of the 5' terminal nucleotides of the sense
and/or antisense strands or regions.
[0074] For a better understanding of the present invention together
with other and further advantages and embodiments, reference is
made to the following description taken in conjunction with the
examples, the scope of the which is set forth in the appended
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0075] The preferred embodiments of the present invention have been
chosen for purposes of illustration and description but are not
intended to restrict the scope of the invention in any way. The
benefits of the preferred embodiments of certain aspects of the
invention are shown in the accompanying figures, wherein:
[0076] FIG. 1A illustrates the functionality of orthoester
modifications on sense and/or antisense strands as measured 24
hours post-transfection. See "Preferred Embodiments" for a
description of modified duplexes. The degree of SEAP silencing
induced by each siRNA was determined by comparing the level of SEAP
expression in cells co-transfected with the siRNA+SEAP expression
plasmid with cells transfected with the SEAP expression plasmid
alone.
[0077] FIG. 1B illustrates the functionality of orthoester
modifications (on sense and/or antisense strands) as measured 48
hours post-transfection.
[0078] FIG. 2A illustrates the functionality of orthoester
modifications (on sense and/or antisense strands) in conjunction
with other modifications, as measured 24 hours
post-transfection.
[0079] FIG. 2B illustrates the functionality of orthoester
modifications on sense and/or antisense strands in conjunction with
other modifications, as measured 72 hours post-transfection.
[0080] FIG. 2C illustrates the functionality of orthoester
modifications on sense and/or antisense strands in conjunction with
other modifications as measured 144 hours post-transfection.
[0081] FIG. 3 illustrates the effects of sense and antisense strand
modifications on siRNA functionality.
[0082] FIG. 4 illustrates the effects of sense and antisense strand
modifications on siRNA functionality.
[0083] FIG. 5 illustrates the effects of thio-based modifications
of an antisense strand in conjunction with various modifications on
the sense strand.
[0084] FIG. 6 illustrates the effects of phosphorothioate
modifications in both sense and antisense strands.
[0085] FIG. 7 illustrates the effects of 2'-O-methyl modifications
(in both sense and antisense strands) in conjunction with a variety
of other modifications.
[0086] FIG. 8 illustrates the effects of siRNAs that are
2'-deoxy-RNA hybrids (on either strand) in conjunction with other
chemical modifications.
[0087] FIG. 9 illustrates the functionality of a cholesterol
conjugate at the 5' end of a sense strand in conjunction with other
chemical modifications.
[0088] FIG. 10 illustrates the functionality of a PEG conjugate at
the 5' end of a sense strand in conjunction with other chemical
modifications.
[0089] FIG. 11 illustrates the increased potency of modified siRNAs
having a cholesterol conjugate at the 5' end of a sense strand.
[0090] FIG. 12 illustrates protected RNA nucleotide
phosphoramidites that can be used for Dharmacon 2'-ACE RNA
synthesis chemistry.
[0091] FIG. 13 illustrates an outline of a Dharmacon RNA synthesis
cycle.
[0092] FIG. 14 illustrates the structure of a preferred 2'-ACE
protected RNA.
[0093] FIG. 15A illustrates functional consequences of a single
2'-deoxy modification (sense or antisense strand) on an otherwise
naked siRNA.
[0094] FIG. 15B illustrates the functional consequences of two
tandem 2'-deoxy modifications (sense or antisense strand) on an
otherwise naked siRNA.
[0095] FIG. 15C illustrates the functional consequences of three
tandem 2'-deoxy modifications (sense or antisense strand) on an
otherwise naked siRNA.
[0096] FIG. 16A illustrates the functionality consequences of a
single 2'-O-methyl modification (sense or antisense strand) on an
otherwise naked siRNA.
[0097] FIG. 16B illustrates the functionality consequences of two
tandem 2'-O-methyl modifications (sense or antisense strand) on an
otherwise naked siRNA.
[0098] FIG. 16C illustrates the functional consequences of three
tandem 2'-O-methyl modifications (sense or antisense strand) on an
otherwise naked siRNA.
[0099] FIG. 17 illustrates the consequences of modifications in the
sense and the antisense strands on duplex stability.
[0100] FIG. 18 illustrates the effect of a conjugate comprising a
5' cholesterol moiety on passive uptake of naked and modified
siRNA.
[0101] FIG. 19 illustrates the functional consequences of two
tandem 2'-deoxy modifications at various positions in a sense
strand.
[0102] FIG. 20 illustrates the functional consequences of three
tandem 2'-deoxy modifications at various positions in a sense
strand.
[0103] FIG. 21 illustrates the functional consequences of a single
2'-deoxy modification at various positions in an antisense
strand.
[0104] FIG. 22 illustrates the functional consequences of two
tandem 2'-deoxy modifications at various positions in an antisense
strand.
[0105] FIG. 23 illustrates the functional consequences of three
tandem 2'-deoxy modifications at various positions in an antisense
strand.
[0106] FIG. 24 illustrates the functional consequences of two
tandem 2'-O-methyl modifications at various positions in a sense
strand.
[0107] FIG. 25 illustrates the functional consequences of three
tandem 2'-O-methyl modifications at various positions in a sense
strand.
[0108] FIG. 26 illustrates the functional consequences of a single
2'-O-methyl modification at various positions in an antisense
strand.
[0109] FIG. 27 illustrates the functional consequences of two
tandem 2'-O-methyl modifications at various positions in an
antisense strand.
[0110] FIG. 28 illustrates the functional consequences of three
tandem 2'-O-methyl modifications at various positions in an
antisense strand.
[0111] FIG. 29 illustrates the functional consequences of two
2'-O-methyl modifications on positions 1 and 2 of (1) the 5' sense,
(2) the 5' antisense, or (3) the 5' sense and antisense strands,
using siRNAs directed against the human cyclophilin gene.
[0112] FIG. 30 illustrates the functional consequences of two
2'-O-methyl modifications on positions 1 and 2 of (1) the 5' sense,
(2) the 5' antisense, and (3) the 5' sense and antisense strands
using siRNAs directed against the firefly luciferase gene. Note: in
this figure, "p" represents phosphorylation of the 5' end of the
antisense strand.
[0113] FIG. 31 illustrates the functional consequences of two
2'-O-methyl modifications on positions 1 and 2 of (1) the 5' sense,
(2) the 5' antisense, and (3) the 5' sense and antisense strands
using siRNAs directed against the firefly luciferase gene. Note: in
this figure, "p" represents phosphorylation of the 5' end of the
antisense strand.
[0114] FIG. 32 illustrates the stability of modified siRNAs in
human serum. Modification "a"=2'-O-methyl modification of all Cs
and Us (in combination with 3' idT capping) of the sense strand.
Modification "b"=2' F modification of all Cs and Us (in combination
with 3' idT capping) of the sense strand.
[0115] FIG. 33 illustrates the affinity of modified
siRNA-cholesterol conjugates for albumin and other serum
proteins.
[0116] FIG. 34 illustrates the effects that small molecule
conjugates have on siRNA potency.
[0117] FIG. 35 illustrates the stability of siRNA conjugates in
human serum. siRNAa carries (1) 2'-O-methyl groups on all Cs and
Us, and (2) a 3' idT cap, on the sense strand. siRNAa-conjugate
carries (1) 2'-O-methyl groups on all Cs and Us, (2) a 3' idT cap,
and (3) a 5' cholesterol conjugate, on the sense strand.
[0118] FIG. 36 illustrates the effects that cholesterol conjugates
have on passive siRNA uptake.
[0119] FIG. 37A is a representation of results from a typical serum
stability experiment that shows an ethidium bromide stained gel
containing siRNA (unmodified, top, and "molecule 1 modifications"
modified, bottom) that have been exposed to serum. For the purposes
of this figure, the term "molecule 1 modifications" refers to
molecules that contain 2'-O-methyl modifications on positions 1 and
2 of the sense strand, 2'-O-methyl modifications on all Cs and Us
of the sense strand, 2'-Fluoro (Fl) modification of all Cs and Us
of the antisense strand, and a phosphate modification on the 5'
terminus of the antisense strand. FIG. 37B is a set of line graphs
that plot the relative stability of four human cyclophilin B siRNAs
in modified and unmodified forms (U1=5'GAA AGA GCA UCU ACG GUG A
(SEQ. ID NO. 315), U2=5'GAA AGG AUU UGG CUA CAA A (SEQ. ID NO.
316), U3=5'ACA GCA AAU UCC AUC GUG U (SEQ. ID NO. 317), and
U4=5'GGA AAG ACU GUU CCA AAA A, (SEQ. ID NO. 318), sense strands).
The X-axis represents time in hours. The Y-axis represents
percentage of duplexes that remain intact. Black squares represent
unmodified sequences. White squares represent modified
sequences.
[0120] FIG. 38 compares the ability of siRNA (U1 and U3) that are
naked or modified (molecule 1 modifications) to silence a given
target at varying concentrations. The Y-axis represents the level
of expression relative to controls (untransfected cells). The
X-axis represents the concentration of siRNA during the
transfection procedures. Black bars represent unmodified sequences.
White bars represent modified sequences. For the purposes of this
figure, the term "molecule 1 modifications" refers to molecules
that contain 2'-O-methyl modifications on positions 1 and 2 of the
sense strand, 2'-O-methyl modifications on all Cs and Us of the
sense strand, 2'-Fluoro (Fl) modification of all Cs and Us of the
antisense strand, and a phosphate modification on the 5' terminus
of the antisense strand.
[0121] FIG. 39 assesses the level of silencing induced by modified
(carrying "molecule 1 modifications) and unmodified siRNA over the
course of seven days. The Y-axis represents the level of gene
expression relative to controls (untransfected cells). The X-axis
represents the number of days after transfection. Black bars
represent unmodified sequences. White bars represent modified
sequences. For the purposes of this figure, the term "molecule 1
modifications" refers to molecules that contain 2'-O-methyl
modifications on positions 1 and 2 of the sense strand, 2'-O-methyl
modifications on all Cs and Us of the sense strand, 2'-Fluoro (Fl)
modification of all Cs and Us of the antisense strand, and a
phosphate modification on the 5' terminus of the antisense
strand.
[0122] FIG. 40 compares the level of cell death induced by four
separate modified (according to molecule 1 modifications) and
unmodified siRNAs transfected into cells at varying concentrations.
The Y-axis represents the relative level of cell viability (as
compared to untransfected cells). The X-axis represents the
concentration of the siRNA during the transfection procedures.
Cultures were tested 24-48 hours after transfection using an Alamar
Blue assay. Black bars represent unmodified sequences. White bars
represent modified sequences. For the purposes of this figure, the
term "molecule 1 modifications" refers to molecules that contain
2'-O-methyl modifications on positions 1 and 2 of the sense strand,
2'-O-methyl modifications on all Cs and Us of the sense strand,
2'-Fluoro (Fl) modification of all Cs and Us of the antisense
strand, and a phosphate modification on the 5' terminus of the
antisense strand.
[0123] FIG. 41 compares the level of off-targeting by four separate
human cyclophilin B siRNAs in modified (according to molecule 1
modifications) and unmodified forms. Data are divided into six
separate groups: number of targets that show decreased expression
by more than four fold (4.times.), number of targets that show
decreased expression by three to four fold (3-4.times.), number of
targets that show decreased expression by 2.5-3 fold
(2.5-3.times.), number of targets that show increased expression by
greater than four fold (>4.times.), number of targets that show
increased expression by three to four fold (3-4.times.), and number
of targets that show increased expression by 2.5-3 fold
(2.5-3.times.). Black bars represent unmodified sequences. White
bars represent modified sequences. For the purposes of this figure,
the term "molecule 1 modifications" refers to molecules that
contain 2'-O-methyl modifications on positions 1 and 2 of the sense
strand, 2'-O-methyl modifications on all Cs and Us of the sense
strand, 2'-Fluoro (Fl) modification of all Cs and Us of the
antisense strand, and a phosphate modification on the 5' terminus
of the antisense strand.
[0124] FIG. 42 shows a histogram comparing the gene silencing
ability of Cyclo14 siRNA containing 1) molecule 2 modifications 2)
naked molecules modified with Cy3, and 3) naked NSC9 molecules
modified with Cy3. The Y-axis depicts the level of human
cyclophilin B expression relative to a control gene (GAPDH).
"Lipid" represents control cells treated with the transfection
reagent, Lipofectamine 2000. "Control" represents cells that are
untreated. NS9 is non-specific sequence #9. For the purposes of
this figure, the term "molecule 2 modifications" refers to
molecules that contain 2'-O-methyl modifications on positions 1 and
2 of the sense strand, 2'-O-methyl modifications on all Cs and Us
of the sense strand, a Cy3 label on the 5' end of the sense strand,
2'-Fluoro (Fl) modifications on all Cs and Us of the antisense
strand, and a phosphate modification on the 5' terminus of the
antisense strand.
[0125] FIGS. 43A and 43B depict the relationship between
modification and function for 2'-O-methylated SEAP-2217 siRNA. The
figures demonstrate the effect of gene silencing of single base
(black bars) and paired (gray) 2'-O-methyl modifications of the
sense strand (FIG. 43A) and antisense strand (FIG. 43B) of
SEAP-2217. The X-axis represents the relative position of the
modification along the siRNA (5'.fwdarw.3'). The Y-axis represents
the percent expression relative to controls.
[0126] FIGS. 44A and 44B show the effects of the addition of
2'-deoxy groups on SEAP-2217 function. Specifically, three
consecutive nucleotides (e.g., positions 1, 2, and 3) are modified
with the 2'-deoxy group. The identification number associated with
each group (e.g., S3D-16 is decoded as follows: "S"=sense strand
modification, "3"=the number of nucleotides that are consecutively
modified, "D"=2' deoxy modification, "16"=the first position of the
modification). FIG. 44A demonstrates sense strand modifications.
FIG. 44B demonstrates antisense strand modifications. Controls
include: no siRNA and non-specific (ns) RNA.
[0127] FIGS. 45A-45F show the effects of 2'-O-methylation with or
without 5' phosphorylation on the antisense strands of six
different luciferase-specific siRNA (luc 8, 18, 56, 58, 63, and
81). "S"=sense strand. "AS"=antisense strand. "*" indicates
2'-O-methylation at positions 1 and 2 of the designated strand. "p"
indicates 5' phosphorylation of the designated strand. The Y-axis
represents the % expression of control (untransfected) cells.
"Control"=mock transfected cells.
[0128] FIGS. 46A-E are representations of the dosage dependence of
five luciferase-specific siRNAs (luc 8, 56, 58, 63, 81) produced
according to a certain embodiment of the present invention.
"S"=sense strand. "AS"=antisense strand. "M" indicates
2'-O-methylation at positions 1 and 2 of the designated strand. "p"
indicates 5'phosphorylation of the designated strand. The Y-axis
represents the % expression of control (untransfected) cells. The
X-axis represents the concentration of the siRNA during the
transfection with 1=200 nM, 2=100 nM, 3=10 nM, 4=1 nM, 5=0.1 nM,
and 6=0.01 nM.
[0129] FIG. 47 is a representation of the possible kinetics of
sense and antisense strand-RISC interactions, and the sensitivity
of this interaction to: (1) 2'-O-methylation (*); and (2) the
relative functionality of the molecule. In highly functional
molecules (e.g. >F90, greater than 90% functionality), RISC
shows a strong preference for association with the antisense strand
(K.sub.overall=100). Modification of the sense strand with
2'-O-methyl groups at nucleotide positions 1 and 2 further biases
the AS strand preference, but the relative percent change is minor.
Sense and antisense strands from poor or moderately functional
duplexes exhibit a more balanced equilibrium with RISC
(K.sub.overall.about.1). In these cases, modification of S
essentially eliminates this strand's ability to associate with RISC
and strongly biases the preferences toward RISC-AS
interactions.
[0130] FIGS. 48A and 48B are representations of competition studies
between cyclo4 and NS4 siRNA. A-I: Cyclo4 siRNA was transfected
into cells at varying concentrations, A-II: Cyclo 4 (at varying
concentrations) plus a Cy3 conjugated 19 bp NS4 duplex modified
with 2'-O-methyl groups on positions 1 and 2 of the sense and
antisense strand, 2'-O-methyl groups on the Cs and Us of the sense
strand, 2' Fl groups on the Cs and Us of the antisense strand, and
a Cy3 group on the 5' end of the sense strand; A-III: Cyclo 4 (at
varying concentrations) plus a Cy3 conjugated 17 bp NS4 duplex
modified with 2'-O-methyl groups on positions 1 and 2 of the sense
and antisense strand, 2'-O-methyl groups on the Cs and Us of the
sense strand, and 2' Fl groups added to the Cs and Us of the
antisense strand; B-I Cyclo 4 (at varying concentrations) plus a 19
bp NS4 duplex; B-II: Cyclo 4 (at varying concentrations) plus a 19
bp NS4 duplex modified with 2'-O-methyl groups on positions 1 and 2
of the sense and antisense strand, 2'-O-methyl groups on the Cs and
Us of the sense strand, and 2' Fl groups on the Cs and Us of the
antisense strand; B-III: Cyclo 4 (at varying concentrations) plus a
17 bp NS4 duplex modified with 2'-O-methyl groups on positions 1
and 2 of the sense and antisense strand, 2'-O-methyl groups on the
Cs and Us of the sense strand, and 2' Fl groups added to the Cs and
Us of the antisense strand. Note: All transfections were performed
using Lipofectamine 2000 (manufacturer's instructions) and total
nucleic acid concentrations of 100 nM. Data was normalized to
internal GAPDH levels.
[0131] FIG. 49 is a representation of competition studies between
GAPDH and Non-specific Control #2. GAPDH siRNA were introduced into
HeLa cells at varying concentrations (0.781-100 nM) along with: (1)
a non-competing siRNA, cyclo14; (2) a competing siRNA (non-specific
sequence #2); or (3) the competing siRNA labeled with 2'-O-methyl
groups on positions 1 and 2 of both the sense and antisense
strand.
DETAILED DESCRIPTION
[0132] The present invention will now be described in connection
with preferred embodiments. These embodiments are presented to aid
in an understanding of the present invention and are not intended,
and should not be construed, to limit the invention in any way. All
alternatives, modifications and equivalents that may become
apparent to those of ordinary skill upon reading this disclosure
are included within the spirit and scope of the present
invention.
[0133] This disclosure is not a primer on compositions and methods
for performing RNA interference. Basic concepts known to those
skilled in the art have not been set forth in detail.
[0134] The present invention is directed to compositions and
methods for performing RNA interference, including siRNA-induced
gene silencing. Through the use of the present invention, modified
polynucleotides, and derivatives thereof, one may improve the
efficiency of RNA interference applications.
[0135] Unless stated otherwise, the following terms and phrases
have the meanings provided below:
[0136] Alkyl
[0137] The term "alkyl" refers to a hydrocarbyl moiety that can be
saturated or unsaturated, and substituted or unsubstituted. It may
comprise moieties that are linear, branched, cyclic and/or
heterocyclic, and contain functional groups such as ethers,
ketones, aldehydes, carboxylates, etc.
[0138] Exemplary alkyl groups include but are not limited to
substituted and unsubstituted groups of methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, eicosyl and alkyl groups of higher number of
carbons, as well as 2-methylpropyl, 2-methyl-4-ethylbutyl,
2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl,
6,6-dimethyloctyl, 6-propyl-6-butyloctyl, 2-methylbutyl,
2-methylpentyl, 3-methylpentyl, and 2-ethylhexyl. The term alkyl
also encompasses alkenyl groups, such as vinyl, allyl, aralkyl and
alkynyl groups.
[0139] Substitutions within an alkyl group can include any atom or
group that can be tolerated in the alkyl moiety, including but not
limited to halogens, sulfurs, thiols, thioethers, thioesters,
amines (primary, secondary, or tertiary), amides, ethers, esters,
alcohols and oxygen. The alkyl groups can by way of example also
comprise modifications such as azo groups, keto groups, aldehyde
groups, carboxyl groups, nitro, nitroso or nitrile groups,
heterocycles such as imidazole, hydrazino or hydroxylamino groups,
isocyanate or cyanate groups, and sulfur containing groups such as
sulfoxide, sulfone, sulfide, and disulfide.
[0140] Further, alkyl groups may also contain hetero substitutions,
which are substitutions of carbon atoms, by for example, nitrogen,
oxygen or sulfur. Heterocyclic substitutions refer to alkyl rings
having one or more heteroatoms. Examples of heterocyclic moieties
include but are not limited to morpholino, imidazole, and
pyrrolidino.
[0141] 2'-O-alkyl Modified Nucleotide
[0142] The phrase "2'-O-alkyl modified nucleotide" refers to a
nucleotide unit having a sugar moiety, for example a deoxyribosyl
moiety that is modified at the 2' position such that an oxygen atom
is attached both to the carbon atom located at the 2' position of
the sugar and to an alkyl group.
[0143] Amine and 2' amine Modified Nucleotide
[0144] The term "amine" refers to moieties that can be derived
directly or indirectly from ammonia by replacing one, two, or three
hydrogen atoms by other groups, such as, for example, alkyl groups.
Primary amines have the general structures RNH.sub.2 and secondary
amines have the general structure R.sub.2NH. The phrase "2' amine
modified nucleotide" refers to a nucleotide unit having a sugar
moiety that is modified with an amine or nitrogen-containing group
attached to the 2' position of the sugar.
[0145] The term amine includes, but is not limited to methylamine,
ethylamine, propylamine, isopropylamine, aniline, cyclohexylamine,
benzylamine, polycyclic amines, heteroatom substituted aryl and
alkylamines, dimethylamine, diethylamine, diisopropylamine,
dibutylamine, methylpropylamine, methylhexylamine,
methylcyclopropylamine, ethylcylohexylamine, methylbenzylamine,
methycyclohexylmethylamine, butylcyclohexylamine, morpholine,
thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine,
piperazine, and heteroatom substituted alkyl or aryl secondary
amines.
[0146] Antisense Strand
[0147] The phrase "antisense strand" as used herein, refers to a
polynucleotide or region of a polynucleotide that is substantially
or 100% complementary to a target nucleic acid of interest. An
antisense strand may be comprised of a polynucleotide region that
is RNA, DNA or chimeric RNA/DNA. For example, an antisense strand
may be complementary, in whole or in part, to a molecule of
messenger RNA, an RNA sequence that is not mRNA (e.g., tRNA, rRNA
and hnRNA) or a sequence of DNA that is either coding or
non-coding. The phrase "antisense strand" includes the antisense
region of both polynucleotides that are formed from two separate
strands, as well as unimolecular siRNAs that are capable of forming
hairpin structures. The phrases "antisense strand" and "antisense
region" are intended to be equivalent and are used interchangeably.
The antisense strand can be modified with a diverse group of small
molecules and/or conjugates.
[0148] 2' Carbon Modification
[0149] The phrase "2' carbon modification" refers to a nucleotide
unit having a sugar moiety, for example a deoxyribosyl moiety that
is modified at the 2' position. A "2'-O-alkyl modified nucleotide"
is modified at this position such that an oxygen atom is attached
both to the carbon atom located at the 2' position of the sugar and
to an alkyl group, e.g., 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl,
2'-O-isopropyl, 2'-O-butyl, 2-O-isobutyl, 2'-O-ethyl-O-methyl
(--OCH.sub.2CH.sub.2OCH.sub.3), and 2'-O-ethyl-OH
(--OCH.sub.2CH.sub.2OH). A "2' carbon sense modification" refers to
a modification at the 2' carbon position of a nucleotide on the
sense strand or within a sense region of polynucleotide. A "2'
carbon antisense modification" refers to a modification at the 2'
carbon position of a nucleotide on the antisense strand or within
an antisense region of polynucleotide.
[0150] Complementary
[0151] The term "complementary" refers to the ability of
polynucleotides to form base pairs with one another. Base pairs are
typically formed by hydrogen bonds between nucleotide units in
antiparallel polynucleotide strands or regions. Complementary
polynucleotide strands or regions can base pair in the Watson-Crick
manner (e.g., A to T, A to U, C to G), or in any other manner that
allows for the formation of stable duplexes.
[0152] Perfect complementarity or 100% complementarity refers to
the situation in which each nucleotide unit of one polynucleotide
strand or region can hydrogen bond with each nucleotide unit of a
second polynucleotide strand or region. Less than perfect
complementarity refers to the situation in which some, but not all,
nucleotide units of two strands or two regions can hydrogen bond
with each other. For example, for two 20-mers, if only two base
pairs on each strand can hydrogen bond with each other, the
polynucleotide strands or regions exhibit 10% complementarity. In
the same example, if 18 base pairs on each strand or each region
can hydrogen bond with each other, the polynucleotide strands
exhibit 90% complementarity. Substantial complementarity refers to
polynucleotide strands or regions exhibiting 90% or greater
complementarity.
[0153] Conjugate and Terminal Conjugate
[0154] The term "conjugate" refers to a molecule or moiety that
alters the physical properties of an siRNA such as those that
increase stability and/or facilitate uptake of siRNA by itself. A
"terminal conjugate" may be attached directly or through a linker
to the 3' and/or 5' end of an siRNA. An internal conjugate may be
attached directly or indirectly through a linker to a base, to the
2' position of the ribose, or to another suitable position or
positions, for example, 5-aminoallyl uridine.
[0155] In an siRNA having two separate strands that are not
attached to one another (i.e., an siRNA that is not a unimolecular
or hairpin siRNA), one or both 5' ends of the strands can bear a
conjugate, and/or one or both 3' ends of the strands comprising the
siRNA can bear a conjugate.
[0156] Conjugates may, for example, be amino acids, peptides,
polypeptides, proteins, antibodies, antigens, toxins, hormones,
lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers
such as polyethylene glycol and polypropylene glycol, as well as
analogs or derivatives of all of these classes of substances.
Additional examples of conjugates also include steroids, such as
cholesterol, phospholipids, di- and tri-acylglycerols, fatty acids,
hydrocarbons that may or may not contain unsaturation or
substitutions, enzyme substrates, biotin, digoxigenin, and
polysaccharides. Still other examples include thioethers such as
hexyl-S-tritylthiol, thiocholesterol, acyl chains such as
dodecandiol or undecyl groups, phospholipids such as
di-hexadecyl-rac-glycerol, triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines,
polyethylene glycol, adamantane acetic acid, palmityl moieties,
octadecylamine moieties, hexylaminocarbonyl-oxycholesterol,
farnesyl, geranyl and geranylgeranyl moieties.
[0157] Conjugates can also be detectable labels. For example,
conjugates can be fluorophores. Conjugates can include fluorophores
such as TAMRA, BODIPY, Cyanine derivatives such as Cy3 or Cy5
Dabsyl, fluoroscein, or any other suitable fluorophore known in the
art.
[0158] A conjugate may be attached to any position on the terminal
nucleotide that is convenient and that does not substantially
interfere with the desired activity of the siRNA(s) that bear it,
for example the 3' or 5' position of a ribosyl sugar. A conjugate
substantially interferes with the desired activity of an siRNA if
it adversely affects its functionality such that the ability of the
siRNA to mediate RNA interference is reduced by greater than 80% in
an in vitro assay employing cultured cells, where the functionality
is measured at 24 hours post transfection.
[0159] Deoxynucleotide
[0160] The term "deoxynucleotide" refers to a nucleotide or
polynucleotide lacking an OH group at the 2' or 3' position of a
sugar moiety with appropriate bonding and/or 2',3' terminal
dideoxy, instead having a hydrogen bonded to the 2' and/or 3'
carbon.
[0161] Deoxyribonucleotide
[0162] The terms "deoxyribonucleotide" and "DNA" refer to a
nucleotide or polynucleotide comprising at least one ribosyl moiety
that has an H at its 2' position of a ribosyl moiety.
[0163] Downstream
[0164] One region of a strand of nucleotides is considered to be
downstream of a second region, if the 5' most portion of the first
region is the closest portion of that region to the 3' end of the
second region.
[0165] Exaequo Agent
[0166] The phrase "exaequo agent" refers to a nucleic acid that is,
from the perspective of its participation in the RNAi pathway, or
ability to compete with other nucleic acids for the ability to
participate in the RNAi pathway, inert or semi-inert. Molecules can
be used as an exaequo agent whereby said agents are used to
equalize or to make level the total amount of nucleic acid in a
solution.
[0167] First 5' Terminal Antisense Nucleotide
[0168] The phrase "first 5' terminal antisense nucleotide" refers
to the nucleotide of the antisense strand or region that is located
at the 5' most position of that strand with respect to the bases of
the antisense strand or region that have corresponding
complementary bases on the sense strand or region. Thus, in an
siRNA that is made of two separate strands (i.e., not a
unimolecular or hairpin siRNA), it refers to the 5' most base other
than bases that are part of any 5' overhang on the antisense
strand. When the first 5' terminal antisense nucleotide is part of
a hairpin molecule, the term "terminal" refers to the 5' most
relative position within the antisense region and thus is the 5'
most nucleotide of the antisense region.
[0169] First 5' Terminal Sense Nucleotide
[0170] The phrase "first 5' terminal sense nucleotide" is defined
in reference to the antisense nucleotide. In molecules that are
comprised of two separate strands (i.e., not a unimolecular or
hairpin siRNA), it refers to the nucleotide of the sense strand
that is located at the 5' most position of that strand with respect
to the bases of the sense strand that have corresponding
complementary bases on the antisense strand. Thus, in an siRNA that
is made of two separate strands (i.e., not a unimolecular or
hairpin siRNA), it is the 5' most base other than bases that are
part of any 5' overhang on the sense strand or region. When the
first 5' terminal sense nucleotide is part of a unimolecular siRNA
that is capable of forming a hairpin molecule, the term "terminal"
refers to the relative position within the sense strand or region
as measured by the distance from the base complementary to the
first 5' terminal antisense nucleotide.
[0171] Functional
[0172] SiRNA may be divided into five (5) groups (non-functional,
semi-functional, functional, highly functional, and
hyper-functional) based on the level or degree of silencing that
they induce in cultured cell lines. As used herein, these
definitions are based on a set of conditions where the siRNA is
transfected into said cell line at a concentration of 100 nM and
the level of silencing is tested at a time of roughly 24 hours
after transfection, and not exceeding 72 hours after transfection.
In this context, "non-functional siRNA" are defined as those siRNA
that induce less than 50% (<50%) target silencing.
"Semi-functional siRNA" induce 50-79% target silencing. "Functional
siRNA" are molecules that induce 80-95% gene silencing.
"Highly-functional siRNA" are molecules that induce greater than
95% gene silencing. "Hyperfunctional siRNA" are a special class of
molecules. For purposes of this document, hyperfunctional siRNA are
defined as those molecules that: (1) induce greater than 95%
silencing of a specific target when they are transfected at
subnanomolar concentrations (i.e., less than one nanomolar); and/or
(2) induce functional (or better) levels of silencing for greater
than 96 hours. These relative functionalities (though not intended
to be absolutes) may be used to compare siRNAs to a particular
target for applications such as functional genomics, target
identification and therapeutics.
[0173] Functional Dose
[0174] A "functional dose" refers to a dose of siRNA that will be
effective at causing a greater than or equal to 95% reduction in
mRNA at levels of 100 nM at 24, 48, 72, and 96 hours following
administration, while a "marginally functional dose" of siRNA will
be effective at causing a greater than or equal to 50% reduction of
mRNA at 100 nM at 24 hours following administration and a
"non-functional dose" of RNA will cause a less than 50% reduction
in mRNA levels at 100 nM at 24 hours following administration.
[0175] Halogen
[0176] The term "halogen" refers to an atom of either fluorine,
chlorine, bromine, iodine or astatine. The phrase "2' halogen
modified nucleotide" refers to a nucleotide unit having a sugar
moiety that is modified with a halogen at the 2' position, attached
directly to the 2' carbon.
[0177] 2' Halogen Modified pyrimidine
[0178] The phrase "2' halogen modified pyrimidine" refers to a
pyrimidine (e.g. cytosine or uracil) that contains a halogen group
attached to the 2' carbon of the sugar of a nucleotide.
[0179] Internucleotide Linkage
[0180] The phrase "internucleotide linkage" refers to the type of
bond or link that is present between two nucleotide units in an
siRNA and may be modified or unmodified. The phrase "modified
internucleotide linkage" includes all modified internucleotide
linkages now known in the art or that come to be known and that,
from reading this disclosure, one skilled in the art will conclude
is useful in connection with the present invention. Internucleotide
linkages may have associated counterions, and the term is meant to
include such counterions and any coordination complexes that can
form at the internucleotide linkages.
[0181] Modifications of internucleotide linkages include, but are
not limited to, phosphorothioates, phosphorodithioates,
methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonate,
3'-alkylene phosphonates, borontrifluoridates, borano phosphate
esters and selenophosphates of 3'-5' linkage or 2'-5' linkage,
phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate
linkages, alkyl phosphonates, alkylphosphonothioates,
arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates,
phosphinates, phosphoramidates, 3'-alkylphosphoramidates,
aminoalkylphosphoramidates, thionophosphoramidates,
phosphoropiperazidates, phosphoroanilothioates,
phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates,
carbamates, methylenehydrazos, methylenedimethylhydrazos,
formacetals, thioformacetals, oximes, methyleneiminos,
methylenemethyliminos, thioamidates, linkages with riboacetyl
groups, aminoethyl glycine, silyl or siloxane linkages, alkyl or
cycloalkyl linkages with or without heteroatoms of, for example, 1
to 10 carbons that can be saturated or unsaturated and/or
substituted and/or contain heteroatoms, linkages with morpholino
structures, amides, polyamides wherein the bases can be attached to
the aza nitrogens of the backbone directly or indirectly, and
combinations of such modified internucleotide linkages within an
siRNA.
[0182] Linker
[0183] A "linker" is a moiety that attaches other moieties to each
other such as a nucleotide and its conjugate. A linker may be
distinguished from a conjugate in that while a conjugate increases
the stability and/or ability of a molecule to be taken up by a
cell, a linker merely attaches a conjugate to the molecule that is
to be introduced into the cell.
[0184] By way of example, linkers can comprise modified or
unmodified nucleotides, nucleosides, polymers, sugars and other
carbohydrates, polyethers such as, for example, polyethylene
glycols, polyalcohols, polypropylenes, propylene glycols, mixtures
of ethylene and propylene glycols, polyalkylamines, polyamines such
as spermidine, polyesters such as poly(ethyl acrylate),
polyphosphodiesters, and alkylenes. An example of a conjugate and
its linker is cholesterol-TEG-phosphoramidites, wherein the
cholesterol is the conjugate and the tetraethylene glycol and
phosphate serve as linkers.
[0185] Nucleotide
[0186] The term "nucleotide" refers to a ribonucleotide or a
deoxyribonucleotide or modified form thereof, as well as an analog
thereof. Nucleotides include species that comprise purines, e.g.,
adenine, hypoxanthine, guanine, and their derivatives and analogs,
as well as pyrimidines, e.g., cytosine, uracil, thymine, and their
derivatives and analogs.
[0187] Nucleotide analogs include nucleotides having modifications
in the chemical structure of the base, sugar and/or phosphate,
including, but not limited to, 5-position pyrimidine modifications,
8-position purine modifications, modifications at cytosine
exocyclic amines, and substitution of 5-bromo-uracil; and
2'-position sugar modifications, including but not limited to,
sugar-modified ribonucleotides in which the 2'-OH is replaced by a
group such as an H, OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2,
or CN, wherein R is an alkyl moiety as defined herein. Nucleotide
analogs are also meant to include nucleotides with bases such as
inosine, queuosine, xanthine, sugars such as 2'-methyl ribose,
non-natural phosphodiester linkages such as methylphosphonates,
phosphorothioates and peptides.
[0188] Modified bases refer to nucleotide bases such as, for
example, adenine, guanine, cytosine, thymine, and uracil, xanthine,
inosine, and queuosine that have been modified by the replacement
or addition of one or more atoms or groups. Some examples of types
of modifications that can comprise nucleotides that are modified
with respect to the base moieties, include but are not limited to,
alkylated, halogenated, thiolated, aminated, amidated, or
acetylated bases, in various combinations. More specific modified
bases include, for example, 5-propynyluridine, 5-propynylcytidine,
6-methyladenine, 6-methylguanine, N,N,-dimethyladenine,
2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine,
3-methyluridine, 5-methylcytidine, 5-methyluridine and other
nucleotides having a modification at the 5 position,
5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,
4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,
3-methylcytidine, 6-methyluridine, 2-methylguanosine,
7-methylguanosine, 2,2-dimethylguanosine,
5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides
such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,
6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as
2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,
pseudouridine, queuosine, archaeosine, naphthyl and substituted
naphthyl groups, any O-- and N-alkylated purines and pyrimidines
such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine
5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and
modified phenyl groups such as aminophenol or 2,4,6-trimethoxy
benzene, modified cytosines that act as G-clamp nucleotides,
8-substituted adenines and guanines, 5-substituted uracils and
thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,
carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated
nucleotides. Modified nucleotides also include those nucleotides
that are modified with respect to the sugar moiety, as well as
nucleotides having sugars or analogs thereof that are not ribosyl.
For example, the sugar moieties may be, or be based on, mannoses,
arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and
other sugars, heterocycles, or carbocycles. The term nucleotide is
also meant to include what are known in the art as universal bases.
By way of example, universal bases include but are not limited to
3-nitropyrrole, 5-nitroindole, or nebularine.
[0189] Further, the term nucleotide also includes those species
that have a detectable label, such as for example a radioactive or
fluorescent moiety, or mass label attached to the nucleotide.
[0190] Nucleotide Unit
[0191] The phrase "nucleotide unit" refers to a single nucleotide
residue and is comprised of a modified or unmodified nitrogenous
base, a modified or unmodified sugar, and a modified or unmodified
moiety that allows for linking of two nucleotides together or a
conjugate that precludes further linkage.
[0192] Off-Target
[0193] The term "off-target" and the phrase "off-target effects"
refer to any instance in which an siRNA or shRNA directed against a
given target causes an unintended effect by interacting either
directly or indirectly with another mRNA sequence, a DNA sequence
or a cellular protein or other moiety. For example, an "off-target
effect" may occur when there is a simultaneous degradation of other
transcripts due to partial homology or complementarity between that
other transcript and the sense and/or antisense strand of the siRNA
or shRNA
[0194] Orthoester
[0195] The term "orthoester protected" or "orthoester modified"
refers to modification of a sugar moiety in a nucleotide unit with
an orthoester. Preferably, the sugar moiety is a ribosyl moiety. In
general, orthoesters have the structure RC(OR').sub.3 wherein R'
can be the same or different, R can be an H, and wherein the
underscored C is the central carbon of the orthoester. The
orthoesters of the invention are comprised of orthoesters wherein a
carbon of a sugar moiety in a nucleotide unit is bonded to an
oxygen, which is in turn bonded to the central carbon of the
orthoester. To the central carbon of the orthoester is, in turn,
bonded two oxygens, such that in total three oxygens bond to the
central carbon of the orthoester. These two oxygens bonded to the
central carbon (neither of which is bonded to the carbon of the
sugar moiety) in turn, bond to carbon atoms that comprise two
moieties that can be the same or different. For example, one of the
oxygens can be bound to an ethyl moiety, and the other to an
isopropyl moiety. In one example, R can be an H, one R' can be a
ribosyl moiety, and the other two R' can be two 2-ethyl-hydroxyl
moieties. Orthoesters can be placed at any position on the sugar
moiety, such as, for example, on the 2', 3' and/or 5' positions.
Preferred orthoesters, and methods of making orthoester protected
polynucleotides, are described in U.S. Pat. Nos. 5,889,136 and
6,008,400.
[0196] Overhang
[0197] The term "overhang" refers to terminal non-base pairing
nucleotide(s) resulting from one strand or region extending beyond
the terminus of the complementary strand to which the first strand
or region forms a duplex. One or both of two polynucleotides or
polynucleotide regions that are capable of forming a duplex through
hydrogen bonding of base pairs may have a 5' and/or 3' end that
extends beyond the 3' and/or 5' end of complementarity shared by
the two polynucleotides or regions. The single-stranded region
extending beyond the 3' and/or 5' end of the duplex is referred to
as an overhang.
[0198] Pharmaceutically Acceptable Carrier
[0199] The phrase "pharmaceutically acceptable carrier" includes
compositions that facilitate the introduction of dsRNA, dsDNA, or
dsRNA/DNA hybrids into a cell and includes but is not limited to
solvents or dispersants, coatings, anti-infective agents, isotonic
agents, and agents that mediate absorption time or release of the
inventive polynucleotides and siRNAs.
[0200] Polynucleotide
[0201] The term "polynucleotide" refers to polymers of nucleotides,
and includes but is not limited to DNA, RNA, DNA/RNA hybrids
including polynucleotide chains of regularly and irregularly
alternating deoxyribosyl moieties and ribosyl moieties (i.e.,
wherein alternate nucleotide units have an --OH, then an --H, then
an --OH, then an --H, and so on at the 2' position of a sugar
moiety), and modifications of these kinds of polynucleotides
wherein the attachment of various entities or moieties to the
nucleotide units at any position are included. Unless otherwise
specified, or clear from context, the term "polynucleotide"
includes both unimolecular siRNAs and siRNAs comprised of two
separate strands.
[0202] Polyribonucleotide
[0203] The term "polyribonucleotide" refers to a polynucleotide
comprising two or more modified or unmodified ribonucleotides
and/or their analogs.
[0204] Ribonucleotide and Ribonucleic Acid
[0205] The term "ribonucleotide" and the phrase "ribonucleic acid"
(RNA), refer to a modified or unmodified nucleotide or
polynucleotide comprising at least one ribonucleotide unit. A
ribonucleotide unit comprises an oxygen attached to the 2' position
of a ribosyl moiety having a nitrogenous base attached in
N-glycosidic linkage at the 1' position of a ribosyl moiety, and a
moiety that either allows for linkage to another nucleotide or
precludes linkage.
[0206] RNA Interference and RNAi
[0207] The phrase "RNA interference" and the term "RNAi" are
synonymous and refer to the process by which a polynucleotide or
siRNA comprising at least one ribonucleotide unit exerts an effect
on a biological process. The process includes, but is not limited
to, gene silencing by degrading mRNA, attenuating translation,
interactions with tRNA, rRNA, hnRNA, cDNA and genomic DNA, as well
as methylation of DNA with ancillary proteins.
[0208] Second 5' Terminal Antisense Nucleotide
[0209] The phrase "second 5' terminal antisense nucleotide" refers
to the nucleotide that is immediately adjacent to the first 5'
terminal antisense nucleotide and attached to the 3' position of
the first 5' terminal antisense nucleotide. Thus, it is the second
most 5' nucleotide of the antisense strand or region within the set
of nucleotides for which there are corresponding sense
nucleotides.
[0210] Second 5' Terminal Sense Nucleotide
[0211] The phrase "second 5' terminal sense nucleotide" refers to
the nucleotide that is immediately adjacent to the first 5'
terminal sense nucleotide and attached to the 3' position of the
first 5' terminal sense nucleotide. Thus, it is the second most 5'
nucleotide of the sense strand or region within the set of
nucleotides for which there are corresponding antisense
nucleotides.
[0212] Sense Strand
[0213] The phrase "sense strand" refers to a polynucleotide or
region that has the same nucleotide sequence, in whole or in part,
as a target nucleic acid such as a messenger RNA or a sequence of
DNA. The phrase "sense strand" includes the sense region of both
polynucleotides that are formed from two separate strands, as well
as unimolecular siRNAs that are capable of forming hairpin
structures. When a sequence is provided, by convention, unless
otherwise indicated, it is the sense strand (or region), and the
presence of the complementary antisense strand (or region) is
implicit. The phrases "sense strand" and "sense region" are
intended to be equivalent and are used interchangeably.
[0214] siRNA or Short Interfering RNA
[0215] The term "siRNA" and the phrase "short interfering RNA"
refer to unimolecular nucleic acids and to nucleic acids comprised
of two separate strands that are capable of performing RNAi and
that have a duplex region that is between 18 and 30 base pairs in
length. Additionally, the term siRNA and the phrase "short
interfering RNA" include nucleic acids that also contain moieties
other than ribonucleotide moieties, including, but not limited to,
modified nucleotides, modified internucleotide linkages,
non-nucleotides, deoxynucleotides and analogs of the aforementioned
nucleotides.
[0216] siRNAs can be duplexes, and can also comprise short hairpin
RNAs, RNAs with loops as long as, for example, 4 to 23 or more
nucleotides, RNAs with stem loop bulges, micro-RNAs, and short
temporal RNAs. RNAs having loops or hairpin loops can include
structures where the loops are connected to the stem by linkers
such as flexible linkers. Flexible linkers can be comprised of a
wide variety of chemical structures, as long as they are of
sufficient length and materials to enable effective intramolecular
hybridization of the stem elements. Typically, the length to be
spanned is at least about 10-24 atoms.
[0217] When the siRNAs are hairpins, the sense strand and antisense
strand are part of one longer molecule.
[0218] Stabilized
[0219] The term "stabilized" refers to the ability of the dsRNAs to
resist degradation while maintaining functionality and can be
measured in terms of its half-life in the presence of, for example,
biological materials such as serum. The half-life of an siRNA in,
for example, serum refers to the time taken for the 50% of the
siRNA to be degraded.
[0220] Substantial Complementarity
[0221] Substantial complementarity refers to polynucleotide strands
exhibiting 90% or greater complementarity.
[0222] Trackability
[0223] The term "trackability" refers to the ability to follow the
movement or localization of a molecule after said molecule has
been, e.g., introduced into a cell. Molecules that are "trackable"
are useful in monitoring the success or failure of, e.g., a
cellular transfection procedure.
Preferred Embodiments
[0224] The present invention will now be described in connection
with preferred embodiments. These embodiments are presented in
order to aid in an understanding of the present invention and are
not intended, and should not be construed, to limit the invention
in any way. All alternatives, modifications and equivalents that
may become apparent to those of ordinary skill upon reading this
disclosure are included within the spirit and scope of the present
invention.
[0225] According to a first embodiment, the present invention
provides an siRNA. The siRNA has a sense strand that comprises a
polynucleotide comprised of at least one orthoester modified
nucleotide, and an antisense strand that comprises a polynucleotide
having at least one 2' modified nucleotide unit. Preferably, the
modified nucleotides are ribonucleotides or their analogs.
Orthoesters can be placed at any position on the sugar moiety, such
as, for example, on the 2', 3' and/or 5' positions. Preferably, the
orthoester moiety is at the 2' position of the sugar moiety.
Preferred orthoesters, and methods of making orthoester protected
polynucleotides, are described in U.S. Pat. Nos. 5,889,136 and
6,008,400. Also, preferably, orthoesters are attached at the 2'
position of a ribosyl moiety. Preferably the orthoester comprises
two 2-ethyl-hydroxyl substituents. The most preferred orthoester is
illustrated below, and is also referred to herein as a 2'-ACE or as
a 2'-bis(hydroxy ethyl) orthoester moiety:
##STR00002##
[0226] The benefits of including orthoester groups on the sense
strand can be seen by reference to FIGS. 1A, 1B, 2A, 2B, and
2C.
[0227] The data of FIG. 1 were generated using an siRNA duplex
targeting SEAP (human secreted alkaline phosphatase) synthesized
using Dharmacon, Inc.'s proprietary ACE chemistry in several
variants. These variants include naked, or unmodified, RNA; ACE
protected RNA, wherein every 2'-OH is modified with an orthoester,
and 2' fluoro modified variants, wherein the fluorine is bonded to
the 2' carbon of each and every C and U.
[0228] Duplexes of siRNA can be comprised of sense and antisense
strands. An array of all possible combinations of sense and
antisense strands was created. With reference to the figures, the
following nomenclature was used: [0229] S--naked sense strand in an
siRNA duplex [0230] AS--naked antisense strand in an siRNA duplex
[0231] pS--2'ACE protected sense strand in an siRNA duplex [0232]
pAS--2'ACE protected antisense strand in an siRNA duplex [0233]
2FS--sense strand in an siRNA duplex with all C and U's modified
such that a fluorine atom is bound to the 2' carbon of each C-
and/or U-bearing nucleotide unit. [0234] 2FAS--antisense strand in
an siRNA duplex with all C and U's modified such that a fluorine
atom is bound to the 2' carbon or each C- and/or U-bearing
nucleotide unit. [0235] S--AS, refers to duplex siRNA formed from
naked sense and naked antisense strands. [0236] pS--AS, refers to
duplex siRNA formed from an ACE modified sense strand and a naked
antisense strand. [0237] P2'F--2'-ACE modification of all
nucleotides except Cs and Us, which have a 2'F modification.
[0238] The duplexes were co-transfected with the pAAV6 plasmid
(SEAP expression plasmid) plus the siRNA of interest, or,
alternatively, the siRNA were transfected into HEK293 cells that
stably expressed SEAP. Under both conditions, standard transfection
protocols were used and results did not vary amongst Hela, MDA 75,
or 3TELi (mouse) cells lines.
[0239] The level of siRNA induced SEAP silencing was determined at
a different time points after transfection. (24, 48, 72, or 144
hours) using SEAP detection kits from Clontech according to the
manufacturer's protocols. The protein reduction levels are in good
correspondence with the mRNA reduction levels (the levels of mRNA
were measured using QuantiGene kits (Bayer)). The level of siRNA
induced toxicity was measured using AlmaBlue toxicity assay or the
levels of expression of housekeeping gene (cyclophilin) or both.
Unless specified, no significant toxicity was observed.
[0240] Each duplex was transfected into the cells at concentrations
varying between 1 and 100 nanomolar (FIG. 1) and 10 picomolar to 1
micromolar (FIG. 2). In FIGS. 1 and 2 the effects of introduction
of the ACE modifications on the sense and antisense strands of the
siRNA duplex in combination with naked and 2' fluoro modifications
are shown.
[0241] The presence of the ACE modifications on the antisense
strand of the oligos significantly interferes with the siRNA duplex
functionality. The ACE modified sense oligos were potent in the
SEAP silencing independent of whether they were used with naked or
2' F modified AS oligos.
[0242] The extent of silencing was comparable at 24, 48, 72 hours
and a detectable reduction in the siRNA silencing was observed
after 144 hours.
[0243] FIGS. 3 and 4 summarize siRNA functionality screens when AS
(FIG. 3) or Sense (FIG. 4) strands were kept constant and screened
in combination with the variety of modifications on the opposite
strand. Specifically, the AS strand was either (1) naked, (2)
modified with 2'F groups at all Cs and Us, (3) modified with
2'-deoxy groups at position 2, 4, 6, 14, 16, and 18 (counting from
the 5'end), (4) modified with 2'-O-methyl groups at all Us and Cs
with the exception of those located at positions 67-10 (counting
from the 5' end of the strand), (5) modified with phosphorothioates
at all positions, or (6) modified with phosphorothioates at
positions 1-6 and 14-19 (counting from the 5' end of the strand).
These AS strands were then paired with sense strands that were (1)
naked, (2) 2'-ACE modified at all positions, (3) 2' F modified on
all Cs and Us, (4) 2' amine (NH.sub.2) modified on all Us, (5)
2'-deoxy modified on all odd numbered nucleotides (e.g. 1,3,5 . . .
), (6) 2'-O-methyl modified at all Cs and Us, or (7) carrying
phosphorothioates at all positions. The results of these studies
demonstrate that while the described 2'-O-methyl modifications of
the antisense strand are not tolerated, other modifications
including phosphorothioate, 2' F, and 2' deoxy modifications are
permitted on this strand in combination with a variety of sense
strand modifications. Similarly, FIG. 4 shows that while amine
modifications are generally not tolerated on the sense strand, all
other modifications including 2'-ACE, 2' F, 2' deoxy, 2'-O-methyl,
and phosphorothioate additions are permitted in combination with a
variety of AS strand modifications without severally disrupting
functionality.
[0244] FIGS. 5, 6, 7 and 8 present a more detailed data grouped
based on the type of modification used. In FIG. 5, naked, 2'O-ACE
modified, or 2'F modified sense strands were paired with either:
2'F-8T AS=four phosphorothioates on each end of the strand,
starting on the second nucleotide in from each end, plus F groups
on the Cs and Us; 8T-AS=four phosphorothioates on each end of the
strand, starting on the second nucleotide in from each end;
4T-AS=Two phosphorothioates on each end of the strand starting on
the second nucleotide in from each end. In FIG. 6 antisense strands
containing phosphorothioate modifications at ever position, or at
twelve positions (AS-Thio12, six phosphorothioate modifications on
either end of the strand) were paired with complementary sense
strands that were naked; 2'-ACE modified at all positions, or
phosphorothioate modified at all positions. Alternatively, sense
strands carrying phosphorothioate modifications at all positions
were paired with antisense strands that were naked; carried 2' F
groups on all Cs and Us, carried phosphorothioates at all
positions, or carried 12 phosphorothioates (modification of six
nucleotides on each end). For FIG. 7: Antisense strands carrying
2'-Ome=2'-O-methyl modifications on all Us and Cs excluding those
that occur between nucleotide positions 7-11 and S-Ome=2'-O-methyl
modifications on Cs and Us of sense; were matched with either
naked, S-2'ACE=2'ACE modifications on all nucleotides;
S-Deoxy-hybrid=2'deoxy modifications on all odd nucleotides across
the strand (1, 3 5 . . . ) S-Ome=2'-O-methyl modification of all Cs
and Us of the sense strand; AS-2'F=2'F modifications of all Cs and
Us on the strand, AS-deoxyHybrid=2' deoxy modification of
nucleotides 2,4,6, 14, 16, 18, or As-Ome=2'-O-methy modification of
all Us and Cs excluding those that occur between nucleotide
positions 7-11. For FIG. 8: AS strands carrying deoxy modifications
(AS-deoxyHybrid=2' deoxy modification of nucleotides 2,4,6, 14, 16,
18.) were combined with sense strands that were naked, S-2'ACE
modified=2' ACE modifications on all the nucleotides;
S-Ome=2'O-methyl modifications on all Us and Cs; or 2' deoxy
(positions 1,3,5 . . . etc.). Similarly, sense-2'deoxy hybrids
(positions 1,3,5 . . . etc.) were paired with antisense strands
that were naked, 2'F modified (Cs and Us), 2'-O-methyl (Us and Cs
excluding those between positions 7 and 11), and 2'deoxy (positions
2,3,6, 14, 16, 18).
[0245] FIG. 5 in particular demonstrates that phosphorothioate
modifications are well tolerated when placed in the antisense
strand in combination with naked, 2'ACE modified and 2'F modified
sense strands.
[0246] FIG. 6 further illustrates that phosphorothioate backbone
modifications are acceptable both on the sense and antisense
strands with the same limitation of nonspecifically induced
toxicity.
[0247] FIG. 7 illustrates that presence of 2'-O-methyl
modifications are well tolerated on sense and but not antisense
strands of the siRNA duplex. It is worth mentioning that the
functional siRNA duplex is formed by the combination of the
2'-O-methyl modified AS strand and deoxyribohybrid in the sense
strand.
[0248] FIG. 8 demonstrates the suitability of the deoxyribohybrid
type modification in RNA interference. Deoxyribohybrids are RNA/DNA
hybrid oligos where deoxy and ribo entities are incorporated
together in an oligo in, for example, a sequence of alternating
deoxy- and ribonucleotides. It is important in the design of these
kinds of oligos to keep the size of continuous DNA/RNA duplex
stretches shorter than 5 nucleotides to avoid the induction of
RNAse H activity. The deoxyribohybrids were functional both in
sense and antisense strands in combination with 2' fluoro and 2'ACE
modified oligos. Also the deoxyribohybrid sense strand was the only
modification (under these conditions) that supported siRNA activity
when the antisense strand was modified with 2'-O-methyl.
[0249] FIG. 9 demonstrates the utility of a conjugate comprising
cholesterol for improvement of the potency of ACE and 2' fluoro
modified siRNAs. Employing a conjugate comprising cholesterol on
the sense strand alleviates negative effects due to modifications
to the sense strand, but does not ameliorate negative effects due
to modifications to the antisense strand.
[0250] FIG. 10 shows equivalent data for a PEG conjugate on the
sense strand.
[0251] FIG. 11 demonstrates that the presence of a conjugate
comprising cholesterol improves the potency of the modified siRNA
oligos.
[0252] FIG. 12 shows the structures of protected RNA nucleotide
phosphoramidites used in Dharmacon's 2'-ACE RNA synthesis
chemistry.
[0253] FIG. 13 outlines an RNA synthesis cycle. Preferably, the
cycle is carried out in an automated fashion on a suitable
synthesizing machine. In step (i), the incoming phosphoramidite
(here, bearing a uridine as nitrogenous base), can bear any
acceptable group on the phosphoramidite moiety at the 3' position
in place of the methyl group shown. For example, an alkyl group or
a cyanoethyl group can be employed at that position. This RNA
synthesis cycle can be carried out, with certain changes, when
synthesizing polynucleotides having modified internucleotide
linkages, and/or when synthesizing polynucleotides having other
modifications, such as at the 2' position, as described
hereinafter.
[0254] FIG. 14 illustrates the structure of a 2'-ACE protected RNA
product immediately prior to 2' deprotection. If it is desired to
retain the orthoester at the 2' position, this 2' deprotection step
is not carried out.
[0255] For a 19-mer duplex having a di-dT overhang at the 3' end,
G2'-N-UGA2'-N-UG2'-N-UA2'-N-UG2'-N-UCAGAGAG2'-NUdTdT with 2' amine
modified nucleotide units at the second, fifth, seventh, ninth,
eleventh, and nineteenth position of the sense strand, significant
loss in functionality occurred whether the antisense strand was
naked, 2' fluoro modified at all C's and U's, was a deoxyhybrid
comprising alternating ribo and deoxyribonucleotide units, or had
2'-O-methyl modifications. Preferably, the sense strand does not
comprise 2' amino modifications at the second, fifth, seventh,
ninth, eleventh, and nineteenth positions.
[0256] On an siRNA 19-mer with a 3' di-dT overhang (see SEQ. ID
NOs. 171-314), replacement of any ribonucleotide unit with a
deoxyribonucleotide unit does not significantly affect the
functionality of the 19-mer in RNAi, whether the modification is on
the sense or the antisense strand (see FIG. 15A). On the same siRNA
19-mer, replacement of two adjacent ribonucleotide units with two
deoxyribonucleotide units in tandem does not significantly affect
the functionality of the 19-mer in RNAi. FIG. 15B illustrates that
when positions 1 and 2, 3 and 4, 5 and 6, and so on, are
independently modified to be deoxyribonucleotides, functionality is
not significantly affected when the modifications are borne on the
sense strand and exhibit only a slight negative effect on
functionality when the modifications are on the antisense strand.
On the same siRNA 19-mer, replacement of three adjacent
ribonucleotide units with three deoxyribonucleotide units in tandem
does not significantly affect the functionality if the
modifications are on the antisense strand, but can significantly
affect functionality (in some instances) if the modified units are
the first through third or seventh through ninth nucleotides of the
sense strand. In this experiment, units 1 to 3, 4 to 6, 7 to 9, and
so on of the polyribonucleotide were independently replaced with
deoxyribonucleotide units (See FIG. 15C).
[0257] On the same siRNA 19-mer polyribonucleotide with 3' di-dT
overhang, modification of any individual unit with a 2'-O-methyl
moiety does not significantly affect the functionality of the
19-mer in RNAi, whether the modification is on the sense or the
antisense strand (see FIG. 16A). Using the same siRNA 19-mer,
replacement of two adjacent ribonucleotide units with two
2'-O-methyl modifications in tandem does not significantly affect
the functionality of the 19-mer in RNAi unless the modifications
are placed at the first and second or thirteenth and fourteenth
positions of the antisense strand, or the seventh and eighth
position of the sense strand (see FIG. 16B). Subsequent experiments
have identified positions of thirteen and fourteen of the antisense
strand and seven and eight of the sense strand to be less critical.
Most notably, in the absence of additional modifications that
compensate for changes in functionality, the first and second
positions of the antisense strand should not bear 2'-O-methyl
modifications if functionality is to be preserved. Using the same
siRNA 19-mer, replacement of three adjacent ribonucleotide units
with 2'-O-methyl modifications in tandem does not significantly
affect the functionality if the modifications are on the antisense
strand at positions other than the first through third positions
(see FIG. 16C). In this experiment, positions 1 to 3, 4 to 6, 7 to
9, and so on of the polyribonucleotide were independently modified
with 2'O-methyl moieties.
[0258] Modification of the same polyribonucleotide with either a
single 2'-deoxy moiety or a single 2'O-methyl moiety has no
significant affect on functionality. Modification of the first and
second or first, second and third positions of the antisense strand
with two or more tandem 2'-O-methyl moieties can significantly
reduce functionality. Positions 7 through 9 on the sense strand and
13 through 15 on the antisense strand are sensitive to two or more
tandem 2'-O-methyl modifications. Thus, preferably the antisense
strand does not comprise 2'-O-methyl modifications at the first and
second; the first, second and third; the thirteenth and fourteenth;
and the thirteenth, fourteenth and fifteenth positions unless
compensating modifications occur at other positions within the
duplex. Subsequent experiments have shown that 2'-O-methyl
modification of positions 1 and 2 (or 1 and 2, and 3) on the
antisense strand are key to duplex functionality. Modifications on
positions 7-9 on the sense strand and 13-15 on the antisense strand
are less critical
[0259] As a matter of practicality it is more economical to
synthesize a sense strand in which all of the nucleotides are
modified by an orthoester group, rather than a sense strand in
which only selected nucleotides are so modified. However, in
theory, if a practical means were developed to synthesize sense
strands in which only certain nucleotides were modified, then those
polynucleotides could be used in the present invention.
[0260] Preferably, the 2' modified nucleotide is selected from the
group consisting of a 2' halogen modified nucleotide, a 2' amine
modified nucleotide, a 2'-O-alkyl modified nucleotide, and a 2'
alkyl modified nucleotide. Where the modification is a halogen, the
halogen is preferably fluorine. When the modification is fluorine,
preferably it is attached to one or more nucleotides comprising a
cytosine or a uracil base moiety. Where the 2' modified nucleotide
is a 2' amine modified nucleotide, the amine is preferably
--NH.sub.2. Where the 2' modified nucleotide is a 2'-O-alkyl
modification, preferably the modification is a 2'-O-methyl, ethyl,
propyl, isopropyl, butyl, or isobutyl moiety and most preferably,
the 2'-O-alkyl modification is a 2'-O-methyl moiety. Where the 2'
modified nucleotide is a 2'-alkyl modification, preferably the
modification is a 2' methyl modification, wherein the carbon of the
methyl moiety is attached directly to the 2' carbon of the sugar
moiety.
[0261] FIG. 2C demonstrates that siRNA effects start to fade out
144 hours after transfection. The dose as well as potency of the
modified oligos were comparable to the naked siRNA duplex.
[0262] According to a second embodiment, the present invention
provides an siRNA comprising a sense strand where the sense strand
comprises a polynucleotide having at least one orthoester modified
nucleotide as provided for according to the first embodiment; an
antisense strand comprising a polynucleotide that has at least one
2' modified nucleotide as provided for according to the first
embodiment; and a conjugate.
[0263] The conjugate within this embodiment is preferably selected
from the group consisting of amino acids, peptides, polypeptides,
proteins, sugars, carbohydrates, lipids, polymers, nucleotides,
polynucleotides, and combinations thereof. More preferably it is
selected from the group consisting of cholesterol, polyethylene
glycol, antigens, antibodies, and receptor ligands. Even more
preferably, the conjugate comprises cholesterol or polyethylene
glycol. Most preferably, the conjugate comprises cholesterol and is
linked to the 5' terminal nucleotide unit of the sense strand at
the 5' carbon position.
[0264] Introduction of a cholesterol-containing conjugate at the 5'
terminus of the sense strand resulted in an increase in potency for
orthoester modified and 2' antisense modified siRNAs that was
comparable to or even superior to the naked, or unmodified,
duplexes. See FIGS. 9 and 11. A 5' cholesterol modification of the
sense strand resulted in a decrease in the functionally effective
dose for orthoester modified and 2' fluorine modified siRNAs that
were comparable or even superior to the corresponding naked
duplexes.
[0265] FIG. 9 demonstrates the utility of the cholesterol
modification for improvement of the potency of ACE and 2' fluoro
modified siRNAs. The positive cholesterol effect was observed with
the modifications introduced mainly on the sense and not antisense
strands.
[0266] FIG. 10 shows equivalent data for PEG sense strand
modifications.
[0267] FIG. 11 demonstrates that the presence of cholesterol
modifications improves not only the potency but the effective dose
of modified siRNA oligos
[0268] Preferably, a single conjugate is employed. Most preferably,
the conjugate is attached to the 5' terminus of the sense strand.
In order of decreasing preference, the single conjugate can be
attached to the 3' terminus of the sense strand, the 3' terminus of
the antisense strand, and the 5' terminus of the antisense
strand.
[0269] Attachment of a conjugate to an siRNA can promote uptake of
the siRNA passively, that is, in the absence of transfection agents
such as lipids or calcium chloride. For example, attachment of a
cholesterol moiety to the 5' end at the 5' position of the sense
strand of SEQ. ID NOs. 1-16 results in RNAi in the absence of
transfection agents (see FIG. 18).
[0270] According to a third embodiment, the present invention
provides an siRNA that has a sense strand comprised of at least one
orthoester modified nucleotide, an antisense strand, and a
conjugate. In this embodiment, the orthoester modification of the
first embodiment may be used in combination with the conjugate of
the second embodiment.
[0271] According to a fourth embodiment, the present invention
provides an siRNA that has a sense strand, an antisense strand, and
a conjugate, wherein the sense strand and/or the antisense strand
has at least one 2' modified nucleotide. The 2' modified nucleotide
of this embodiment is preferably selected according to the same
parameters as the 2' modified nucleotide of the first embodiment.
Similarly, the conjugate is preferably selected according to the
same parameters by which the conjugate is selected in the above
described second embodiment.
[0272] According to a fifth embodiment, the present invention
provides an siRNA having a sense strand comprised of at least one
orthoester modified nucleotide, an antisense strand comprised of at
least one 2' modified nucleotide selected from the group consisting
of a 2' halogen modified nucleotide, a 2' amine modified
nucleotide, a 2'-O-alkyl modified nucleotide, and a 2' alkyl
modified nucleotide, and a conjugate selected from the group
consisting of amino acids, peptides, polypeptides, proteins,
sugars, carbohydrates, lipids, polymers, nucleotides,
polynucleotides, and combinations thereof, wherein the
polyribonucleotide comprises between 18 and 30 nucleotide base
pairs.
[0273] The orthoester of this embodiment is selected according to
the criteria for selecting the orthoester of the first embodiment.
Where the 2' modification is a halogen, preferably it is fluorine
and is attached to at least one C- and/or U-containing nucleotide
units of the antisense strand. Where the 2' modified nucleotide is
a 2' amine modified nucleotide, the amine is preferably --NH.sub.2.
Where the 2' modified nucleotide is a 2'-O-alkyl modification,
preferably it is a 2'-O-methyl, ethyl, propyl, isopropyl, butyl, or
isobutyl moiety and most preferably, the 2'-O-alkyl modification is
a 2'-O-methyl moiety. Where the 2' modified nucleotide is a 2'
alkyl modification, preferably it is a 2' methyl modification,
wherein the carbon of the methyl moiety is attached directly to the
2' carbon of the sugar moiety.
[0274] According to a sixth embodiment, the present invention
includes a composition comprising the structures below:
##STR00003##
wherein each of B.sub.1 and B.sub.2 is a nitrogenous base,
heterocycle or carbocycle; X is selected from the group consisting
of O, S, C, and N; W is selected from the group consisting of an
OH, a phosphate, a phosphate ester, a phosphodiester, a
phosphotriester, a modified internucleotide linkage, a conjugate, a
nucleotide, and a polynucleotide; R1 is an orthoester; R2 is
selected from the group consisting of a 2'-O-alkyl group, an alkyl
group, an amine, and a halogen; and Y is a nucleotide or
polynucleotide. Where R2 is a halogen, the halogen is preferably a
fluorine. Where R2 is a fluorine, the fluorine is preferably
attached to one or more C- and/or U-containing nucleotide units.
Where R2 is an amine, the amine is preferably --NH.sub.2. Where R2
is a 2'-O-alkyl modification, preferably it is a 2'-O-methyl,
ethyl, propyl, isopropyl, butyl, or isobutyl moiety and most
preferably a 2'-O-methyl moiety. Where R2 is a 2' alkyl
modification, preferably it is a 2' methyl modification, wherein
the carbon of the methyl moiety is attached directly to the 2'
carbon of the sugar moiety.
[0275] R1, the orthoester, of this embodiment is selected according
to the parameters for selecting the orthoester of the first
embodiment.
[0276] The dashed lines in the formula indicate interaction by
hydrogen bonding between nitrogenous bases. Preferably, B.sub.1 and
B.sub.2 are naturally occurring nitrogenous bases such as, for
example, adenine, thymine, guanine, cytosine, uracil, xanthine,
hypoxanthine, and queuosine or analogs thereof. Preferably, X is an
O.
[0277] With respect to each of the above-described embodiments, the
siRNAs can be of any length, but preferably are 18-30 nucleotide
base pairs, more preferably 18-19 base pairs, excluding any
overhang. By using siRNAs of less than about 30 base pairs in
length one can avoid nonspecific processes, such as
interferon-related responses, which can reduce the functionality of
an siRNA application, while retaining a functional response in RNA
interference applications. Additionally, preferably the nucleotides
are ribonucleotides.
[0278] In the above-described embodiments, overhangs can be present
on either or both strands, at either or both ends. Preferably, if
an siRNA has an overhang, it is one to six nucleotide units in
length, more preferably two to three, and most preferably two, and
is located at the 3' end of each strand of the siRNA. However,
siRNAs with blunt ends are functional. Overhangs of two nucleotides
are most preferred.
[0279] Similarly in the above-described embodiments, either or both
strands of the siRNA can have one or more modified internucleotide
linkages. Preferably, the modified internucleotide linkages are
selected from the group consisting of phosphorothioates and
phosphorodithioates. Additionally, preferably, the polynucleotides
comprise more than 4 modified internucleotide linkages. More
preferably, the polynucleotides of the invention comprise more than
8 modified internucleotide linkages. Most preferably, about 10
modified internucleotide linkages are employed. For the greatest
amount of stability, complete modification is preferred; however, a
number of factors affect how many modified linkages can be employed
in practice. These factors include the degree of stability
conferred by the linkage, the degree to which the linkage affects
functionality, the ability to introduce the linkage chemically, and
the toxicity of the linkage. Preferably, modifications are
localized on the 3' and 5' ends to protect against exonuclease
activity.
[0280] The polynucleotides of the aforementioned embodiments of the
present invention are stabilized. The half-lives of these
stabilized siRNA are from 20 seconds to 100 or more hours.
Preferably, the stabilized siRNAs of the invention display
half-lives of greater than one hour. More preferably, the
stabilized siRNAs of the invention display half-lives of greater
than 10 hours. Most preferably, the stabilized siRNAs of the
invention display half-lives in excess of 100 hours. Additionally,
preferably the effect of the siRNAs will survive cell division for
at least one or more generations.
[0281] The polynucleotides of the invention exhibit enhanced
stability in the presence of human serum. Preferably the half life
of, for example, a 19-mer duplex of the present invention in human
serum is from several minutes to 24 hours. More preferably, the
half life of a 19-mer duplex in human serum is from 24 hours to 3
days. Most preferably, the half life of a 19-mer duplex in human
serum if from 3 to 20 or more days.
[0282] For a 19-mer polyribonucleotide duplex comprising an
antisense strand with deoxyribonucleic modifications at the second,
fourth, sixth, fourteenth, sixteenth, and eighteenth positions,
exposure to fetal bovine serum for half an hour at 37 degrees
Centigrade resulted in protection of the fourth and sixth positions
from degradation, presumably by serum nucleases. Similarly, for a
19-mer polyribonucleotide duplex comprising 2'-O-methyl
modifications on the antisense strand at the second through sixth,
twelfth, fourteenth, sixteenth and seventeenth, and nineteenth
positions resulted in protection of these positions from
degradation by serum nucleases. Introduction of phosphorothioate
modifications in the antisense strand for a 19-mer
polyribonucleotide duplex at between nucleotide units one through
six and thirteen through nineteen rendered the modified
internucleotide linkages resistant to serum nuclease degradation.
However, a 19-mer modified with an ACE orthoester moiety at each 2'
position of an antisense strand did not confer stability in human
serum, presumably due to the action not of serum ribonucleases but
of serum phosphodiesterases.
[0283] Modifications at the 2' position in the antisense strand of
a polyribonucleotide duplex, at C and U nucleotide units, greatly
enhance the stability of the polyribonucleotide duplex in serum.
FIG. 17 illustrates stability as a function of type of modification
at the 2' position on both the sense and antisense strands for
2'-O-methyl (SEQ. ID NO. 13), for 2'F (5'-2' G fU G A fU G fU A fU
G fU fC A G A G A G fU dT dT-3') (SEQ. ID NO. 17); for
phosphorothioate internucleotide linkages (SEQ. ID. NOs. 10 and 11)
and for ACE-protected (SEQ. ID. NOs. 3 and 4). The vertical axis
represents the percent of nondegraded polynucleotide versus a
control. Thus, the higher the percent stability relative to
control, the less degradation observed. From FIG. 17 it is apparent
that modifying the sense strand is sufficient to achieve
stabilization.
[0284] Modification of each C and each U with either a 2'-O-methyl
moiety or a 2' fluoro moiety results in complete stabilization of
the sense and the antisense strand. Annealing a stable sense
strand, such as one having 2' fluoro or 2'-O-methyl modifications,
to a naked antisense strand results in improved stability.
[0285] The compositions of the invention can be made according to
Dharmacon's RNA synthesis chemistry, which is based on a novel
protecting group scheme. A class of silyl ethers is used to protect
the 5'-hydroxyl (5'-SIL) in combination with an acid-labile
orthoester protecting group on the 2'-hydroxyl (2'-ACE). This set
of protecting groups is then used with standard phosphoramidite
solid-phase synthesis technology. The structures of some protected
and functionalized ribonucleotide phosphoramidites are as
illustrated in FIG. 12. As an alternative, compositions of the
invention can be made using any other suitable RNA synthesis
chemistry.
[0286] According to a seventh embodiment, the present invention
provides a method of performing RNA interference. This method is
comprised of exposing an siRNA to a target nucleic acid in order to
perform RNAi. Under this method, the siRNA is comprised of a sense
strand and an antisense strand, and at least one of said sense
strand and said antisense strand comprises at least one orthoester
modified nucleotide.
[0287] Preferably, the polynucleotides of the antisense strand
exhibit 90% or more complementarity to the target nucleic acid of
interest. More preferably, the polynucleotides antisense strand of
the invention exhibit 99% or more complementarity to the target
nucleic acid of interest. Most preferably, the polynucleotides of
the invention are perfectly complementary to the target nucleic
acid of interest over at least 18 to 19 contiguous bases.
[0288] Preferably, the at least one orthoester modified nucleotide
is located on the sense strand, and the composition of the
orthoester is defined by the parameters described above for the
first embodiment.
[0289] In addition to the orthoester modification, any of the above
described other modifications may also be present when using this
method. For example, the antisense strand preferably comprises at
least one modified nucleotide selected from the group consisting of
a 2' halogen modified nucleotide, a 2' amine modified nucleotide, a
2'-O-alkyl modified nucleotide and a 2' alkyl modified nucleotide.
Where the modified nucleotide is a 2' halogen modified nucleotide,
the halogen is preferably a fluorine. Where the halogen is a
fluorine, the fluorine is preferably attached to C- or U-containing
nucleotide units. Where the 2' modification is an amine, preferably
the amine is --NH.sub.2. Where the 2' modification is a 2'-O-alkyl
group, preferably the group is methoxy, --OCH.sub.3. Where the 2'
modification is an alkyl group, preferably the modification is a
methyl group, --CH.sub.3. Further, preferably none of these
modifications occur at nucleotides 8-11, and more preferably none
of the occur at positions 7-12 of the antisense strand.
[0290] The method can also be carried out wherein the siRNA
comprises a 5' conjugate. The conjugate can be selected according
to the above-described criteria for selecting conjugates.
[0291] When using these methods, the siRNA can be of any number of
base pairs, but is preferably is 18-30 base pairs, and more
preferably is 19 base pairs. Additionally preferably the
polynucleotide comprises an antisense strand and a sense strand of
ribonucleotides.
[0292] Overhangs of one or more base pairs at the 3' and/or 5'
terminal nucleotide units on either or both strands can also be
present according to the above-described parameters for
overhangs.
[0293] According to an eighth embodiment, the present invention
provides a method of performing RNA interference, comprised of
exposing an siRNA to a target nucleic acid, wherein the siRNA is
comprised of a sense strand, an antisense strand, and a conjugate,
where either the sense strand or the antisense strand comprises a
2' modified nucleotide. Preferably, the siRNAs of this embodiment
of the invention exhibit the same degree of complementarity as in
the previous example.
[0294] According to this embodiment, the antisense strand
preferably comprises at least one nucleotide selected from the
group consisting of a 2' halogen modified nucleotide, a 2' amine
modified nucleotide, a 2'-O-alkyl modified nucleotide and a 2'
alkyl modified nucleotide. The modification may be on the antisense
strand and/or on the sense strand. Where the modified nucleotide is
a 2' halogen modified nucleotide, the halogen is preferably
fluorine. Where the halogen is fluorine, the fluorine is preferably
attached to at least one C- or U-containing nucleotides. The
preferred 2' amine modification is --NH.sub.2. The preferred
2'-O-alkyl modification is --OCH.sub.3. The preferred 2' alkyl
modification is --CH.sub.3.
[0295] The method can also be carried out wherein the siRNA
comprises a conjugate. The conjugate is selected according to the
parameters for selecting the above-described conjugates. The siRNA
can be of any number of base pairs, but as with the previous
embodiment is preferably 18-30 base pairs, most preferably 18-19
base pairs. Similarly, overhangs of one or more base pairs on the
3' and/or 5' terminal nucleotide units on either or both strands
can be present. Further, either the sense or antisense strand can
comprise at least one modified internucleotide linkage, which
preferably is selected from the group consisting of
phosphorothioate linkages and phosphorodithioate linkages.
Preferably the sense and antisense strands are
polyribonucleotides.
[0296] Each of the aforementioned embodiments permits the
conducting of efficient RNAi interference because the
polynucleotide is more stable than naked polynucleotides. Unlike
naked polynucleotides, the polynucleotides of the present invention
will resist degradation by nucleases and other substances that are
present in blood, serum and other biological media.
[0297] An additional surprising benefit of the present invention is
that it minimizes nonspecific RNA interference, also referred to as
"off-targeting". Nonspecific RNA interference occurs when a sense
strand silences or partially silences the function of untargeted
genes. Orthoester modifications and the other modifications
described herein, alone or in combination with one another, can be
employed in the sense strand, the antisense strand, or both, to
reduce or prevent such nonspecific RNA interference.
[0298] In reducing nonspecific RNA interference, preferably sense
strand modifications are made at the 2' position at the 8.sup.th,
9.sup.th, 10.sup.th, or 11.sup.th nucleotide from the 5' terminus,
with the 5' terminal nucleotide designated as the 1.sup.st. More
preferably, all of the 8.sup.th, 9.sup.th, 10.sup.th and 11.sup.th
nucleotides are modified at the 2' position. Most preferably, the
8.sup.th, 9.sup.th, 10.sup.th and 11.sup.th nucleotides are all
modified at the 2' position and the modification is an
orthoester.
[0299] According to a ninth embodiment, the invention provides a
method of performing RNA interference, said method comprising
exposing an siRNA to a target nucleic acid, wherein said siRNA is
comprised of a sense strand and an antisense strand, and wherein
said sense strand is substantially nonfunctional. By "substantially
nonfunctional" is meant that the sense strand is incapable of
inhibiting expression of any non-targeted gene by 50% or more.
Thus, a "substantially nonfunctional" sense strand is one that
inhibits expression of non-target mRNAs by less than 50%. An added
advantage of the invention is an enhanced stability in
serum-containing media and serum.
[0300] According to this embodiment, the sense strand can comprise
at least one 2'-O-alkyl modification, at least one cytosine- or
uracil-containing nucleotide base, wherein the at least one
cytosine- or uracil-containing nucleotide base has a 2'-O-methyl
modification. Preferably, the 2'-O-alkyl modification is a
2'-O-methyl modification. More preferably, the 2'O-alkyl
modification is a 2'-O-methyl modification is on the first, second,
eighteenth and/or nineteenth nucleotide base.
[0301] The sense strand can further comprise a conjugate.
Preferably, the conjugate is cholesterol. Preferably, the
cholesterol is attached to the 5' and/or 3' end of the sense
strand. Modification of an siRNA duplex with cholesterol
drastically increases the duplex's affinity for albumin and other
serum proteins, thus altering the biodistribution of the duplex
without any significant toxicity.
[0302] The sense strand can comprise a cap on its 3' end.
Preferably, the cap is an inverted deoxythymidine or two
consecutive 2'O-methyl modified bases at the end positions
(nucleotides 18 and 19).
[0303] The antisense strand can comprise at least one modified
nucleotide. Preferably, the at least one modified nucleotide is a
2'-halogen modified nucleotide. Most preferably, the modified
nucleotide is a 2'-fluorine modified nucleotide.
[0304] Where the sense strand comprises one or more cytosine-
and/or uracil-containing nucleotide bases, each of the one or more
cytosine- and/or uracil-containing nucleotide bases can be
2'-fluorine modified.
[0305] According to a tenth embodiment, the invention provides a
method of performing RNA interference, said method comprising
exposing an siRNA to a target nucleic acid, wherein said siRNA
comprises: (a) a conjugate; (b) a sense strand comprising at least
one 2'-O-alkyl modification, wherein said sense strand is
substantially nonfunctional; and (c) an antisense strand comprising
at least one 2'-fluorine modification, wherein said sense and
antisense strands form a duplex of 18-30 base pairs. Preferably,
the least one 2'-O-alkyl modification is on the first, second,
eighteenth and/or nineteenth nucleotide base. Preferably, the
conjugate is cholesterol. Preferably, the cholesterol is attached
to the 5' and/or 3' end of the sense strand.
[0306] The sense strand can further comprise a cap on its 3' end.
Preferably, the cap is an inverted deoxythymidine (idT) or two
consecutive 2'O-methyl modified bases at the end positions
(nucleotides 18 and 19).
[0307] The advantages of the above described embodiments of the
present invention include allowing modifications of the sense
strand of the siRNA duplex that promote the directionality of RISC
complex assembly and prevent the sense strand from participating in
gene silencing. The inventors have systematically studied the
effects of using siRNAs having various modifications on the
efficiency of siRNA-mediated silencing. The inventors have found
that modification of each position on a sense and antisense strand
with a 2'-deoxy or a 2'-O-methyl modification did not interfere
with siRNA function. Where tandem blocks of 2 or 3 such
modifications were used, patterns of well-tolerated modifications
are different between the sense and antisense strands. siRNA
duplexes having positions 1 and 2 of the sense strand modified with
2-O-methyl were fully functional. But modification of the same
positions in the antisense strand resulted in completely
nonfunctional siRNAs. See FIGS. 19-31. Phosphorylation of the
antisense strand at its 5' end partially recovered antisense strand
functionality.
[0308] The modifications described herein are an inexpensive,
reliable, and non-toxic method of modifying siRNA duplexes such
that a sense strand will be substantially unable to function as an
antisense strand. The practical effect of this is that siRNA
specificity and potency will be increased. Recent microarray
analysis has suggested that the presence of 11 nucleotides is
sufficient to induce nonspecific silencing, and that the homology
between the sense strand of an siRNA duplex and other mRNA
transcripts can, in some cases, generate at least half of
non-specific activity (Jackson, A. L., et al. (2003). Expression
profiling reveals off-target gene regulation by RNAi. Nature
Biotechnology 21, 635-637). Thus, if the nonspecific activity of
the sense strand is blocked, the duplex specificity can be
increased. This would also have the effect of shifting the
equilibrium toward a functional RISC formation, lowering the siRNA
concentration required as well.
[0309] The inventors provide modifications that are well tolerated
and increase the stability of an siRNA duplex in the presence of
serum, such as human serum. Stabilizing modification of the sense
strand of an siRNA duplex, alone, can confer some stability to a
non-modified, or naked, antisense strand. Modification of every C
or U of a sense strand with a 2'-O-alkyl modification, such as a
2'-O-methyl moiety, is very effective for stabilization of some
sequences but not for others. Furthermore, the inventors discovered
that 2'-O-methyl modification of the 5' terminal nucleotides of the
sense strand is important. Modification in this manner is also
expected to result in a low level of non-specific effects compared
to fully modified siRNAs. As the data herein describe, modification
at positions 1, 2, 18 and 19 of the sense strand does not interfere
with duplex performance. One example of duplex stabilization that
takes place with sense strand modification is shown in FIG. 32.
This figure demonstrates that the half-life of the anti SEAP siRNA
2217 was increased from 10 minutes to 4 hours when the sense strand
of the duplex was modified with O-methyls on all Us and Cs.
Modification of the 3' end by idT is important because the dTdT
version of the antisense strand was two-fold less stable. This mode
of modification can be applied to any sequence, because the
antisense strand is left naked.
[0310] A half-life of several hours in serum should be sufficient
to insure effective delivery of an siRNA, since intracellular siRNA
is stabilized by the RISC complex. FIG. 35 shows the stability of
an siRNA duplex when the Cs and Us of the sense strand are modified
with 2'-O-methyls and an idT cap is placed on the 3' end. The
functionality of this type of formulation is sequence dependent,
but is significantly improved by the presence of cholesterol on the
5' end of the sense strand. Additional modifications (e.g.,
2'-O-methyl modifications of sense strand Cs and Us, plus
2'-O-methyl modification of positions 1 and 2 of the sense strand,
2' F modifications of Cs and Us of the antisense strand, and a
phosphate group on the 5' end of the antisense strand, see FIG. 37)
can further increase the serum stability of such molecules to 5
days without appreciably altering silencing functionality.
[0311] Modification of an siRNA with a cholesterol conjugate has
another unexpected feature. siRNAs modified with cholesterol
display very high affinity for albumin and other serum proteins.
See FIG. 33. Serum protein affinity has proven useful in previous
studies of antisense biodistribution in the mouse. The presence of
phosphothio modifications is responsible for the majority of
nonspecific antisense binding activity, but was proven beneficial
for in vivo antisense applications, mainly because of high affinity
to serum proteins and thus altered pharmacokinetic behavior.
Cholesterol modified siRNAs display the advantage of serum protein
affinity without the disadvantage of increased nonspecificity of
phosphothio modifications.
[0312] According to the eleventh embodiment, the present invention
provides an siRNA comprised of a sense strand and an antisense
strand. The sense strand has a first 5' terminal sense nucleotide
and a second 5' terminal sense nucleotide, both of which have
2'carbon modifications, preferably 2'-O-alkyl modifications. In
some embodiments, the siRNA also comprises a label, which if
present is preferably located on the first 5' terminal sense
nucleotide. Further, at least one and preferably each of the
pyrimidines (e.g., cytosine or uracil) on the sense strand has a 2'
carbon modifications, preferably 2'-O-alkyl modifications. These
modifications are in addition to any modification of the first or
second 5' terminal sense nucleotide, which may, depending on the
sequence of the siRNA, be a pyrimidine.
[0313] The antisense strand of the eleventh embodiment comprises at
least one 2'-halogen modified pyrimidine, preferably, a fluoro
modified pyrimidine, and a first 5' terminal antisense nucleotide
that has been phosphorylated. Preferably all of the pyrimidines on
the antisense strand have 2'-fluoro modifications.
[0314] Preferably, the siRNA comprises from 18-30 base pairs, more
preferably from 19-25 base pairs, and most preferably from 19-23
base pairs, exclusive of overhangs or loop or stem structures when
present in unimolecular siRNAs that are capable of forming
hairpins. Preferably, the sense strand and antisense strand are,
exclusive of overhangs, loop or stem structures at least 79%
complementary, more preferably at least 90% complementary over the
range of base pairs, and most preferably 100% complementary over
this range. Similarly preferably, the antisense region is
preferably at least 79%, more preferably at least 90%, and most
preferably at least 100% complementary to the target region.
Preferably, the polynucleotide is RNA.
[0315] The siRNA may also contain overhangs at either the 5' or 3'
end of either the sense strand or the antisense strand. However, in
the case of polynucleotides comprised of two separate strands
preferably, if there are any overhangs, they are only on the 3' end
of the sense strand and/or the antisense strand. Additionally,
preferably any overhangs are six or fewer bases in length, more
preferably two or fewer bases in length. Most preferably, there are
either no overhangs, or overhangs of two bases on one or both of
the sense strand and antisense strand at the 3' end. According to
the eleventh embodiment it is preferable not to have overhangs on
the 5' end of the antisense strand. Overhanging nucleotides are
frequently removed by one or more intracellular enzymatic processes
or events, which may leave an unphosphorylated 5'-nucleotide.
Therefore, it is preferable not to have overhangs on the 5' end of
the antisense strand.
[0316] The phosphorylation of the first 5' terminal antisense
nucleotide refers to the presence of one or more phosphate groups
attached to the 5' carbon of the sugar moiety of the nucleotide.
Preferably, there is only one phosphate group.
[0317] The 2'-O-alkyl modifications, regardless of on which bases
they appear are preferably selected from the group consisting of
2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-isopropyl, 2'-O-butyl,
2-O-isobutyl, 2'-O-ethyl-O-methyl (--CH.sub.2CH.sub.2OCH.sub.3),
and 2'-O-ethyl-OH (--OCH.sub.2CH.sub.2OH). Most preferably, the
2'-O-alkyl modification is a 2'-O-methyl moiety. Further, there is
no requirement that the modification be the same on each of the
first 5' terminal sense nucleotide and the second 5' terminal sense
nucleotide. However, as a matter of practicality with respect to
synthesizing the molecules of the present invention, it may be
desirable to use the same modification throughout.
[0318] With respect to the 2'-O-alkyl pyrimidine modifications, at
least one pyrimidine other than any pyrimidine within the first two
5' terminal sense nucleotides is modified with a 2'-O-alkyl group.
Preferably, all pyrimidines on the sense strand within the duplex
forming region are modified. Further, preferably, the 2'-O-alkyl
modifications are 2'-O-methyl groups. As with the 2'-O-alkyl
modifications, the same alkyl modification need not be used on each
nucleotide that has a 2'-O-alkyl group. When overhangs, loops or
stems are present, the pyrimidines of those regions may or may not
contain 2'-O-alkyl groups. However, at least one of the 2'-O-alkyl
groups is preferably in the sense region.
[0319] With respect to the 2' halogen modified nucleotides, the
modification appears on at least one of the pyrimidines of the
antisense strand and more preferably on all of the pyrimidines of
the antisense strand. Further, preferably the halogen is fluorine.
When overhangs, stems or loops are presented, the pyrimidines of
those regions may or may not contain halogen groups but preferably
any stem or loop structure would not contain this modification, and
the at least one halogen group is within the sense region. It
should be noted that 2' halogen modifications, including 2' fluoro
modifications may be used to increase the stability of the siRNA
independent of the other modifications described herein. Thus, they
may be used independently, as well as in connection with these
other modifications in siRNA applications.
[0320] Other types of nucleotide modifications of the sense and/or
antisense strands may be included if they do not greatly negate the
benefits of the present invention, including stability and with
respect to the eleventh embodiment, functionality. For example, the
use of pyrimidine modified nucleotides such a halogen, preferably
fluorine, modified and additional 2'-O-alkyl modified pyrimidines
provide a certain level of nuclease resistance.
[0321] In addition to chemical modifications, it is postulated that
base pair mismatches or bulges can be added to the sense and/or
antisense strands that alter the ability of these strands to
participate in RISC-mediated association with targets that share
less than 100% homology. Examples of such mismatches include (but
are not limited to) purine-pyrimidine mismatches (e.g., G-U, C-A)
and purine-purine or pyrimidine-pyrimidine mismatches (e.g., G-A,
U-C). The introduction of these types of modification may be
combined with the above-described modifications, and evaluated to
determine whether they alter other attributes of functionality
(e.g., off-target effects) without detracting from the benefits of
the present invention.
[0322] In some applications, it may be desirable to use a label,
for example, a fluorescent label. When the label is fluorescent,
preferably, the fluorescent label is Cy3.TM., Cy5.TM., or the Alexa
dyes (Molecular Probes, Eugene, Oreg.). These labels are preferably
added to the 5' end of the sense strand, more preferably at the 5'
terminal sense nucleotide. They may be used to enable users to
visualize the distribution of the labeled siRNA within, e.g., a
transfected cell, and allow one to assess the success of any given
transfection. The use of labeled nucleotides is well known to
persons of ordinary skill, and labels other than fluorescent
labels, e.g., mass or radioactive labels, may be used in
applications in which such types of labels would be
advantageous.
[0323] The fluorescent modifications can be used to segregate
transfected cells from untransfected cells. Specifically, a
population of cells can be transfected with the molecules of the
eleventh embodiment and subsequently sorted by FACS to segregate
transfected cells from untransfected cells. This enables one to
obtain a purer population (rather than a mixed one), which in turn
improves ones ability to clearly identify phenotypes that result
from gene silencing.
[0324] The molecules of the eleventh embodiment have a variety of
uses including, for example, the molecules may be used as
transfection control reagents or stable silencing reagents. When
these molecules contain labels, transfection of these molecules
into cells allows the user to visualize and to determine what
fraction of the cells have been successfully transfected. In
addition, these modifications do not appreciably alter siRNA
function; thus, the molecules of the eleventh embodiment can
simultaneously be transfection controls and silencing reagents.
Further, because the above-described modifications do not place
limitations on the sequences that may be used, they may be used in
diverse siRNA and RNAi applications and are not target sequence
dependent.
[0325] Thus, molecules of the eleventh embodiment, with their
unique set of modifications, provide stability and "trackability"
without altering functionality. They can also be used to isolate a
pure population of cells that have been transfected. If 2'-O-alkyl
groups are added to the first two or first three nucleotides of the
antisense strand, the additional benefit of reducing off-target
effects can be realized. Further if 2'-O-alkyl groups are added to
the first two or three nucleotides of the sense strand, they can
have the additional benefit of reducing sense strand off-target
effects.
[0326] According to a twelfth embodiment, the present invention
provides another siRNA comprised of a sense strand and an antisense
strand. The sense strand is defined according to the same
parameters as the sense strand for the eleventh embodiment,
including the 2' carbon modification, preferably 2'-O-alkyl
modifications of the first and second 5' terminal sense nucleotide
and at the at least one 2' carbon modification, preferably
2'-O-alkyl modification of pyrimidines. However, instead of the
above-described modifications to the antisense strand, the
antisense strand of the twelfth embodiment comprises first and
second, or first, second and third, 5' terminal antisense
nucleotides, each of which have 2'-O-alkyl modifications. Further,
there is at least one 2'-O-alkyl modified pyrimidine on the
antisense strand other than the modification on the first and
second 5' terminal antisense nucleotides. Still further, preferably
there are no 2' Fl modifications and there is no phosphorylation of
the first 5' terminal antisense nucleotide. The absence of
phosphorylation renders the molecule of limited functionality as
compared to corresponding functional polynucleotides. As another
variation to the above description, molecules of the twelfth
embodiment can, in addition to all of the above attributes, contain
5'-blocking groups on the first nucleotide of the sense, antisense,
or both sense and antisense strands. Such blocking groups will
further eliminate the possibility that this molecule will be
phosphorylated and enter RISC, and can consist of 5'-alkyl,
5'-O-alkyl, azide (N3), amines, and other moieties.
[0327] The other preferred parameters with respect to size,
overhangs and other modifications are the same as for the eleventh
embodiment. However, because these compositions are not used for
silencing of genes, they could be 16-28 base pairs in length,
thought are preferably 18-30 base pairs in length.
[0328] The molecules of the twelfth embodiment may, in addition to
being a transfection control, also act as a potential "filler" or
exaequo agent, which is particularly useful in dosage experiments
or as a negative control in microarray studies. Many experiments
test the effects of a given siRNA at different concentrations
(e.g., 100 nM, 50 nM, 25 nM, and 1 nM). Optimally, when
transfection experiments are performed, one wants to have a
constant concentration of "total siRNA" to avoid any anomalies that
result from transfection of different levels of nucleic acids.
Unfortunately, addition of any unmodified control siRNA (e.g., a
non-specific control) has the potential downside of competing with
the siRNA under study for interaction with RISC. Molecules of the
twelfth embodiment alleviate this problem. The addition of
2'-O-methyl modifications on positions 1 and 2 (or 1, 2 and 3) of
both the sense and antisense strands in the absence of a
phosphorylated 5' terminal antisense nucleotide, limits the ability
of this molecule to interact with RISC. Thus, this molecule can be
transfected, but it cannot compete with other siRNA for RISC. As
was the case with the molecules of the eleventh embodiment, the
addition of 2'-O-methyl groups on the pyrimidines (Cs and Us)
minimizes the possibility of nuclease digestion.
[0329] The molecules of the twelfth embodiment, unlike other
"non-specific" sequences or controls, interact poorly with RISC.
Thus, these molecules should generate only limited off-targeting
side effects, and have the ability of being trackable without
competing for sites on RISC.
[0330] The above-described embodiments (both the eleventh and the
twelfth) may apply to siRNA that do not have contiguous sense and
antisense strands (i.e., two separate strands), as well as to
unimolecular siRNAs that are capable of forming hairpins (shRNA).
The term "siRNA" includes both types of RNA. For example, the
hairpin may comprise a loop structure, which preferably comprises
from four to ten bases, and a sense region, wherein the sense
region and antisense regions are independently 19-23 base pairs in
length and substantially complementary to each other. Preferable
sequences of the loop structure include, for example, 5'-UUCG (SEQ.
ID NO. 319), 5'-UUUGUGUAG (SEQ. ID NO. 320), and 5'-CUUCCUGUCA
(SEQ. ID NO. 321).
[0331] The hairpin is preferably constructed with the loop region
downstream of the antisense region. This construction is desirable
particularly with respect to the eleventh embodiment, because it is
easier to phosphorylate the terminal antisense nucleotide. Thus,
when designing the unimolecular siRNAs, it is preferable that there
are no overhangs upstream of the 5' terminal antisense
nucleotide.
[0332] When designing a unimolecular siRNA, specifically a
left-handed unimolecular structure (e.g., 5'-AS-Loop-S) according
to the present invention, preferably, the first 5' terminal sense
nucleotide is defined as the nucleotide that is the 18.sup.th,
19.sup.th or 20.sup.th base of the sense region counting from the
base that is complementary to the first 5' terminal antisense
nucleotide (i.e. counting from the 3' end of the sense region). The
first 5' terminal sense nucleotide is defined in this manner
because when unimolecular siRNA that are capable of forming
hairpins enter a cell, typically, Dicer will process hairpin siRNAs
that contain lengthier duplex regions, into molecules that are
comprised of two separate strands (siRNA) of approximately 18-20
base pairs, and it is desirable for these molecules to have the
sense strand modifications associated with the end of this
processed molecule. Most preferably, the first 5' terminal sense
nucleotide is defined as the nucleotide that is the 19.sup.th base
of the sense region from the 3' end of the sense region. Further,
preferably, the polynucleotide is capable of forming a left-handed
hairpin.
[0333] The shRNA can further comprise a stem region, wherein the
stem region comprises one or more nucleotides or modified
nucleotides immediately adjacent to the 5' end and the 3' end of
the loop structure, and wherein the one or more nucleotides or
modified nucleotides of the stem region are or are not
target-specific. Preferably, the entire length of the unimolecular
siRNA contains fewer than 100 bases, more preferably fewer than 85
bases.
[0334] The unimolecular siRNAs of the present invention may
ultimately be processed by cellular machinery such that they are
converted into two separate strands. Alternatively, the molecules
may bypass one or more steps in the RNAi pathway (e.g., Dicer
processing) and enter RISC as unimolecular hairpin molecules.
Further, these unimolecular siRNAs may be introduced into the cell
with less than all modifications, and modified in the cell itself
through the use of natural processes or processing molecules that
have been introduced (e.g., with respect to the eleventh
embodiment, phosphorylation in the cell by native kinases).
However, preferably the polynucleotide is introduced with all
modifications already present. (Similarly, when the siRNA is
comprised of two separate strands, preferably those strands contain
all modifications when introduced into the cell with all
modifications, though the antisense strand could e.g., be modified
with a phosphorylation group after introduction.)
[0335] Although the above-described embodiments (eleventh and
twelfth) are directed to increased stability, it is important to
note that these and other types of modifications may also affect
other parameters, such as specificity. Further, these stability and
functionality modifications may be combined (e.g., 2' carbon
modifications (preferably-O-methyl modifications) of the first and
second (or first, second and third) 5' terminal sense and antisense
nucleotides in conjunction with an additional 2' carbon
modifications (preferably-O-methyl modifications) of at least one
sense pyrimidine, preferably all sense pyrimidine(s) other than the
first two (or three) that have 2' carbon modifications
(preferably-O-methyl modifications), at least one, preferably all,
2'-Fl modifications of antisense pyrimidine(s) other than the first
two (or three that have 2' carbon modifications
(preferably-O-methyl modifications) and phosphorylation of the 5'
terminal antisense nucleotide.) Further, the above described
modifications of the present invention may be combined with siRNA
that contain sequences that were selected at random, or according
to rationale design as described in, for example, U.S. patent
application Ser. No. 10/714,333.
[0336] Additionally stabilization modifications that are addressed
to the phosphate backbone may be included in the polynucleotides
for some applications of the present invention. For example, at
least one phosphorothioate and/or methylphosphonate may be
substituted for the phosphate group at some or all 3' positions of
any or all pyrimidines in the sense and/or antisense strands of the
oligonucleotide backbone, as well as in any overhangs, loop
structures or stem structures that may be present. Phosphorothioate
(and methylphosphonate) analogues arise from modification of the
phosphate groups in the oligonucleotide backbone. In the
phosphorothioate, the phosphate O.sup.- is replaced by a sulfur
atom. In methylphosphonates, the oxygen is replaced with a methyl
group. In one embodiment the phosphorothioate modification or
methylphosphonate is located at the 3' positions of all antisense
strand nucleotides that also contains 2' fluoro (or other halogen)
modified nucleotides. Additionally, phosphorothioate 3'
modifications may be used instead of and independent of 2' fluoro
modifications to increase stability of an siRNA molecule. These
modifications may be used in combination with the other
modifications disclosed herein, or independent of those
modifications in siRNA applications.
[0337] Nucleases typically use both the oxygen groups on the
phosphate moiety and the 2'OH position of the ribose ring to
mediate attack on RNA. Substitution of a sulfur group for one of
the oxygens eliminates the ability of the phosphate to participate
in this reaction, thus limiting the sensitivity of this site to
nuclease digestion. However, it should be noted that
phosphorothioates are typically toxic, thus, they would be
beneficial primarily when any toxic effects are negated, which it
is postulated might be accomplished by limiting the use of this
modification to e.g., every other nucleotide, every third
nucleotide, or every fourth nucleotide.
[0338] The molecules of the twelfth embodiment are of limited
functionality, but as noted above, may be used in negative control
studies, and as exaequo agents. The set of modifications associated
with the molecules make them poor substrates for intracellular
kinases that typically add an essential phosphate group to the 5'
end of the antisense strand of siRNA. While the modifications
strongly inhibit phosphorylation, it is possible that some small
fraction of molecules carrying these modifications might still be
phosphorylated at C5 of the 5'-antisense position and generate a
small amount of silencing. In these cases, it may be desirable to
modify the 5' carbon position of the 5' end of the sense and/or the
antisense strand of these molecules with a blocking group that
prevents kinase phosphorylation. The blocking group may for example
be any group that prevents phosphorylation of the 5' carbon
position of the terminal nucleoside, including, but not limited to
5'-alkyl , 5'-O-methyl, and 5'-amine groups.
[0339] Another example of a control or exaequo agent contain only
the following 2' carbon modifications: (i) a first and second, or
first, second and third, 5' terminal sense nucleotides that each
comprises 2'modifications, such as the 2'modifications described
above, for example 2'-O-methyl modifications; and (ii) a first and
second, or first, second and third, 5' terminal antisense
nucleotides that each comprises 2'modifications, such as the
2'modifications described above, for example 2'-O-methyl
modifications, and the 5' terminal antisense nucleotide has not
been phosphorylated.
[0340] The polynucleotides of the present invention may be
synthesized by any method that is now known or that comes to be
known and that from reading this disclosure a person of ordinary
skill in the art would appreciate would be useful to synthesize the
molecules of the present invention. siRNA duplexes containing the
specified modifications may be chemically synthesized using
compositions of matter and methods described in Scaringe, S. A.
(2000) "Advanced 5'-silyl-2'-orthoester approach to RNA
oligonucleotide synthesis," Methods Enzymol. 317, 3-18; Scaringe,
S. A. (2001) "RNA oligonucleotide synthesis via
5'-silyl-2'-orthoester chemistry," Methods 23, 206-217; Scaringe,
S. and Caruthers, M. H. (1999) U.S. Pat. No. 5,889,136; Scaringe,
S. and Caruthers, M. H. (1999) U.S. Pat. No. 6,008,400; Scaringe,
S. (2000) U.S. Pat. No. 6,111,086; Scaringe, S. (2003) U.S. Pat.
No. 6,590,093. The synthesis method utilizes nucleoside
base-protected 5'-O-silyl-2'-O-orthoester-3'-O-phosphoramidites to
assemble the desired unmodified siRNA sequence on a solid support
in the 3' to 5' direction. Briefly, synthesis of the required
phosphoramidites begins from standard base-protected
ribonucleosides (uridine, N.sup.4-acetylcytidine,
N.sup.2-isobutyrylguanosine and N.sup.6-isobutyryladenosine).
Introduction of the 5'-O-silyl and 2'-O-orthoester protecting
groups, as well as the reactive 3'-O-phosphoramidite moiety is then
accomplished in five steps, including: [0341] 1. Simultaneous
transient blocking of the 5'- and 3'-hydroxyl groups of the
nucleoside sugar with Markiewicz reagent
(1,3-dichloro-1,1,3,3,-tetraisopropyldisiloxane [TIPS--Cl.sub.2])
in pyridine solution {Markiewicz, W. T. (1979)
"Tetraisopropyldisiloxane-1,3-diyl, a Group for Simultaneous
Protection of 3'- and 5'-Hydroxy Functions of Nucleosides," J.
Chem. Research(S), 24-25}, followed by chromatographic
purification; [0342] 2. Regiospecific conversion of the 2'-hydroxyl
of the TIPS-nucleoside sugar to the bis(acetoxyethyl)orthoester
[ACE derivative] using tris(acetoxyethyl)-orthoformate in
dichloromethane with pyridinium p-toluenesulfonate as catalyst,
followed by chromatographic purification; [0343] 3. Liberation of
the 5'- and 3'-hydroxyl groups of the nucleoside sugar by specific
removal of the TIPS-protecting group using hydrogen fluoride and
N,N,N''N'-tetramethylethylene diamine in acetonitrile, followed
chromatographic purification; [0344] 4. Protection of the
5'-hydroxyl as a 5'-O-silyl ether using
benzhydroxy-bis(trimethylsilyloxy)silyl chloride [BzH--Cl] in
dichloromethane, followed by chromatographic purification; and
[0345] 5. Conversion to the 3'-O-phosphoramidite derivative using
bis(N,N-diisopropylamino)methoxyphosphine and
5-ethylthio-1H-tetrazole in dichloromethane/acetonitrile, followed
by chromatographic purification.
[0346] The phosphoramidite derivatives are typically thick,
colorless to pale yellow syrups. For compatibility with automated
RNA synthesis instrumentation, each of the products is dissolved in
a pre-determined volume of anhydrous acetonitrile, and this
solution is aliquoted into the appropriate number of serum vials to
yield a 1.0-mmole quantity of phosphoramidite in each vial. The
vials are then placed in a suitable vacuum desiccator and the
solvent removed under high vacuum overnight. The atmosphere is then
replaced with dry argon, the vials are capped with rubber septa,
and the packaged phosphoramidites are stored at -20.degree. C.
until needed. Each phosphoramidite is dissolved in sufficient
anhydrous acetonitrile to give the desired concentration prior to
installation on the synthesis instrument.
[0347] The synthesis of the desired oligoribonucleotide is carried
out using automated synthesis instrumentation. It begins with the
3'-terminal nucleoside covalently bound via its 3'-hydroxyl to a
solid beaded polystyrene support through a cleavable linkage. The
appropriate quantity of support for the desired synthesis scale is
measured into a reaction cartridge, which is then affixed to
synthesis instrument. The bound nucleoside is protected with a
5'-O-dimethoxytrityl moiety, which is removed with anhydrous acid
(3% [v/v] dichloroacetic acid in dichloromethane) in order to free
the 5'-hydroxyl for chain assembly.
[0348] Subsequent nucleosides in the sequence to be assembled are
sequentially added to the growing chain on the solid support using
a four-step cycle, consisting of the following general reactions:
[0349] 1. Coupling: the appropriate phosphoramidite is activated
with 5-ethylthio-1H-tetrazole and allowed to react with the free
5'-hydroxyl of the support bound nucleoside or oligonucleotide.
Optimization of the concentrations and molar excesses of these two
reagents, as well as of the reaction time, results in coupling
yields generally in excess of 98% per cycle. [0350] 2. Oxidation:
the internucleotide linkage formed in the coupling step leaves the
phosphorous atom in its P(III) [phosphite] oxidation state. The
biologically-relevant oxidation state is P(V) [phosphate]. The
phosphorous is therefore oxidized from P(III) to P(V) using a
solution of tert-butylhydroperoxide in toluene. [0351] 3. Capping:
the small quantity of residual un-reacted 5'-hydroxyl groups must
be blocked from participation in subsequent coupling cycles in
order to prevent the formation of deletion-containing sequences.
This is accomplished by treating the support with a large excess of
acetic anhydride and 1-methylimidazole in acetonitrile, which
efficiently blocks residual 5'-hydroxyl groups as acetate esters.
[0352] 4. De-silylation: the silyl-protected 5'-hydroxyl must be
deprotected prior to the next coupling reaction. This is
accomplished through treatment with triethylamine trihydrogen
fluoride in N,N-dimethylformamide, which rapidly and specifically
liberates the 5'-hydroxyl without concomitant removal of other
protecting groups (2'-O-ACE, N-acyl base-protecting groups, or
phosphate methyl).
[0353] It should be noted that in between the above four reaction
steps are several washes with acetonitrile, which are employed to
remove the excess of reagents and solvents prior to the next
reaction step. The above cycle is repeated the necessary number of
times until the unmodified portion of the oligoribonucleotide has
been assembled. The above synthesis method is only exemplary and
should not be construed as limited the means by which the molecules
may be made. Any method that is now known or that comes to be known
for synthesizing siRNA and that from reading this disclosure one
skilled in the art would conclude would be useful in connection
with the present invention may be employed.
[0354] The siRNA duplexes of certain embodiments of the eleventh
and twelfth embodiments include two modified nucleosides (e.g.,
2'-O-methyl derivatives) at the 5'-end of each strand. The
5'-O-silyl-2'-O-methyl-3'-O-phosphoramidite derivatives required
for the introduction of these modified nucleosides are prepared
using procedures similar to those described previously (steps 4 and
5 above), starting from base-protected 2'-O-methyl nucleosides
(2'-O-methyl-uridine, 2'-O-methyl-N.sup.4-acetylcytidine,
2'-O-methyl-N.sup.2-isobutyrylguanosine and
2'-O-methyl-N.sup.6-isobutyryladenosine). The absence of the
2'-hydroxyl in these modified nucleosides eliminates the need for
ACE protection of these compounds. As such, introduction of the
5'-O-silyl and the reactive 3'-O-phosphoramidite moiety is
accomplished in two steps, including: [0355] 1. Protection of the
5'-hydroxyl as a 5'-O-silyl ether using
benzhydroxy-bis(trimethylsilyloxy)silyl chloride (BzH--Cl) in
N,N-dimethylformamide, followed by chromatographic purification;
and [0356] 2. Conversion to the 3'-O-phosphoramidite derivative
using bis(N,N-diisopropylamino)methoxyphosphine and
5-ethylthio-1H-tetrazole in dichloromethane/acetonitrile, followed
by chromatographic purification.
[0357] Post-purification packaging of the phosphoramidites is
carried out using the procedures described previously for the
standard nucleoside phosphoramidites. Similarly, the incorporation
of the two 5'-O-silyl-2'-O-methyl nucleosides via their
phosphoramidite derivatives is accomplished by twice applying the
same four-step cycle described previously for the standard
nucleoside phosphoramidites.
[0358] The siRNA duplexes of certain embodiments of the eleventh
embodiment of this invention include a phosphate moiety at the
5'-end of the antisense strand. This phosphate is introduced
chemically as the final coupling to the antisense sequence. The
required phosphoramidite derivative
(bis(cyanoethyl)-N,N-diisopropylamino phosphoramidite) is
synthesized as follows in brief: phosphorous trichloride is treated
one equivalent of N,N-diisopropylamine in anhydrous tetrahydrofuran
in the presence of excess triethylamine. Then, two equivalents of
3-hydroxypropionitrile are added and allowed to react completely.
Finally, the product is purified by chromatography.
Post-purification packaging of the phosphoramidite is carried out
using the procedures described previously for the standard
nucleoside phosphoramidites. Similarly, the incorporation of the
phosphoramidite at the 5'-end of the antisense strand is
accomplished by applying the same four-step cycle described
previously for the standard nucleoside phosphoramidites.
[0359] The modified, protected oligoribonucleotide remains linked
to the solid support at the finish of chain assembly. A two-step
rapid cleavage/deprotection procedure is used to remove the
phosphate methyl protecting groups, cleave the oligoribonucleotide
from the solid support, and remove the N-acyl base-protecting
groups. It should be noted that this procedure also removes the
cyanoethyl protecting groups from the 5'-phosphate on the antisense
strand. Additionally, the procedure removes the acetyl
functionalities from the ACE orthoester, converting the 2'-O-ACE
protecting group into the bis(2-hydroxyethyl)orthoester. This new
orthoester is significantly more labile to mild acid as well as
more hydrophilic than the parent ACE group. The two-step procedure
is briefly as follows: [0360] 1. The support-bound
oligoribonucleotide is treated with a solution of disodium
2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in
N,N-dimethylformamide. This reagent rapidly and efficiently removes
the methyl protecting groups from the internucleotide phosphate
linkages without cleaving the oligoribonucleotide from the solid
support. The support is then washed with water to remove excess
dithiolate. [0361] 2. The oligoribonucleotide is cleaved from the
solid support with 40% (w/v) aqueous methylamine at room
temperature. The methylamine solution containing the crude
oligoribonucleotide is then heated to 55.degree. C. to remove the
protecting groups from the nucleoside bases. The crude
orthoester-protected oligoribonucleotide is obtained following
solvent removal in vacuo.
[0362] Removal of the 2'-orthoesters is the final step in the
synthesis process. This is accomplished by treating the crude
oligoribonucleotide with an aqueous solution of acetic acid and
N,N,N',N'-tetramethyl ethylene diamine, pH 3.8, at 55.degree. C.
for 35 minutes. The completely deprotected oligoribonucleotide is
then desalted by ethanol precipitation and isolated by
centrifugation.
[0363] In addition, incorporation of fluorescent labels at the
5'-terminus of a polynucleotide is a common and well-understood
manipulation for those skilled in the art. In general, there are
two methods that are employed to accomplish this incorporation, and
the necessary materials are available from several commercial
sources (e.g., Glen Research Inc., Sterling, Va., USA; Molecular
Probes Inc., Eugene, Oreg., USA; TriLink BioTechnologies Inc., San
Diego, Calif., USA; and others). The first method utilizes a
fluorescent molecule that has been derivatized with a
phosphoramidite moiety similar to the phosphoramidite derivatives
of the nucleosides described previously. In such case, the
fluorescent dye is appended to the support-bound polynucleotide in
the final cycle of chain assembly. The fluorophore-modified
polynucleotide is then cleaved from the solid support and
deprotected using the standard procedures described above. This
method has been termed "direct labeling." Alternatively, the second
method utilizes a linker molecule derivatized with a
phosphoramidite moiety that contains a protected reactive
functional group (e.g., amino, sulfhydryl, carbonyl, carboxyl, and
others). This linker molecule is appended to the support-bound
polynucleotide in the final cycle of chain assembly. The
linker-modified polynucleotide is then cleaved from the solid
support and deprotected using the standard procedures described
above. The functional group on the linker is deprotected either
during the standard deprotection procedure, or by utilizing a
subsequent group-specific treatment. The crude linker-modified
polynucleotide is then reacted with an appropriate fluorophore
derivative that will result in formation of a covalent bond between
a site on the fluorophore and the functional group of the linker.
This method has been termed "indirect labeling."
[0364] Once synthesized, the polynucleotides of the present
invention may immediately used or be stored for future use.
Preferably, the polynucleotides of the invention are stored as
duplexes in a suitable buffer. Many buffers are known in the art
suitable for storing siRNAs. For example, the buffer may be
comprised of 100 mM KCl, 30 mM HEPES-pH 7.5, and 1 mM MgCl2.
Preferably, the siRNAs of the present invention retain 30% to 100%
of their activity when stored in such a buffer at 4.degree. C. for
one year. More preferably, they retain 80% to 100% of their
biological activity when stored in such a buffer at 4.degree. C.
for one year. Alternatively, the compositions can be stored at
-20.degree. C. in such a buffer for at least a year or more.
Preferably, storage for a year or more at -20.degree. C. results in
less than a 50% decrease in biological activity. More preferably,
storage for a year or more at -20.degree. C. results in less than a
20% decrease in biological activity after a year or more. Most
preferably, storage for a year or more at -20.degree. C. results in
less than a 10% decrease in biological activity.
[0365] In order to ensure stability of the siRNA pools prior to
usage, they may be retained in dried-down form at -20.degree. C.
until they are ready for use. Prior to usage, they should be
resuspended; however, once resuspended, for example, in the
aforementioned buffer, they should be kept at -20.degree. C. until
used. The aforementioned buffer, prior to use, may be stored at
approximately 4.degree. C. or room temperature. Effective
temperatures at which to conduct transfection are well known to
persons skilled in the art, and include for example, room
temperature.
[0366] In developing the present invention, two or more different
modifications were added to a duplex to increase stability.
Applicants appreciate that other modifications and combinations may
be discovered in the future that assist in improving stability.
Additionally, the modifications of the present invention could be
combined with modifications that are desired for other purposes.
For example, in some instances, one modification could stabilize
the molecule against one particular set of conditions (e.g., one
type of nuclease) while a second modification could stabilize the
molecule against a second set of conditions (e.g., a different
family of nucleases). Alternatively, two separate modifications
could act additively or synergistically to stabilize a molecule
towards a certain set of conditions. In still other instances, one
modification could stabilize the molecule, but have detrimental
consequences on other desirable properties, e.g., the potency or
toxicity of the siRNA. In cases such as these, additional
modifications could be added that restore these aspects of
functionality of the molecule.
[0367] A variety of approaches can be used to identify both the
type of molecule and the key position needed to enhance stability.
In one non-limiting example, a modification-function walk is
performed. In this procedure, a single type of modification is
added to one or more nucleotides across the sense and/or antisense
strand. Subsequently, modified and unmodified molecules are tested
for (1) functionality and (2) stability, by one of several methods.
Thus, for example, 2'-O-Me groups can be added to positions 1 and
2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15
and 16, 17 and 18, or 18 and 19 of either the sense and/or
antisense strand and tested for functionality (e.g., by measuring
the ability of these molecules to silence specific targets) and
stabilize the molecule against actions by, e.g., nucleases. If key
positions are identified that enhance stability, but result in a
loss of duplex functionality, then a second round of modification
walks, whereby additional chemical groups (e.g., 5' phosphate on
the 5' end of the antisense strand), mismatches, or bulges that are
suspected to increase duplex functionality can be added to
molecules that already contain the modification that enhance
stability.
[0368] According to the thirteenth embodiment, the present
invention is directed to an siRNA, comprising: [0369] (a) an
antisense strand, wherein said antisense strand is comprised of a
first 5' terminal antisense nucleotide and said first 5' terminal
antisense nucleotide is phosphorylated at said first 5' terminal
antisense nucleotide's 5' carbon position; and [0370] (b) a sense
strand, wherein said sense strand is comprised of a first 5'
terminal sense nucleotide and a second 5' terminal sense
nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification.
[0371] Preferably, the siRNA comprises from 18-30 base pairs, more
preferably from 19-25 base pairs, and most preferably from 19-23
base pairs, exclusive of overhangs. Preferably, the sense strand
and antisense strand are at least substantially complementary over
the range of base pairs, and more preferably 100% complementary
over this range. Preferably the polynucleotide is RNA.
[0372] The siRNA may also contain overhangs at either the 5' or 3'
end of either the sense strand or the antisense strand. However,
preferably if there are any overhangs, they are on the 3' end of
the sense strand and/or the antisense strand. Additionally,
preferably any overhangs are six or fewer bases in length, more
preferably two or fewer bases in length. Most preferably, there are
either no overhangs, or overhangs of two bases on one or both of
the sense strand and antisense strand at the 3' end. Because
overhanging nucleotides are frequently removed by one or more
intracellular enzymatic processes or events, thus leaving an
unphosphorylated 5'-nucleotide, it is preferable not to have
overhangs on the 5' end of the antisense strand.
[0373] The phosphorylation of the first 5' terminal antisense
nucleotide refers to the presence of one or more phosphate groups
attached to the 5' carbon of the sugar moiety of the nucleotide.
Preferably there is only one phosphate group.
[0374] The 2' carbon modifications refer to modifications on the 2'
carbon of the nucleotide's sugar moiety. The "first 2' carbon sense
modification" refers to the modification that is on the first 5'
terminal sense nucleotide. The "second 2' carbon sense
modification" refers to the modification that is on the second 5'
terminal sense nucleotide.
[0375] It is known that addition of chemical modifications to key
positions along an RNA-RNA, RNA-DNA, or DNA-DNA duplex can
significantly alter the chemical and functional properties of these
molecules. Applicants appreciate that modifications can be added to
the sense strand of a siRNA to prevent that strand from entering
RISC and inducing sense strand specific off-target effects. Such
modifications can include addition of any number of chemical
moieties. Preferably, the moiety is attached to the 2' position of
the nucleotide's ribose ring (i.e., the 2' carbon). According to
the present embodiment, preferably the modification is a 2'-O-alkyl
group. However, it may be any other modification that when used in
the context of the present invention minimizes off-target effects
by this strand. For example, the 2' modified nucleotide may be
selected from the group consisting of a 2' halogen modified
nucleotide, a 2' amine modified nucleotide and a 2' alkyl modified
nucleotide if such modifications are included under conditions that
minimize off-target effects. Where the modification is a halogen,
the halogen is preferably fluorine. Where the 2' modified
nucleotide is a 2' amine modified nucleotide, the amine is
preferably --NH.sub.2. Where the 2' modified nucleotide is a
2'-alkyl modification, preferably the modification is a 2' methyl
modification, wherein the carbon of the methyl moiety is attached
directly to the 2' carbon of the sugar moiety.
[0376] As noted above, preferably the modification is a 2'-O-alkyl
group. More preferably the modification is selected from the group
consisting of 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-isopropyl,
2'-O-butyl, 2-O-isobutyl, 2'-O-ethyl-O-methyl
(--OCH.sub.2CH.sub.2OCH.sub.3), and 2'-O-ethyl-OH
(--OCH.sub.2CH.sub.2OH). Most preferably, the 2'-O-alkyl
modification be the same on each of the first 5' terminal sense
nucleotide and the second 5' terminal sense nucleotide. However, as
a matter of practicality with respect to synthesizing the molecules
of the present invention, it may be desirable to use the same
modification throughout.
[0377] The above-described modifications should not be construed to
suggest that no other moieties may be modified. Other types of
modifications are permissible so long as they do not increase
off-target effects. For example, in certain embodiments, additional
modifications can be added to one, two, three, or more consecutive
nucleotides or every-other nucleotide of the sense strand.
Alternatively, additional modifications can be confined to specific
positions that have been identified as being key to sense strand
entrance into the RISC complex. Further, additional 2'-O-methyl
groups (or other 2' modifications) can be added to one or more,
preferably all, pyrimidines (e.g., C and/or U nucleotides) of the
sense strand and/or 2' Fl modifications (or other halogen
modifications) can be added to one or more, preferably all
pyrimidines (e.g., C and/or U nucleotides) of the antisense strand.
(If the halogen modification is used in connection with the
sixteenth and seventeenth embodiments below, preferably all
pyrimidines other than those that fall within the first two 5'
terminal antisense nucleotides are halogen modified, or the first
three 5' terminal antisense nucleotides, if the first three contain
other modifications.)
[0378] In addition to chemical modifications, it is postulated that
base pair mismatches or bulges can be added to the sense and/or
antisense strands that alter the ability of these strands to
participate in RISC-mediated association with targets that share
less than 100% homology. Examples of such mismatches include (but
are not limited to) purine-pyrimidine mismatches (e.g., G-U, C-A)
and purine-purine or pyrimidine-pyrimidine mismatches (e.g., G-A,
U-C). The introduction of these types of modifications may be
combined with the above-described modifications, and evaluated to
determine whether they further reduce off-target effects.
[0379] In order to determine what modifications are permissible,
several non-limiting assays can be performed to identify
modifications that limit off-target effects. In one non-limiting
example, the sense strand (carrying the modification being tested)
can be labeled with one of many labeled nucleotides. Subsequently,
a binding assay can be performed whereby the affinity of RISC for
the modified sense strand can be compared with that of the
unmodified form.
[0380] According to a fourteenth embodiment, the present invention
is directed to a unimolecular siRNA that is capable of forming a
hairpin siRNA, said unimolecular siRNA comprising: [0381] (a) an
antisense region, wherein said antisense region is comprised of a
first 5' terminal antisense nucleotide and wherein said first 5'
terminal antisense nucleotide is phosphorylated at said first 5'
terminal antisense nucleotide's 5' carbon position; [0382] (b) a
sense region, wherein said sense region is comprised of a first 5'
terminal sense nucleotide and a second 5' terminal sense
nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification; and [0383] (c) a loop region, wherein said loop
region is located between said sense region and said antisense
region.
[0384] According to this embodiment, the range of modifications is
the same as those for the thirteenth embodiment. However, because
the polynucleotide is unimolecular and is capable of forming a
hairpin, and not two separate strands, there is one contiguous
molecule that comprises both a sense region and an antisense
region. Preferably, the sense region and the antisense region are
at least substantially complementary, more preferably 100%
complementary. As with the thirteenth embodiment, preferably the
sense region and the antisense region comprise 18-30 base pairs,
more preferably from 19-25 base pairs, and most preferably from
19-23 base pairs. Preferably the nucleotide is RNA.
[0385] When designing a unimolecular siRNA, specifically a
left-handed unimolecular structure (e.g., 5'-AS-Loop-S) according
to the present invention, preferably, the first 5' terminal sense
nucleotide is defined as the nucleotide that is the 18.sup.th,
19.sup.th or 20.sup.th base of the sense region counting from the
base that is complementary to the first 5' terminal antisense
nucleotide (i.e., counting from the 3' end of the sense region).
The first 5' terminal sense nucleotide is defined in this manner
because when unimolecular siRNAs that are capable of forming
hairpins enter a cell, typically, Dicer will process hairpin siRNAs
that contain lengthier duplex regions, into molecules that are
comprised of two separate strands (siRNA) of approximately 18-20
base pairs, and it is desirable for these molecules to retain the
sense strand modifications associated with the 5' end of the sense
strand of this processed molecule. Most preferably, the first 5'
terminal sense nucleotide is defined as the nucleotide that is the
19.sup.th base of the sense region from the 3' end of the sense
region. Further, preferably, the polynucleotide is capable of
forming a left-handed hairpin.
[0386] The hairpin may comprise a loop structure, which preferably
comprises from four to ten bases, and a sense region, wherein the
sense region and antisense region are independently 19-23 base
pairs in length and substantially complementary to each other.
Preferable sequences of the loop structure include, for example,
5'-UUCG (SEQ. ID NO. 323), 5'-UUUGUGUAG (SEQ. ID NO. 324), and
5'-CUUCCUGUCA (SEQ. ID NO. 325).
[0387] The hairpin is preferably constructed with the loop region
downstream of the antisense region. This construction is desirable
because it is easier to phosphorylate the terminal antisense
nucleotide. Thus, when designing the unimolecular siRNA, it is
preferable that there are no overhangs upstream of the 5' terminal
antisense nucleotide. At the 3' end of the sense region in some
embodiments, it may be desirable to have an overhang of, e.g., one,
two or three nucleotides. Additionally, preferably the unimolecular
siRNA is capable of forming a left handed hairpin.
[0388] The shRNA can further comprise a stem region, wherein the
stem region comprises one or more nucleotides or modified
nucleotides immediately adjacent to the 5' end and the 3' end of
the loop structure, and wherein the one or more nucleotides or
modified nucleotides of the stem region are (or are not)
target-specific. Preferably, the entire length of the unimolecular
siRNA contains fewer than 100 bases, more preferably fewer than 85
bases.
[0389] The unimolecular siRNA of the present invention may
ultimately be processed by cellular machinery such that they are
converted into two separate strands. Further, these unimolecular
siRNA may be introduced into the cell with less than all
modifications, and modified in the cell itself through the use of
natural processes or processing molecules that have been introduced
(e.g., phosphorylation in the cell). However, preferably the siRNA
is introduced with all modifications already present. (Similarly,
the strands of the first embodiment are preferably introduced into
the cell with all modifications, though the antisense strand could,
e.g., be modified after introduction.)
[0390] According to a fifteenth embodiment, the present invention
is directed to a method for minimizing off-target effects, said
method comprising exposing a modified siRNA to a target nucleic
acid, or a cell or organism that is either expressing the target
nucleic acid or capable of expressing the target nucleic acid,
wherein said siRNA comprises an antisense strand and a sense
strand, wherein: [0391] (a) said sense strand is comprised of a
first 5' terminal sense nucleotide and a second 5' terminal sense
nucleotide, wherein said first 5' terminal sense nucleotide
comprises a first 2' carbon sense modification and said second 5'
terminal sense nucleotide comprises a second 2' carbon sense
modification; and [0392] (b) said antisense strand is comprised of
a first 5' terminal antisense nucleotide and said first 5' terminal
antisense nucleotide is phosphorylated at said first 5' terminal
antisense nucleotide's 5' position.
[0393] The modified siRNAs of the fifteenth embodiment are
preferably the siRNAs of the thirteenth embodiment.
[0394] According to a sixteenth embodiment, the present invention
is directed to a method for minimizing off-target effects, said
method comprising exposing a unimolecular siRNA capable of forming
a hairpin to a target nucleic acid, or a cell or organism that is
either expressing the target nucleic acid or capable of expressing
the target nucleic acid, wherein said siRNA comprises an antisense
region and a sense region, wherein: [0395] (a) said sense region is
comprised of a first 5' terminal sense nucleotide and a second 5'
terminal sense nucleotide, wherein said first 5' terminal sense
nucleotide comprises a first 2' carbon sense modification and said
second 5' terminal sense nucleotide comprises a second 2' carbon
sense modification; [0396] (b) said antisense region is comprised
of a first 5' terminal antisense nucleotide and said first 5'
terminal antisense nucleotide is phosphorylated at said first 5'
terminal antisense nucleotide's 5' carbon position; and [0397] (c)
a loop region, wherein said loop region is located between said
sense region and said antisense region.
[0398] The modified siRNAs of the sixteenth embodiment are
preferably the siRNAs of the fourteenth embodiment.
[0399] In other embodiments there can still be additional
modifications such that the combination of the modifications can be
described as follows: (1) positions 1 and 2 (or 1, 2, and 3) of the
sense strand (or sense region in an shRNA), counting from the 5'
end of that strand are modified with 2' modifying groups (carbon 2
on the ribose sugar); (2) positions 1 and 2 (or 1, 2, and 3) of the
antisense strand (or antisense region in an shRNA), counting from
the 5' end of that strand are modified with 2' modifying groups
(carbon 2 on the ribose sugar); and (3) the 5' end of the antisense
strand is phosphorylated (carbon 5 on the ribose sugar). A
combination of all three types of modifications may be beneficial
in substantially reducing both sense and antisense strand induced
off-target effects.
[0400] According to the seventeenth embodiment, the present
invention provides an siRNA comprising: [0401] (a) an antisense
strand, wherein said antisense strand is comprised of a first 5'
terminal antisense nucleotide and a second 5' terminal antisense
nucleotide, wherein said first 5' terminal antisense nucleotide
comprises a first 2' carbon antisense modification and said second
5' terminal antisense nucleotide comprises a second 2' carbon
antisense modification and said first 5' terminal antisense
nucleotide is phosphorylated at said first 5' terminal antisense
nucleotide's 5' carbon position; and [0402] (b) a sense strand,
wherein said sense strand is comprised of a first 5' terminal sense
nucleotide and a second 5' terminal sense nucleotide, wherein said
first 5' terminal sense nucleotide comprises a first 2' carbon
sense modification and said second 5' terminal sense nucleotide
comprises a second 2' carbon sense modification.
[0403] According to this seventeenth embodiment, the modifications
are defined similarly to those of the thirteenth embodiment with
the addition of 2' carbon modifications on the first 5' terminal
antisense nucleotide and the second 5' terminal antisense
nucleotide. Alternatively, the modifications are defined similarly
to those of the thirteenth embodiment, with the addition of 2'
carbon modifications on the second terminal antisense nucleotide.
These 2' carbon modifications are defined in the same way as the
modifications for the first 5' terminal sense nucleotide and the
second 5' terminal sense nucleotide. Additionally, there may be a
further 2' carbon modification on the third 5' terminal antisense
nucleotide.
[0404] According to an eighteenth embodiment, the present invention
provides a unimolecular siRNA that is capable of forming a hairpin
molecule. The siRNA is comprised of: [0405] (a) an antisense
region, wherein said antisense region is comprised of a first 5'
terminal antisense nucleotide and a second 5' terminal antisense
nucleotide, wherein said first 5' terminal antisense nucleotide
comprises a first 2' carbon antisense modification and said second
5' terminal antisense nucleotide comprises a second 2' carbon
antisense modification and said first 5' terminal antisense
nucleotide is phosphorylated at said first 5' terminal antisense
nucleotide's 5' carbon position; [0406] (b) a sense region, wherein
said sense region is comprised of a first 5' terminal sense
nucleotide and a second 5' terminal sense nucleotide, wherein said
first 5' terminal sense nucleotide comprises a first 2' carbon
sense modification and said second 5' terminal sense nucleotide
comprises a second 2' carbon sense modification; and [0407] (c) a
loop region, wherein said loop region is located between said sense
region and said antisense region.
[0408] According to this eighteenth embodiment, the modifications
are defined similarly to those of the seventeenth embodiment with
the addition of 2' carbon modifications on the first 5' terminal
antisense nucleotide and the second 5' terminal antisense
nucleotide. These 2' carbon modifications are defined in the same
way as the modifications for the first 5' terminal sense nucleotide
and the second 5' terminal sense nucleotide. Additionally, there
may be a further 2' carbon modification on the third 5' terminal
antisense nucleotide.
[0409] According to a nineteenth embodiment, the present invention
provides a method for reducing off-target effects during gene
silencing. This method comprises administering the siRNA of the
seventeenth embodiment to a target nucleic acid or to a cell or
organism that is expressing or is capable of expressing the target
nucleic acid.
[0410] According to an twentieth embodiment, the present invention
provides a method for reducing off-target effects during gene
silencing. This method comprises administering the siRNA of the
eighteenth embodiment to a target nucleic acid or to a cell or
organism that is expressing or is capable of expressing the target
nucleic acid.
[0411] Although the above-described embodiments are directed to
increased specificity, it is important to note that siRNA may be
modified with other types and combinations of modifications that
may be designed to affect other parameters, such as those described
above that affect stability.
[0412] However, some stability directed modifications render siRNA
of limited functionality and should not be used except for example,
as a control or exaequo agent. (E.g. a sense strand, wherein said
sense strand is comprised of a first 5' terminal sense nucleotide
and a second 5' terminal sense nucleotide, wherein said first 5'
terminal sense nucleotide comprises a first 2'-O-alkyl (preferably
methyl) sense modification and said second 5' terminal sense
nucleotide comprises a second 2'-O-alkyl (preferably methyl) sense
modification; at least one (preferably all) 2'-O-alkyl (preferably
methyl) pyrimidine modified sense nucleotide(s), wherein said at
least one 2'-O-alkyl (preferably methyl) pyrimidine modified sense
nucleotide(s) is a nucleotide other than said first 5' terminal
sense nucleotide or said second 5' terminal sense nucleotide; and
an antisense strand, wherein said antisense strand is comprised of
a first 5' terminal antisense nucleotide and a second 5' terminal
antisense nucleotide, wherein said first 5' terminal antisense
nucleotide comprises and a first 2'-O-alkyl (preferably methyl)
antisense modification and said second 5' terminal sense nucleotide
comprises a second 2'-O-alkyl (preferably methyl) antisense
modification; and 2' labeled pyrimidines on the antisense strand,
preferably, at least one 2'-O-alkyl (preferably methyl) pyrimidine
modified antisense nucleotide, wherein said at least one
(preferably all) 2'-O-alkyl (preferably methyl) modified antisense
nucleotide is a nucleotide other than said first 5' terminal
antisense nucleotide or said second 5' terminal antisense
nucleotide, and optionally a label such as a fluorescent label on
the 5' terminal sense nucleotide).
[0413] Further, certain other related combinations of modifications
can be used to develop siRNA that may be used as controls or
exaequo agents. For example, an siRNA or shRNA may be developed
similar to the seventeenth and eighteenth embodiments described in
this disclosure, without the first 5' terminal antisense
nucleotide's having been phosphorylated. Thus, under one embodiment
there are 2' modifications that are limited to: (i) a first and
second, or first, second and third, 5' terminal sense nucleotides
that each comprises 2'modifications, such as the 2'modifications
described above, for example 2'-O-methyl modifications; and (ii) a
first and second, or first, second and third, 5' terminal antisense
nucleotides that each comprises 2'modifications, such as the
2'modifications described above, for example 2'-O-methyl
modifications.
[0414] When using exaequo agents or controls, it may be desirable
to modify the 5' carbon position of the 5' end of the sense and/or
the antisense strand with a blocking group. The blocking group may
for example be an alkyl group or any other group that prevents
phosphorylation of the 5' carbon position of the nucleotide.
Phosphorylation may occur in a cell due to the activity of kinases
that are present in cells. Exemplary blocking groups include but
are not limited to methyl, O-methyl, and amine groups.
[0415] These molecules are not functional, but may be used in
negative control studies, and as exaequo agents. Further, if a
label such as the labels described above are used, these agents can
be useful as tracking agents, which would assist in detection of
transfection, as well as detection of where in the cell the
molecule is present. Because these agents are not used for
silencing, they can contain from 16-28 base pairs, though
preferably contain 18-30 base pairs.
[0416] Additionally stabilization modifications that are addressed
to the phosphate backbone may be included in the siRNAs for some
applications of the present invention. For example, at least one
phosphorothioate and/or methylphosphonate may be substituted for
the phosphate group at some or all 3' positions of any or all
pyrimidines in the sense and/or antisense strands of the
oligonucleotide backbone, as well as in any overhangs, loop
structures or stem structures that may be present. Phosphorothioate
(and methylphosphonate) analogues arise from modification of the
phosphate groups in the oligonucleotide backbone. In the
phosphorothioate, the phosphate O-- is replaced by a sulfur atom.
In methylphosphonates, the oxygen is replaced with a methyl group.
In one embodiment the phosphorothioate modification or
methylphosphonate is located at the 3' positions of all antisense
strand nucleotides that also contains 2' fluoro (or other halogen)
modified nucleotides. Additionally, phosphorothioate 3'
modifications may be used instead of and independent of 2' fluoro
modifications to increase stability of an siRNA molecule. These
modifications may be used in combination with the other
modifications disclosed herein, or independent of those
modifications in siRNA applications.
[0417] Nucleases typically use both the oxygen groups on the
phosphate moiety and the 2'OH position of the ribose ring to
mediate attack on RNA. Substitution of a sulfur group for one of
the oxygens eliminates the ability of the phosphate to participate
in this reaction, thus limiting the sensitivity of this site to
nuclease digestion. However, it should be noted that
phosphorothioates are typically toxic, thus, they would be
beneficial primarily when any toxic effects are negated, which it
is postulated might be accomplished by limiting the use of this
modification to, e.g., every other nucleotide, every third
nucleotide, or every fourth nucleotide.
[0418] Furthermore, for some applications it may be desirable to
incorporate a label into the nucleotides of the present invention,
e.g., a fluorescent label, a radioactive label or a mass label.
[0419] The above described modifications of the present invention
may be combined with siRNA that contains sequences that were
selected at random, or according to rationale design as described
in, for example, U.S. patent application Ser. No. 10/714,333.
Additionally, it may be desirable to select sequences in whole or
in part based on internal thermal stability, which may facilitate
processing by cellular machinery.
[0420] In developing the thirteenth through twentieth embodiments
of the present invention, two or more different modifications were
added to a duplex to minimize off-target effects. As noted above,
Applicants appreciate that other modifications and combinations
that assist in minimizing off-target effects may be discovered in
the future. Additionally, the modifications of the present
invention could be combined with modifications that are desired for
other purposes. For example, in some instances, one modification
could affect one particular step of off-target silencing (e.g.
sense strand association with RISC) while a second modification
could affect a completely different step e.g. altering the ability
of sense/antisense strands to associate with targets that have less
than 100% homology. Alternatively, two separate modifications could
affect the same step. In some cases, two or more modifications
could act additively or synergistically, limiting off-target
effects by minimizing undesirable interactions or processes at one
or more steps. In still other instances, one modification could
eliminate off-target effects, but have detrimental consequences on
more desirable properties e.g. the potency or stability of the
siRNA. In cases such as these, additional modifications could be
added that restore functionality of the molecule.
[0421] A variety of approaches can be used to identify both the
type of molecule and the key position needed to eliminate sense
and/or antisense strand off-targeting effects. In one non-limiting
example, a modification-function walk is performed. In this
procedure, a single type of modification is added to one or more
nucleotides across the sense and/or antisense strand. Subsequently,
modified and unmodified molecules are tested for: (1)
functionality; and (2) off-targeting effects, by one of several
methods. Thus, for example, 2'-O-Me groups can be added to
positions 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12,
13 and 14, 15 and 16, 17 and 18, or 18 and 19 of either the sense
and/or antisense strand and tested for functionality (e.g., by
measuring the ability of these molecules to silence specific
targets) and off-target effects (e.g., by microarray analysis). If
key positions are identified that eliminate some or all
off-targeting, but result in a loss of duplex functionality, then a
second round of modification walks, whereby additional chemical
groups (e.g., 5' phosphate on the 5' end of the antisense strand),
mismatches, or bulges that are suspected to increase duplex
functionality can be added to molecules that already contain the
modification that eliminates off-targeting.
[0422] It should be noted that the modifications of the thirteenth
through twentieth embodiment of the present invention may have
different effects depending on the functionality of the siRNA that
are employed. Thus, in highly functional siRNA, the modifications
of the present invention may cause a molecule to lose a certain
amount of functionality, but would nonetheless be desirable because
off-target effects are reduced. By contrast when moderately or
poorly functional siRNA are used, there is very little
functionality decrease and in some cases, functionality can
increase.
[0423] Because the ability of the dsRNA of the present invention to
retain functionality and to resist degradation is not dependent on
the sequence of the bases, the cell type, or the species into which
it is introduced, the present invention is applicable across a
broad range of organisms, including but not limited plants,
animals, protozoa, bacteria, viruses and fungi. The present
invention is particularly advantageous for use in mammals such as
cattle, horse, goats, pigs, sheep, canines, rodents such as
hamsters, mice, and rats, and primates such as, gorillas,
chimpanzees, and humans.
[0424] The present invention may be used advantageously with
diverse cell types, including but not limited to primary cells,
germ cell lines and somatic cells. The cells may be, for example,
stem cells or differentiated cells. For example, the cell types may
be embryonic cells, oocytes, sperm cells, adipocytes, fibroblasts,
myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells,
megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils,
basophils, mast cells, leukocytes, granulocytes, keratinocytes,
chondrocytes, osteoblasts, osteoclasts, hepatocytes and cells of
the endocrine or exocrine glands.
[0425] The present invention is applicable for use for employing
RNA interference (and/or using as a control) directed against a
broad range of genes, including but not limited to the 45,000 genes
of a human genome, such as those implicated in diseases such as
diabetes, Alzheimer's and cancer, as well as all genes in the
genomes of the humans, mice, hamsters, chimpanzees, goats, sheep,
horses, camels, pigs, dogs, cats, nematodes (e.g., C. elegans),
flies (e.g., D. melanogaster), and other vertebrates and
invertebrates.
[0426] The siRNAs of the present invention may be administered to a
cell by any method that is now known or that comes to be known and
that from reading this disclosure, one skilled in the art would
conclude would be useful with the present invention. For example,
the siRNAs may be passively delivered to cells.
[0427] Passive uptake of modified siRNAs can be modulated, for
example, by the presence of a conjugate such as a polyethylene
glycol moiety or a cholesterol moiety at the 5' terminal of the
sense strand and/or, in appropriate circumstances, a
pharmaceutically acceptable carrier.
[0428] Other methods for delivery include, but are not limited to,
transfection techniques employing DEAE-Dextran, calcium phosphate,
cationic lipids/liposomes, microinjection, electroporation,
immunoporation, and coupling of the siRNAs to specific conjugates
or ligands such as antibodies, antigens, or receptors.
[0429] Preferably, the siRNAs comprise duplexes when they are
administered.
[0430] Further, the method of assessing the level of gene silencing
is not limited. Thus, the silencing ability of any given siRNA can
be studied by one of any number of art tested procedures including
but not limited to Northern analysis, Western Analysis, RT PCR,
expression profiling, and others.
[0431] Further, the siRNA of the present invention may be used in a
diverse set of applications, including but not limited to basic
research, drug discovery and development, diagnostics and
therapeutics. For example, the present invention may be used to
validate whether a gene product is a target for drug discovery or
development. In this application, the mRNA that corresponds to a
target nucleic acid sequence of interest is identified for targeted
degradation. Inventive siRNAs that are specific for targeting the
particular gene are introduced into a cell or organism, preferably
in duplex form. The cell or organism is maintained under conditions
allowing for the degradation of the targeted mRNA, resulting in
decreased activity or expression of the gene. The extent of any
decreased expression or activity of the gene is then measured,
along with the effect of such decreased expression or activity, and
a determination is made that if expression or activity is
decreased, then the nucleic acid sequence of interest is an agent
for drug discovery or development. In this manner, phenotypically
desirable effects can be associated with RNA interference of
particular target nucleic acids of interest, and in appropriate
cases toxicity and pharmacokinetic studies can be undertaken and
therapeutic preparations developed.
[0432] The present invention may also be used in RNA interference
applications that induce transient or permanent states of disease
or disorder in an organism by, for example, attenuating the
activity of a target nucleic acid of interest believed to be a
cause or factor in the disease or disorder of interest. Increased
activity of the target nucleic acid of interest may render the
disease or disorder worse, or tend to ameliorate or to cure the
disease or disorder of interest, as the case may be. Likewise,
decreased activity of the target nucleic acid of interest may cause
the disease or disorder, render it worse, or tend to ameliorate or
cure it, as the case may be. Target nucleic acids of interest can
comprise genomic or chromosomal nucleic acids or extrachromosomal
nucleic acids, such as viral nucleic acids.
[0433] Further, the present invention may be used in RNA
interference applications that determine the function of a target
nucleic acid or target nucleic acid sequence of interest. For
example, knockdown experiments that reduce or eliminate the
activity of a certain target nucleic acid of interest. This can be
achieved by performing RNA interference with one or more siRNAs
targeting a particular target nucleic acid of interest. Observing
the effects of such a knockdown can lead to inferences as to the
function of the target nucleic acid of interest. RNA interference
can also be used to examine the effects of polymorphisms, such as
biallelic polymorphisms, by attenuating the activity of a target
nucleic acid of interest having one or the other allele, and
observing the effect on the organism or system studied.
Therapeutically, one allele or the other, or both, may be
selectively silenced using RNA interference where selective allele
silencing is desirable.
[0434] Still further, the present invention may be used in RNA
interference applications, such as diagnostics, prophylactics, and
therapeutics including use of the compositions in the manufacture
of a medicament in animals, preferably mammals, more preferably
humans in the treatment of diseases, or over or under expression of
a target. Preferably, the disease or disorder is one that arises
from the malfunction of one or more proteins, the disease or
disorder of which is related to the expression of the gene product
of the one or more proteins. For example, it is widely recognized
that certain cancers of the human breast are related to the
malfunction of a protein expressed from a gene commonly known as
the "bcl-2" gene. A medicament can be manufactured in accordance
with the compositions and teachings of the present invention,
employing one or more siRNAs directed against the bcl-2 gene, and
optionally combined with a pharmaceutically acceptable carrier,
diluent and/or adjuvant, which medicament can be used for the
treatment of breast cancer. Applicants have established the utility
of the methods and compositions in cellular models. Methods of
delivery of polynucleotides, such as siRNAs, to cells within
animals, including humans, are well known in the art. Any delivery
vehicle now known in the art, or that comes to be known, and has
utility for introducing polynucleotides, such as siRNAs, to
animals, including humans, is expected to be useful in the
manufacture of a medicament in accordance with the present
invention, so long as the delivery vehicle is not incompatible with
any modifications that may be present a composition made according
to the present invention. A delivery vehicle that is not compatible
with a composition made according to the present invention is one
that reduces the efficacy of the composition by greater than 95% as
measured against efficacy in cell culture.
[0435] Animal models exist for many, many disorders, including, for
example, cancers, diseases of the vascular system, inborn errors or
metabolism, and the like. It is within ordinary skill in the art to
administer nucleic acids to animals in dosing regimens to arrive at
an optimal dosing regimen for particular disease or disorder in an
animal such as a mammal, for example, a mouse, rat or non-human
primate. Once efficacy is established in the mammal by routine
experimentation by one of ordinary skill, dosing regimens for the
commencement of human trials can be arrived at based on data
arrived at in such studies.
[0436] Dosages of medicaments manufactured in accordance with the
present invention may vary from micrograms per kilogram to hundreds
of milligrams per kilogram of a subject. As is known in the art,
dosage will vary according to the mass of the mammal receiving the
dose, the nature of the mammal receiving the dose, the severity of
the disease or disorder, and the stability of the medicament in the
serum of the subject, among other factors well known to persons of
ordinary skill in the art.
[0437] For these applications, an organism suspected of having a
disease or disorder that is amenable to modulation by manipulation
of a particular target nucleic acid of interest is treated by
administering siRNA. Results of the siRNA treatment may be
ameliorative, palliative, prophylactic, and/or diagnostic of a
particular disease or disorder. Preferably, the siRNA is
administered in a pharmaceutically acceptable manner with a
pharmaceutically acceptable carrier or diluent.
[0438] Therapeutic applications of the present invention can be
performed with a variety of therapeutic compositions and methods of
administration. Pharmaceutically acceptable carriers and diluents
are known to persons skilled in the art. Methods of administration
to cells and organisms are also known to persons skilled in the
art. Dosing regimens, for example, are known to depend on the
severity and degree of responsiveness of the disease or disorder to
be treated, with a course of treatment spanning from days to
months, or until the desired effect on the disorder or disease
state is achieved. Chronic administration of siRNAs may be required
for lasting desired effects with some diseases or disorders.
Suitable dosing regimens can be determined by, for example,
administering varying amounts of one or more siRNAs in a
pharmaceutically acceptable carrier or diluent, by a
pharmaceutically acceptable delivery route, and amount of drug
accumulated in the body of the recipient organism can be determined
at various times following administration. Similarly, the desired
effect (for example, degree of suppression of expression of a gene
product or gene activity) can be measured at various times
following administration of the siRNA, and this data can be
correlated with other pharmacokinetic data, such as body or organ
accumulation. Those of ordinary skill can determine optimum
dosages, dosing regimens, and the like. Those of ordinary skill may
employ EC.sub.50 data from in vivo and in vitro animal models as
guides for human studies.
[0439] Still further, the present invention may be used in RNA
interference applications, such as diagnostics, prophylactics, and
therapeutics. For these applications, an organism suspected of
having a disease or disorder that is amenable to modulation by
manipulation of a particular target nucleic acid of interest is
treated by administering siRNA. Results of the siRNA treatment may
be ameliorative, palliative, prophylactic, and/or diagnostic of a
particular disease or disorder. Preferably, the siRNA is
administered in a pharmaceutically acceptable manner with a
pharmaceutically acceptable carrier or diluent.
[0440] Further, the siRNAs can be administered in a cream or
ointment topically, an oral preparation such as a capsule or tablet
or suspension or solution, and the like. The route of
administration may be intravenous, intramuscular, dermal,
subdermal, cutaneous, subcutaneous, intranasal, oral, rectal, by
eye drops, by tissue implantation of a device that releases the
siRNA at an advantageous location, such as near an organ or tissue
or cell type harboring a target nucleic acid of interest.
[0441] According to another embodiment, the present invention
comprises an siRNA, comprising a sense strand and an antisense
strand, wherein the sense strand and the antisense strand each
comprises at least one orthoester modification at a 2'
position.
[0442] According to another embodiment, the present invention
comprises an siRNA, comprising a sense strand and an antisense
strand, wherein said antisense strand comprises at least one
orthoester modification and/or at least one modification selected
from the group consisting of a 2'-halogen modification, a 2'-alkyl
modification, a 2'-O-alkyl modification, a 2'-amine modification,
and a 2'-deoxy modification.
[0443] According to another embodiment, the present invention
comprises an siRNA, comprising a sense strand and an antisense
strand, wherein the sense strand and/or the antisense strand
comprises at least one orthoester modification, and wherein the
sense strand and/or the antisense strand comprises at least one 2'
modification selected from the group consisting of a 2'-halogen
modification, a 2'-alkyl modification, a 2'-O-alkyl modification, a
2'-amine modification, and a 2'-deoxy modification.
[0444] In yet another embodiment, the present invention comprises
an siRNA, comprising: (a) a sense strand, wherein said sense strand
is comprised of a first 5' terminal sense nucleotide and a second
5' terminal sense nucleotide, wherein said first 5' terminal sense
nucleotide comprises a first 2' carbon sense modification and said
second 5' terminal sense nucleotide comprises a second 2' carbon
sense modification; and (b) an antisense strand, wherein said
antisense strand is comprised of a first 5' terminal antisense
nucleotide and a second 5' terminal antisense nucleotide, wherein
said first 5' terminal antisense nucleotide comprises a first 2'
carbon antisense modification and a 5' modification, and said
second 5' terminal antisense nucleotide comprises a second 2'
carbon antisense modification.
[0445] In yet another embodiment, the present invention comprises
an siRNA, comprising a sense strand and an antisense strand,
wherein the antisense strand comprises at least one 2'-orthoester
modification, and wherein the antisense strand further modified
with one or more modifications selected from the group consisting
of a 2' orthoester modification, a 2'-alkyl modification, a
2'-halogen modification, a 2'-O-alkyl modification, a 2'-amine
modification, and a 2'-deoxy modification, wherein the 2'-alkyl
modification, the 2'-O-methyl modification, and the 2' halogen
modification is on one or more pyrimidines of the antisense strand,
and wherein the siRNA further comprises a 3' cap. The 2' carbon
modification can be a 2'-O-alkyl modification such as, for example,
a 2'-O-methyl modification. The first 5' terminal sense nucleotide
and/or the first 5' terminal antisense nucleotide can be further
modified with a 5' blocking group. The 5' blocking group can be
selected from the group consisting of a 5'-methyl modification, a
5'-O-methyl modification, and a 5' azide modification.
[0446] In another embodiment, the first and second 2'-O-alkyl
modifications of the 5' terminal sense nucleotide and/or the 5'
terminal antisense nucleotide is a 2'-O-methyl modification, and
the first 5' terminal sense and/or antisense nucleotide can further
comprise a 5' blocking group. The 5' blocking group is selected
from the group consisting of a 5'-methyl modification, a
5'-O-methyl modification, or a 5' azide modification.
[0447] In other embodiments, the invention provides siRNA,
comprising an antisense strand and a sense strand, wherein the
sense strand is comprised of a first 5' terminal sense nucleotide
and a second 5' terminal sense nucleotide, wherein the first 5'
terminal sense nucleotide comprises a first 2' carbon sense
modification and the second 5' terminal sense nucleotide comprises
a second 2' carbon sense modification.
[0448] In other embodiments, the present invention provides an
siRNA comprising (a) a sense strand, wherein the sense strand is
comprised of a first 5' terminal sense nucleotide and a second 5'
terminal sense nucleotide, wherein the first 5' terminal sense
nucleotide comprises a first 2' carbon sense modification and the
second 5' terminal sense nucleotide comprises a second 2' carbon
sense modification; and (b) an antisense strand, wherein the
antisense strand is comprised of a first 5' terminal antisense
nucleotide and a second 5' terminal antisense nucleotide, wherein
the first 5' terminal antisense nucleotide comprises a 5' phosphate
modification and the second 5' terminal antisense nucleotide
comprises a 2' carbon antisense modification.
[0449] In other embodiments, any of the compositions of the present
invention can further comprise a 3' cap. The 3' cap can be, for
example, an inverted deoxythymidine.
[0450] In other embodiments, the compositions of the present
invention can comprise at least one of a 2'-deoxy modification
and/or a methylphosphonate internucleotide linkage. The
compositions of the present invention can also comprise, on one or
more pyrimidines, modification selected from the group consisting
of a 2'-alkyl modification, a 2'-O-alkyl modification, and a
2'-halogen modification. The 2'-alkyl modification can be a
2'-methyl modification, such as, for example, a 2'-O-methyl
modification. The 2'-halogen modification can be, for example, a
2'-fluorine modification.
[0451] In other embodiments, the compositions of the present
invention can comprise at least one of a 2'-amine modification
and/or a 2'-halogen modification. The 2'-amine modification can be,
for example, a 2'-NH.sub.2 modification. The 2'-halogen
modification can be, for example, a 2'-fluorine modification. The
compositions of the present invention can have one or more modified
internucleotide linkages, including, for example, one or more
phosphorothioate links, phosphorodithioate links, methylphosphonate
links, and combinations thereof.
[0452] In other embodiments of the present invention, any of the
compositions can comprise a conjugate. The conjugate can be
selected from the group consisting of amino acids, peptides,
polypeptides, proteins, sugars, carbohydrates, lipids, polymers,
nucleotides, polynucleotides, and combinations thereof The
conjugate can be cholesterol or PEG. The conjugate can further
comprise a label, such as, for example, a fluorescent label. The
fluorescent label can be selected from the group consisting of
TAMRA, BODIPY, Cy3, Cy5, fluoroscein, and Dabsyl. Alternatively,
the fluorescent label can be any fluorescent label known in the
art.
[0453] In other embodiments, the compositions of the present
invention can comprise at least one 2'-orthoester modification,
wherein the 2'-orthoester modification is preferably a
2'-bis(hydroxy ethyl)orthoester modification.
[0454] In other embodiments, any of the compositions of the present
invention can be used in a method of performing RNA interference,
comprising administering one or more of the compositions of the
invention to a cell. Preferably, where an orthoester modification
is present on an siRNA, the orthoester modification is a
2'-bis(hydroxy ethyl)orthoester.
[0455] Having described the invention with a degree of
particularity, examples will now be provided. These examples are
not intended to and should not be construed to limit the scope of
the claims in any way. Although the invention may be more readily
understood through reference to the following examples, they are
provided by way of illustration and are not intended to limit the
present invention unless specified.
Examples
Example 1
Synthesizing siRNAs
[0456] RNA oligonucleotides were synthesized in a stepwise fashion
using the nucleotide addition reaction cycle illustrated in FIG.
13. The synthesis is preferably carried out as an automated process
on an appropriate machine. Several such synthesizing machines are
known to those of skill in the art. Each nucleotide is added
sequentially (3'- to 5'-direction) to a solid support-bound
oligonucleotide. Although polystyrene supports are preferred, any
suitable support can be used. The first nucleoside at the 3'-end of
the chain is covalently attached to a solid support. The nucleotide
precursor, an activated ribonucleotide such as a phosphoramidite or
H-phosphonate, and an activator such as a tetrazole, for example,
S-ethyl-tetrazole (although any other suitable activator can be
used) are added (step i in FIG. 13), coupling the second base onto
the 5'-end of the first nucleoside. The support is washed and any
unreacted 5'-hydroxyl groups are capped with an acetylating reagent
such as but not limited to acetic anhydride or phenoxyacetic
anhydride to yield unreactive 5'-acetyl moieties (step ii). The
P(III) linkage is then oxidized to the more stable and ultimately
desired P(V) linkage (step iii), using a suitable oxidizing agent
such as, for example, t-butyl hydroperoxide or iodine and water. At
the end of the nucleotide addition cycle, the 5'-silyl group is
cleaved with fluoride ion (step iv), for example, using
triethylammonium fluoride or t-butyl ammonium fluoride. The cycle
is repeated for each subsequent nucleotide. It should be emphasized
that although FIG. 13 illustrates a phosphoramidite having a methyl
protecting group, any other suitable group may be used to protect
or replace the oxygen of the phosphoramidite moiety. For example,
alkyl groups, cyanoethyl groups, or thio derivatives can be
employed at this position. Further, the incoming activated
nucleoside in step (i) can be a different kind of activated
nucleoside, for example, an H-phosphonate, methyl phosphonamidite
or a thiophosphoramidite. It should be noted that the initial, or
3', nucleoside attached to the support can have a different 5'
protecting group such as a dimethoxytrityl group, rather than a
silyl group. Cleavage of the dimethoxytrityl group requires acid
hydrolysis, as employed in standard DNA synthesis chemistry. Thus,
an acid such as dichloroacetic acid (DCA) or trichloroacetic acid
(TCA) is employed for this step alone. Apart from the DCA cleavage
step, the cycle is repeated as many times as necessary to
synthesize the polynucleotide desired.
[0457] Following synthesis, the protecting groups on the
phosphates, which are depicted as methyl groups in FIG. 13, but
need not be limited to methyl groups, are cleaved in 30 minutes
utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate
trihydrate (dithiolate) in DMF (dimethylformamide). The
deprotection solution is washed from the solid support bound
oligonucleotide using water. The support is then treated with 40%
methylamine for 20 minutes at 55.degree. C. This releases the RNA
oligonucleotides into solution, deprotects the exocyclic amines and
removes the acetyl protection on the 2'-ACE groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0458] The 2'-orthoester groups are the last protecting groups to
be removed, if removal is desired. The structure of the 2'-ACE
protected RNA immediately prior to 2'-deprotection is as
represented in FIG. 14.
[0459] For automated procedures, solid supports having the initial
nucleoside are installed in the synthesizing instrument. The
instrument will contain all the necessary ancillary reagents and
monomers needed for synthesis. Reagents are maintained under argon,
since some monomers, if not maintained under an inert gas, can
hydrolyze. The instrument is primed so as to fill all lines with
reagent. A synthesis cycle is designed that defines the delivery of
the reagents in the proper order according to the synthesis cycle,
delivering the reagents in the order specified in FIG. 13. Once a
cycle is defined, the amount of each reagent to be added is
defined, the time between steps is defined, and washing steps are
defined, synthesis is ready to proceed once the solid support
having the initial nucleoside is added.
[0460] For the RNA analogs described herein, modification is
achieved through three different general methods. The first, which
is implemented for carbohydrate and base modifications, as well as
for introduction of certain linkers and conjugates, employs
modified phosphoramidites in which the modification is
pre-existing. An example of such a modification would be the
carbohydrate 2'-modified species (2'-F, 2'-NH2, 2'-O-alkyl, etc.)
wherein the 2' orthoester is replaced with the desired modification
3' or 5' terminal modifications could also be introduced such as
fluoroscein derivatives, Dabsyl, cholesterol, cyanine derivatives
or polyethylene glycol. Certain inter-nucleotide bond modifications
would also be introduced via the incoming reactive nucleoside
intermediate. Examples of the resultant internucleotide bond
modification include but are not limited to methylphosphonates,
phosphoramidates, phosphorothioates or phoshorodithioates.
[0461] Many modifiers can be employed using the same or similar
cycles. Examples in this class would include, for example,
2-aminopurine, 5-methyl cytidine, 5-aminoallyl uridine,
diaminopurine, 2-O-alkyl, multi-atom spacers, single monomer
spacers, 2'-aminonucleosides, 2'-fluoro nucleosides, 5-iodouridine,
4-thiouridine, acridines, 5-bromouridine, 5-fluorocytidine,
5-fluorouridine, 5-iodouridine, 5-iodocytidine, 5-biotin-thymidine,
5-fluoroscein-thymidine, inosine, pseudouridine, abasic monomer,
nebularane, deazanucleoside, pyrene nucleoside, azanucleoside, etc.
Often the rest of the steps in the synthesis would remain the same
with the exception of modifications that introduce substituents
that are labile to standard deprotection conditions. Here modified
conditions would be employed that do not effect the substituent.
Second, certain internucleotide bond modifications require an
alteration of the oxidation step to allow for their introduction.
Examples in this class include phosphorothioates and
phosphorodithioates wherein oxidation with elemental sulfur or
another suitable sulfur transfer agent is required. Third, certain
conjugates and modifications are introduced by "post-synthesis"
process, wherein the desired molecule is added to the biopolymer
after solid phase synthesis is complete. An example of this would
be the addition of polyethylene glycol to a pre-synthesized
oligonucleotide that contains a primary amine attached to a
hydrocarbon linker. Attachment in this case can be achieved by
using a N-hydroxy-succinimidyl ester of polyethylene glycol in a
solution phase reaction.
[0462] While this outlines the most preferred method for synthesis
of synthetic RNA and its analogs, any nucleic acid synthesis method
which is capable of assembling these molecules could be employed in
their assembly. Examples of alternative methods include
5'-DMT-2'-TBDMS and 5'-DMT-2'-TOM synthesis approaches. Some
2'-O-methyl, 2'-F and backbone modifications can be introduced in
transcription reactions using modified and wild type T7 and SP6
polymerases, for example.
[0463] Synthesizing Modified RNA
[0464] The following guidelines are provided for synthesis of
modified RNAs, and can readily be adapted to use on any of the
automated synthesizers known in the art.
[0465] 3' Terminal Modifications
[0466] There are several methods for incorporating 3'
modifications. The 3' modification can be anchored or "loaded" onto
a solid support of choice using methods known in the art.
Alternatively, the 3' modification may be available as a
phosphoramidite. The phosphoramidite is coupled to a universal
support using standard synthesis methods where the universal
support provides a hydroxyl at which the 3' terminal modification
is created by introduction of the activated phosphoramidite of the
desired terminal modification. Alternatively, the 3' modification
could be introduced post synthetically after the polynucleotide is
removed from the solid support. The free polynucleotide initially
has a 3' terminal hydroxyl, amino, thiol, or halogen that reacts
with an appropriately activated form of the modification of choice.
Examples include but are not limited to N-hydroxy succinimidyl
ester, thioether, disulfide, maliemido, or haloalkyl reactions.
This modification now becomes the 3' terminus of the
polynucleotide. Examples of modifications that can be conjugated
post synthetically can be but are not limited to fluorosceins,
acridines, TAMRA, dabsyl, cholesterol, polyethylene glycols,
multi-atom spacers, cyanines, lipids, carbohydrates, fatty acids,
steroids, peptides, or polypeptides.
[0467] 5' Terminal Modifications
[0468] There are a number of ways to introduce a 5' modification
into a polynucleotide. For example, a nucleoside having the 5'
modification can be purchased and subsequently activated to a
phosphoramidite. The phosphoramidite having the 5' modification may
also be commercially available. Then, the activated nucleoside
having the 5' modification is employed in the cycle just as any
other activated nucleoside may be used. However, not all 5'
modifications are available as phosphoramidites. In such an event,
the 5' modification can be introduced in an analogous way to that
described for 3' modifications above.
[0469] Thioates
[0470] Polynucleotides having one or more thioate moieties, such as
phosphorothioate linkages, were made in accordance with the
synthesis cycle described above and illustrated in FIG. 13.
However, in place of the t-butyl hydroperoxide oxidation step,
elemental sulfur or another sulfurizing agent was used.
[0471] 5'-Thio Modifications
[0472] Monomers having 5' thiols can be purchased as
phosphoramidites from commercial suppliers such as Glen Research.
These 5' thiol modified monomers generally bear trityl protecting
groups. Following synthesis, the trityl group can be removed by any
method known in the art.
[0473] Other Modifications
[0474] For certain modifications, the steps of the synthesis cycle
will vary somewhat. For example, where the 3' end has an inverse dT
(wherein the first base is attached to the solid support through
the 5'-hydroxyl and the first coupling is a 3'-3' linkage)
detritylation and coupling occurs more slowly, so extra
detritylating reagent, such as dichloroactetic acid (DCA), should
be used and coupling time should be increased to 300 seconds. Some
5' modifications may require extended coupling time. Examples
include cholesterol, fluorophores such as Cy3 or Cy5 biotin,
dabsyl, amino linkers, thio linkers, spacers, polyethylene glycol,
phosphorylating reagent, BODIPY, or photocleavable linkers.
[0475] It should be noted that if a polynucleotide is to have only
a single modification, that modification can be most efficiently
carried out manually by removing the support having the partially
built polynucleotide on it, manually coupling the monomer having
the modification, and then replacing the support in the automated
synthesizer and resuming automated synthesis.
Example 2
Deprotection and Cleavage of Synthesized Oligos from the
Support
[0476] Cleaving can be done manually or in an automated process on
a machine. Cleaving of the protecting moiety from the
internucleotide linkage, for example a methyl group, can be
achieved by using any suitable cleaving agent known in the art, for
example, dithiolate or thiophenol. One molar dithiolate in DMF is
added to the solid support at room temperature for 10 to 20
minutes. The support is then thoroughly washed with, for example,
DMF, then water, then acetonitrile. Alternatively a water wash
followed by a thorough acetonitrile will suffice to remove any
residual dithioate.
[0477] Cleavage of the polynucleotide from the support and removal
of exocyclic base protection can be done with 40% aqueous
N-methylamine (NMA), followed by heating to 55 degrees Centigrade
for twenty minutes. Once the polynucleotide is in solution, the NMA
is carefully removed from the solid support. The solution
containing the polynucleotide is then dried down to remove the NMA
under vacuum. Further processing, including duplexing, desalting,
gel purifying, quality control, and the like can be carried out by
any method known in the art.
[0478] For some modifications, the NMA step may vary. For example,
for a 3' amino modification, the treatment with NMA should be for
forty minutes at 55 degrees Centigrade. Puromycin, 5' terminal
amino linker modifications, and 2' amino nucleoside modifications
are heated for 1 hour after addition of 40% NMA. Oligonucleotides
modified with Cy5 are treated with ammonium hydroxide for 24 hours
while protected from light.
[0479] Preparation of Cleave Reagents
[0480] HPLC grade water and synthesis grade acetonitrile are used.
The dithiolate is pre-prepared as crystals. Add 4.5 grams of
dithiolate crystals to 90 mL of DMF. Forty percent NMA can be
purchased, ready to use, from a supplier such as Sigma Aldrich
Corporation.
[0481] Annealing Single Stranded Polynucleotides
[0482] Single stranded polynucleotides can be annealed by any
method known in the art, employing any suitable buffer. For
example, equal amounts of each strand can be mixed in a suitable
buffer, such as, for example, 50 mM HEPES pH 7.5, 100 mM potassium
chloride, 1 mM magnesium chloride. The mixture is heated for one
minute at 90 degrees Centigrade, and allowed to cool to room
temperature. In another example, each polynucleotide is separately
prepared such that each is at 50 micromolar concentration. Thirty
microliters of each polynucleotide solution is then added to a tube
with 15 microliters of 5.times. annealing buffer, wherein the
annealing buffer final concentration is 100 mM potassium chloride,
30 mM HEPES-KOH pH 7.4 and 2 mM magnesium chloride. Final volume is
75 microliters. The solution is then incubated for one minute at 90
degrees Centigrade, spun in a centrifuge for 15 seconds, and
allowed to incubate at 37 degrees Centigrade for one hour, then
allowed to come to room temperature. This solution can then be
stored frozen at minus 20 degrees Centigrade and freeze thawed up
to five times. The final concentration of the duplex is 20
micromolar. An example of a buffer suitable for storage of the
polynucleotides is 20 mM KCl, 6 mM HEPES pH 7.5, 0.2 mM MgCl.sub.2.
All buffers used should be RNase free.
[0483] Removal of the Orthoester Moiety
[0484] If desired, the orthoester moiety or moieties may be removed
from the polynucleotide by any suitable method known in the art.
One such method employs a volatile acetic
acid-tetramethylenediamine (TEMED) pH 3.8 buffer system that can be
removed by lyophilization following removal of the orthoester
moiety or moieties. Deprotection at a pH higher than 3.0 helps
minimize the potential for acid-catalyzed cleavage of the
phosphodiester backbone. For example, deprotection can be achieved
using 100 mM acetic acid adjusted to pH 3.8 with TEMED by
suspending the orthoester protected polynucleotide and incubating
it for 30 minutes at 60 degrees Centigrade. The solution is then
lyophilized or subjected to a SpeedVac to dryness prior to use. If
necessary, desalting following deprotection can be performed by any
method known in the art, for example, ethanol precipitation or
desalting on a reversed phase cartridge.
Example 3
siRNAs Synthesized for Use in RNA Interference
[0485] The following is a list of 19-mer siRNAs having a di-dT
overhang that were synthesized using Dharmacon, Inc.'s proprietary
ACE chemistry, and were designed and used in accordance with the
invention described herein. "SEAP" refers to human secreted
alkaline phosphatase; "human cyclo" refers to human cyclophilin; an
asterisk between nucleotide units refers to a modified
internucleotide linkage that is a phosphorothioate linkage; the
structure 2'-F-C or 2'-F-U refers to a nucleotide unit having a
fluorine atom attached to the 2' carbon of a ribosyl moiety; the
structure 2'-N-C or 2'-N-U refers to a nucleotide unit having an
--NH.sub.2 group attached to the 2' carbon of a ribosyl moiety; the
structure 2'-OME-C or 2'-OME-U refers to a nucleotide unit having a
2'-O-methyl modification at the 2' carbon of a ribosyl moiety of
either Cs or Us, respectively; dG, dU, dA, dC, and dT refer to a
nucleotide unit that is deoxy with respect to the 2' position, and
instead has a hydrogen attached to the 2' carbon of the ribosyl
moiety. Unless otherwise indicated, all nucleotide units in the
list below are ribosyl with an --OH at the 2' carbon.
TABLE-US-00001 TABLE 1 SEAP Constructs Identifier Sequence SEQ. ID
NO. SP-2217-s gugauguaugucagagagudtdt 1 SP-2217-as
acucucugacauacaucacdtdt 2 SP-2217-s-p gugauguaugucagagagudtdt(ace
on) 3 SP-2217-as-p acucucugacauacaucacdtdt(ace on) 4 SP-2217-as4
ac*u*cucugacauacau*c*acdtdt 5 SP-2217-as8
ac*u*c*u*cugacauac*a*u*c*acdtdt 6 SP-2217-as8F
a2'-F-c*2'-F-u*2'-F-c*2'-F-u*2'-F- 7 c2'-F-uga2'-F-ca2'-F-ua2'-F-
c*a*2'-F-u*2'-F-c*a2'-F-cdtdt SP-s-N
g2'-N-uga2'-N-ug2'-N-ua2'-N-ug2'- 8 N-u2'-N-cagagag2'-N-udtdt
SP-as-N-12 a2'-N-c2'-N-u2'-N-c2'-N-u2'-N-cug 9
aca2'-N-ua2'-N-ca2'-N-u2'-N-ca2'- N-cdtdt SP-s-thio
g*u*g*a*u*g*u*a*u*g*u*c*a*g*a*g*a* 10 g*udtdt SP-as-thio
a*c*u*c*u*c*u*g*a*c*a*u*a*c*a*u*c* 11 a*cdtdt SP-as-thio12
a*c*u*c*u*c*ugacaua*c*a*u*c*a*cdtdt 12 SP-s-M
g2'-OMe-uga2'-OMe-ug2'-OMe-ua2'- 13 OMe-ug2'-OMe-u2'-OMe-cagagag2'-
OMe-udtdt SP-as-M10 a 2'-OMe-c 2'-OMe-u 2'-OMe-c 2'- 14 OMe-u
2'-OMe-c u g a c a 2'-OMe-u a 2'-OMe-c a 2'-OMe-u 2'-OMe-c a
2'-OMe-c dt dt SP-2217-s dgudgadugduadugducdagdagdagdudtdt 15
SP-2217-as adcudcudcugacauadcaducdacdtdt 16 SP-2217-sF
g2'-F-uga2'-F-ug2'-F-ua2'-F-ug2'- 17 F-u2'-F-cagagag2'-F-udtdt
TABLE-US-00002 TABLE 2 Human Cyclophylin Constructs SEQ. ID
Identifier Sequence NO. H-cyclo-476-s ugguguuuggcaaaguucudtdt 18
H-cyclo-476-as agaacuuugccaaacaccadtdt 19 H-cyc-F-s
(2'-F-u)gg(2'-F-u)g(2'-F- 20 u)(2'-F-u)(2'-F-u)gg(2'-F-
c)aaag(2'-F-u)(2'-F-u)(2'-F- c)(2'-F-u)dtdt H-cyc-F-as9
agaa(2'-F-c)(2'-F-u)(2'-F- 21 u)(2'-F-u)g(2'-F-c)(2'-F-
c)aaa(2'-F-c)a(2'-F-c)(2'-F- c)adtdt H-cyc-F-as8
agaa(2'-F-c)(2'-F-u)(2'-F- 22 u)ug(2'-F-c)(2'-F-c)aaa(2'-F-
c)a(2'-F-c)(2'-F-c)adtdt H-cyclo-476-as6 agaa(2'-F-c)(2'-F-u)(2'-F-
23 u)ugccaaa(2'-F-c)a(2'-F- c)(2'-F-c)adtdt H-cyclo-476-as1
agaacuu(2'-Fu)gccaaacaccadtdt 24
TABLE-US-00003 TABLE 3 Firefly Luciferase Constructs SEQ. ID
Identifier Sequence NO. Luc-1188-2'F-s ga2'F-u2'F-ua2'F-ug2'F-u2'F-
25 c2'F-cgg2'F-u2'F-ua2'F-ug2'F- uadtdt Luc-1188-2'F-as
2'F-ua2'F-ca2'F-uaa2'F-c2'F- 26 cgga2'F-ca2'F-uaa2'F-u2'F-
cdtdt
Example 4
Performing RNA Interference
[0486] Transfection
[0487] SiRNA duplexes were annealed using standard buffer (50
millimolar HEPES pH 7.5, 100 millimolar KCl, 1 mM MgCl.sub.2). The
transfections are done according to the standard protocol described
below.
[0488] Standard Transfection Protocol for 96 Well and 6 Well
Plates: siRNAs [0489] 1. Protocols for 293 and Calu6, HeLa, MDA 75
are identical. [0490] 2. Cell are plated to be 95% confluent on the
day of transfection. [0491] 3. SuperRNAsin (Ambion) is added to
transfection mixture for protection against RNAses. [0492] 4. All
solutions and handling have to be carried out in RNAse free
conditions. Plate 1 0.5-1 ml in 25 ml of media in a small flask or
1 ml in 50 ml in a big flask.
96 Well Plate
[0492] [0493] 1. Add 3 ml of 0.05% trypsin-EDTA in a medium flask
(6 in a big) incubate 5 min at 37 degrees C. [0494] 2. Add 7 ml (14
ml big) of regular media and pipet 10 times back and forth to
re-suspend cells. [0495] 3. Take 25 microliters of the cell
suspension from step 2 and 75 microliters of trypan blue stain
(1:4) and place 10 microliters in a cell counter. [0496] 4. Count
number of cells in a standard hemocytometer. [0497] 5. Average
number of cells.times.4.times.10000 is number of cells per ml.
[0498] 6. Dilute with regular media to have 350 000/ml. [0499] 7.
Plate 100 microliters (35,000 cell for HEK293) in a 96 well
plate.
[0500] Transfection for 2.times.96 Well Plates (60 Well Format)
[0501] 1. OPTI-MEM 2 ml+80 microliters Lipofectamine 2000 (1:25)+15
microliters of SuperRNAsin (AMBION). [0502] 2. Transfer siRNA
aliquots (0.8 microliters of 100 micromolar to screen (total
dilution factor is 1:750, 0.8 microliters of 100 micromolar
solution will give 100 nanomolar final) to the deepdish in a
desired order (Usually 3 columns.times.6 for 60 well format or four
columns by 8 for 96 well). [0503] 3. Transfer 100 microliters of
OPTI-MEM. [0504] 4. Transfer 100 microliters of OPTI-MEM with
Lipofectamine 2000 and SuperRNAsin to each well. [0505] 5. Leave
for 20-30 min RT. [0506] 6. Add 0.55 ml of regular media to each
well. Cover plate with film and mix. [0507] 7. Array out
100.times.3.times.2 directly to the cells (sufficient for two
plates).
[0508] Transfection for 2.times.6 Well Plates [0509] 8. 8 ml
OPTI-MEM+160 microliters Lipofectamine 2000 (1:25). 30 microliters
of SuperRNAsin (AMBION). [0510] 9. Transfer siRNA aliquots (total
dilution factor is 1:750, 5 microliters of 100 micromolar solution
will give 100 nanomolar final) to polystyrene tubes. [0511] 10.
Transfer 1,300 microliters of OPTI-MEM with Lipofectamine 2000 and
SuperRNAsin (AMBION). [0512] 11. Leave for 20-30 min RT. [0513] 12.
Add 0.55 ml of regular media to each well. Cover plate with film
and mix. [0514] 13. Transfer 2 ml to each well (sufficient for two
wells).
[0515] The mRNA or protein levels are measured 24, 48, 72, and 96
hours post transfection with standard kits or Custom B-DNA sets and
Quantigene kits (Bayer).
Example 5
Measurement of Activity/Detection
[0516] The level of siRNA-induced RNA interference, or gene
silencing, was estimated by assaying the reduction in target mRNA
levels or reduction in the corresponding protein levels. Assays of
mRNA levels were carried out using B-DNA.TM. technology (Quantagene
Corp.). Protein levels for fLUC and rLUC were assayed by STEADY
GLO.TM. kits (Promega Corp.). Human alkaline phosphatase levels
were assayed by Great EscAPe SEAP Fluorescence Detection Kits
(#K2043-1), BD Biosciences, Clontech.
Example 6
2'-Deoxy Modifications/Firefly Luciferase Gene
[0517] The functional effect on an siRNA of having two tandem
2'-deoxy modifications, and three tandem 2'-deoxy modifications in
a sense strand and in an antisense strand were systematically
examined by introducing the modifications into a 21-mer siRNA
having a 19-mer region of complementarity and a di-dT overhang on
the 3' ends of the duplex. The siRNAs were directed against the
firefly luciferase gene (fLUC5) transfected into HEK293 cells.
siRNA functionality was measured as described above. Toxicity was
measured by ALMAR blue, and appeared unaffected. Functionality was
assessed at three concentrations: 1, 10 and 100 nM final. The
sequences of the siRNAs used, and the placement of the 2'-deoxy
modifications, are indicated in Table 4. The results of these
experiments are shown in FIGS. 19-23.
TABLE-US-00004 TABLE 4 Constructs for 2'-Deoxy Modifications/fLUC
Identifier Sequence SEQ. ID NO. fLUC5-AS 3D19
uuuaugaggaucucucdudgdadtdt 27 fLUC5-AS 3D16
uuuaugaggaucucudcdudgadtdt 28 fLUC5-AS 3D13
uuuaugaggaucdudcducugadtdt 29 fLUC5-AS 3D10
uuuaugaggdadudcucucugadtdt 30 fLUC5-AS 3D7
uuuaugdadgdgaucucucugadtdt 31 fLUC5-AS 3D4
uuudadudgaggaucucucugadtdt 32 fLUC5-AS 3D1
dududuaugaggaucucucugadtdt 33 fLUC5-AS 2D19
uuuaugaggaucucucudgdadtdt 34 fLUC5-AS 2D17
uuuaugaggaucucucdudgadtdt 35 fLUC5-AS 2D15
uuuaugaggaucucdudcugadtdt 36 fLUC5-AS 2D13
uuuaugaggaucdudcucugadtdt 37 fLUC5-AS 2D11
uuuaugaggadudcucucugadtdt 38 fLUC5-AS 2D9 uuuaugagdgdaucucucugadtdt
39 fLUC5-AS 2D7 uuuaugdadggaucucucugadtdt 40 fLUC5-AS 2D5
uuuadudgaggaucucucugadtdt 41 fLUC5-AS 2D3 uududaugaggaucucucugadtdt
42 fLUC5-AS 2D1 duduuaugaggaucucucugadtdt 43 fLUC5-AS 1D19
uuuaugaggaucucucugdadtdt 44 fLUC5-AS 1D18 uuuaugaggaucucucudgadtdt
45 fLUC5-AS 1D17 uuuaugaggaucucucdugadtdt 46 fLUC5-AS 1D16
uuuaugaggaucucudcugadtdt 47 fLUC5-AS 1D15 uuuaugaggaucucducugadtdt
48 fLUC5-AS 1D14 uuuaugaggaucudcucugadtdt 49 fLUC5-AS 1D13
uuuaugaggaucducucugadtdt 50 fLUC5-AS 1D12 uuuaugaggaudcucucugadtdt
51 fLUC5-AS 1D11 uuuaugaggaducucucugadtdt 52 fLUC5-AS 1D10
uuuaugaggdaucucucugadtdt 53 fLUC5-AS 1D9 uuuaugagdgaucucucugadtdt
54 fLUC5-AS 1D8 uuuaugadggaucucucugadtdt 55 fLUC5-AS 1D7
uuuaugdaggaucucucugadtdt 56 fLUC5-AS 1D6 uuuaudgaggaucucucugadtdt
57 fLUC5-AS 1D5 uuuadugaggaucucucugadtdt 58 fLUC5-AS 1D4
uuudaugaggaucucucugadtdt 59 fLUC5-AS 1D3 uuduaugaggaucucucugadtdt
60 fLUC5-AS 1D2 uduuaugaggaucucucugadtdt 61 fLUC5-AS 1D1
duuuaugaggaucucucugadtdt 62 fLUC5-S 3D19 ucagagagauccucaudadadadtdt
63 fLUC5-S 3D16 ucagagagauccucadudadaadtdt 64 fLUC5-S 3D13
ucagagagauccdudcdauaaadtdt 65 fLUC5-S 3D10
ucagagagadudcdcucauaaadtdt 66 fLUC5-S 3D7
ucagagdadgdauccucauaaadtdt 67 fLUC5-S 3D4
ucadgdadgagauccucauaaadtdt 68 fLUC5-S 3D1
dudcdagagagauccucauaaadtdt 69 fLUC5-S 2D19
ucagagagauccucauadadadtdt 70 fLUC5-S 2D17 ucagagagauccucaudadaadtdt
71 fLUC5-S 2D15 ucagagagauccucdaduaaadtdt 72 fLUC5-S 2D13
ucagagagauccdudcauaaadtdt 73 fLUC5-S 2D11 ucagagagaudcdcucauaaadtdt
74 fLUC5-S 2D9 ucagagagdaduccucauaaadtdt 75 fLUC5-S 2D7
ucagagdadgauccucauaaadtdt 76 fLUC5-S 2D5 ucagdadgagauccucauaaadtdt
77 fLUC5-S 2D3 ucdadgagagauccucauaaadtdt 78 fLUC5-S 2D1
dudcagagagauccucauaaadtdt 79 fLUC5-S 1D19 ucagagagauccucauaadadtdt
80 fLUC5-S 1D18 ucagagagauccucauadaadtdt 81 fLUC5-S 1D17
ucagagagauccucaudaaadtdt 82 fLUC5-S 1D16 ucagagagauccucaduaaadtdt
83 fLUC5-S 1D15 ucagagagauccucdauaaadtdt 84 fLUC5-S 1D14
ucagagagauccudcauaaadtdt 85 fLUC5-S 1D13 ucagagagauccducauaaadtdt
86 fLUC5-S 1D12 ucagagagaucdcucauaaadtdt 87 fLUC5-S 1D11
ucagagagaudccucauaaadtdt 88 fLUC5-S 1D10 ucagagagaduccucauaaadtdt
89 fLUC5-S 1D9 ucagagagdauccucauaaadtdt 90 fLUC5-S 1D8
ucagagadgauccucauaaadtdt 91 fLUC5-S 1D7 ucagagdagauccucauaaadtdt 92
fLUC5-S 1D6 ucagadgagauccucauaaadtdt 93 fLUC5-S 1D5
ucagdagagauccucauaaadtdt 94 fLUC5-S 1D4 ucadgagagauccucauaaadtdt 95
fLUC5-S 1D3 ucdagagagauccucauaaadtdt 96 fLUC5-S 1D2
udcagagagauccucauaaadtdt 97 fLUC5-S 1D1 ducagagagauccucauaaadtdt 98
A "d" indicates that the nucleotide following the "d" is deoxy at
the 2' position.
Example 7
2'-O-Methyl Modifications/Firefly Luciferase Gene
[0518] The functional effect on an siRNA of having a single
2'-O-methyl modification, two tandem 2'-O-methyl modifications, and
three tandem 2'-O-methyl modifications in a sense strand and in an
antisense strand were systematically examined by introducing the
modifications into a 21-mer siRNA having a 19-mer region of
complementarity and a di-dT overhang on the 3' ends of the duplex.
The siRNAs were directed against the firefly luciferase gene
(fLUC5) transfected into HEK293 cells. siRNA functionality was
measured as described above. Functionality was assessed at three
concentrations: 1, 10 and 100 nM final. Toxicity was measured by
ALMAR blue, and appeared unaffected. The sequences of the siRNAs
used, and the placement of the 2'-O-methyl modifications, are
indicated in Table 5. The results of these experiments are shown in
FIGS. 24-28.
TABLE-US-00005 TABLE 5 Constructs for 2'-O-Methyl
Modifications/fLUC Identifier Sequence SEQ. ID NO. fLUC5-AS 3M19
uuuaugaggaucucucmumgmadtdt 99 fLUC5-AS 3M16
uuuaugaggaucucumcmumgadtdt 100 fLUC5-AS 3M13
uuuaugaggaucmumcmucugadtdt 101 fLUC5-AS 3M10
uuuaugaggmamumcucucugadtdt 102 fLUC5-AS 3M7
uuuaugmamgmgaucucucugadtdt 103 fLUC5-AS 3M4
uuumamumgaggaucucucugadtdt 104 fLUC5-AS 3M1
mumumuaugaggaucucucugadtdt 105 fLUC5-AS 2M19
uuuaugaggaucucucumgmadtdt 106 fLUC5-AS 2M17
uuuaugaggaucucucmumgadtdt 107 fLUC5-AS 2M15
uuuaugaggaucucmumcugadtdt 108 fLUC5-AS 2M13
uuuaugaggaucmumcucugadtdt 109 fLUC5-AS 2M11
uuuaugaggamumcucucugadtdt 110 fLUC5-AS 2M9
uuuaugagmgmaucucucugadtdt 111 fLUC5-AS 2M7
uuuaugmamggaucucucugadtdt 112 fLUC5-AS 2M5
uuuamumgaggaucucucugadtdt 113 fLUC5-AS 2M3
uumumaugaggaucucucugadtdt 114 fLUC5-AS 2M1
mumuuaugaggaucucucugadtdt 115 fLUC5-AS 1M19
uuuaugaggaucucucugmadtdt 116 fLUC5-AS 1M18 uuuaugaggaucucucumgadtdt
117 fLUC5-AS 1M17 uuuaugaggaucucucmugadtdt 118 fLUC5-AS 1M16
uuuaugaggaucucumcugadtdt 119 fLUC5-AS 1M15 uuuaugaggaucucmucugadtdt
120 fLUC5-AS 1M14 uuuaugaggaucumcucugadtdt 121 fLUC5-AS 1M13
uuuaugaggaucmucucugadtdt 122 fLUC5-AS 1M12 uuuaugaggaumcucucugadtdt
123 fLUC5-AS 1M11 uuuaugaggamucucucugadtdt 124 fLUC5-AS 1M10
uuuaugaggmaucucucugadtdt 125 fLUC5-AS 1M9 uuuaugagmgaucucucugadtdt
126 fLUC5-AS 1M8 uuuaugamggaucucucugadtdt 127 fLUC5-AS 1M7
uuuaugmaggaucucucugadtdt 128 fLUC5-AS 1M6 uuuaumgaggaucucucugadtdt
129 fLUC5-AS 1M5 uuuamugaggaucucucugadtdt 130 fLUC5-AS 1M4
uuumaugaggaucucucugadtdt 131 fLUC5-AS 1M3 uumuaugaggaucucucugadtdt
132 fLUC5-AS 1M2 umuuaugaggaucucucugadtdt 133 fLUC5-AS 1M1
muuuaugaggaucucucugadtdt 134 fLUC5-S 3M19
ucagagagauccucaumamamadtdt 135 fLUC5-S 3M16
ucagagagauccucamumamaadtdt 136 fLUC5-S 3M13
ucagagagauccmumcmauaaadtdt 137 fLUC5-S 3M10
ucagagagamumcmcucauaaadtdt 138 fLUC5-S 3M7
ucagagmamgmauccucauaaadtdt 139 fLUC5-S 3M4
ucamgmamgagauccucauaaadtdt 140 fLUC5-S 3M1
mumcmagagagauccucauaaadtdt 141 fLUC5-S 2M19
ucagagagauccucauamamadtdt 142 fLUC5-S 2M17
ucagagagauccucamumaaadtdt 143 fLUC5-S 2M15
ucagagagauccumcmauaaadtdt 144 fLUC5-S 2M13
ucagagagaucmcmucauaaadtdt 145 fLUC5-S 2M11
ucagagagamumccucauaaadtdt 146 fLUC5-S 2M9 ucagagamgmauccucauaaadtdt
147 fLUC5-S 2M7 ucagamgmagauccucauaaadtdt 148 fLUC5-S 2M5
ucagmamgagauccucauaaadtdt 149 fLUC5-s 2M3 ucmamgagagauccucauaaadtdt
150 fLUC5-S 2M1 mumcagagagauccucauaaadtdt 151 fLUC5-S 1M19
ucagagagauccucauaamadtdt 152 fLUC5-S 1M18 ucagagagauccucauamaadtdt
153 fLUC5-S 1M17 ucagagagauccucaumaaadtdt 154 fLUC5-S 1M16
ucagagagauccucamuaaadtdt 155 fLUC5-S 1M15 ucagagagauccucmauaaadtdt
156 fLUC5-S 1M14 ucagagagauccumcauaaadtdt 157 fLUC5-S 1M13
ucagagagauccmucauaaadtdt 158 fLUC5-S 1M12 ucagagagaucmcucauaaadtdt
159 fLUC5-S 1M11 ucagagagaumccucauaaadtdt 160 fLUC5-S 1M10
ucagagagamuccucauaaadtdt 161 fLUC5-S 1M9 ucagagagmauccucauaaadtdt
162 fLUC5-S 1M8 ucagagamgauccucauaaadtdt 163 fLUC5-S 1M7
ucagagmagauccucauaaadtdt 164 fLUC5-S 1M6 ucagamgagauccucauaaadtdt
165 fLUC5-S 1M5 ucagmagagauccucauaaadtdt 166 fLUC5-S 1M4
ucamgagagauccucauaaadtdt 167 fLUC5-S 1M3 ucmagagagauccucauaaadtdt
168 fLUC5-S 1M2 umcagagagauccucauaaadtdt 169 fLUC5-S 1M1
mucagagagauccucauaaadtdt 170 The letter "m" indicates that the
nucleotide following the "m" is modified with a 2'-O-methyl
moiety.
Example 8
[0519] 2'-Deoxy and 2'-O-Methyl Modifications/Sense vs. Antisense
Strands
[0520] Fifteen duplexes, five directed against the human
cyclophylin gene, and 10 were directed against the firefly
luciferase gene were tested with various modifications (see FIGS.
29-31). For human cyclophilin B, duplexes tested included: (1)
unmodified, (2) 2'-O-methyl modifications at the first and second
positions of the sense strand, and (3) 2'-O-methyl modifications at
the first and second positions of the antisense strand, and (4)
2'-O-methyl modifications on the sense and antisense strands. For
luciferase, all the modifications described for human cyclophilin B
plus: (1) 2'-O-methyl modifications on the AS strand in conjunction
with 5' phosphorylation of the AS strand, and (2) 2'-O-methyl
modification of the sense and antisense strands in conjunction with
5' phosphorylation of the antisense strand, were tested. For all 15
duplexes, modifications at positions 1 and 2 of the sense strand
with 2'O-methyl moieties did not interfere with functionality. The
same modifications of the antisense strand limited the
functionality of the duplexes. This decrease in functionality was
partially reduced where the antisense strand was phosphorylated at
its 5' end (see FIGS. 30 and 31). Taking the above data together,
2'-O-methylaltion of positions 1 and 2 of the sense strand in
combination with 5' phosphorylation of the antisense strand is an
inexpensive, reliable, and non-toxic method of modifying an siRNA
duplex to limit sense strand off-targeting without altering duplex
functionality. This information is of commercial value because it
helps increase siRNA specificity and potency. Recent microarray
data indicates that the presence of just 11 nucleotides is
sufficient to induce nonspecific silencing. The homology present
within a sense strand of an siRNA duplex typically constitutes at
least half nonspecific functionality. If the inherent nonspecific
functionality is blocked, the sense strand will not be able to
contribute to off-targeting and the siRNA's specificity should
increase. Shifting of the equilibrium toward a functional
RISC-antisense strand complex will also lower the effective
concentration of siRNA.
Example 9
Modified siRNAs with 5' Conjugates
[0521] The effects of various modifications on duplex stability,
functionality, and passive uptake were assessed. FIG. 33
demonstrates the effects of cholesterol conjugation and (2'O-methyl
modification on positions 1 and 2 of the sense and antisense strand
plus 2'-O-methyl modification of all Cs and Us of the sense strand,
plus 2' F modification of all Cs and Us of the antisense strand,
plus 5' phosphorylation of the antisense strand) on duplex
stability. Naked or modified siRNA were 32P-labeled (AS strand, T4
Kinase, Promega), incubated with serum (or serum albumin), and run
in gel shift assays (denaturing, 15% PAGE). Results of these
studies show that while unmodified duplexes are quickly degraded,
siRNA carrying cholesterol and the before described modifications
are stable in the presence of serum or serum albumin. Similarly,
siRNA carrying 2'-O-methyl modification of Cs and Us (sense) in
combination with 3' idT capping and CHO (5' sense) conjugation,
also exhibit enhanced stability (FIG. 35).
[0522] Studies on similarly modified molecules (e.g., cholesterol
modification of the 5' end of the sense strand in conjugation with
2' Fl modification of the sense strand) reveal that such
modifications can accentuate functionality (FIG. 34) and passive
uptake (FIG. 36, modification=2' F on Cs and Us of both the sense
and antisense with cholesterol).
Example 10
2'Deoxy and 2'-O-Methyl Modification Walks on SEAP 2217 Target
[0523] The constructs used for the 2'-deoxy and 2'-O-methyl walks
using siRNAs targeted against the SEAP construct (see FIGS. 31 and
32) are listed in Table 6.
TABLE-US-00006 TABLE 6 Constructs for 2'-Deoxy and 2'-O-Methyl
Walks Identifier Sequence SEQ. ID NO. 2217-S 2M1
mgmugauguaugucagagagudtdt 171 2217-AS 3D19
acucucugacauacaudcdadcdtdt 172 2217-AS 3D16
acucucugacauacadudcdacdtdt 173 2217-AS 3D13
acucucugacaudadcdaucacdtdt 174 2217-AS 3D10
acucucugadcdaduacaucacdtdt 175 2217-AS 3D7
acucucdudgdacauacaucacdtdt 176 2217-AS 3D4
acudcdudcugacauacaucacdtdt 177 2217-AS 3D1
dadcducucugacauacaucacdtdt 178 2217-AS 2D19
acucucugacauacaucdadcdtdt 179 2217-AS 2D17
acucucugacauacaudcdacdtdt 180 2217-AS 2D15
acucucugacauacdaducacdtdt 181 2217-AS 2D13
acucucugacaudadcaucacdtdt 182 2217-AS 2D11
acucucugacdaduacaucacdtdt 183 2217-AS 2D9 acucucugdadcauacaucacdtdt
184 2217-AS 2D7 acucucdudgacauacaucacdtdt 185 2217-AS 2D5
acucdudcugacauacaucacdtdt 186 2217-AS 2D3 acdudcucugacauacaucacdtdt
187 2217-AS 2D1 dadcucucugacauacaucacdtdt 188 2217-AS 3M19
acucucugacauacaumcmamcdtdt 189 2217-AS 3M16
acucucugacauacamumcmacdtdt 190 2217-AS 3M13
acucucugacaumamcmaucacdtdt 191 2217-AS 3M10
acucucugamcmamuacaucacdtdt 192 2217-AS 3M7
acucucmumgmacauacaucacdtdt 193 2217-AS 3M4
acumcmumcugacauacaucacdtdt 194 2217-AS 3M1
mamcmucucugacauacaucacdtdt 195 2217-AS 2M19
acucucugacauacaucmamcdtdt 196 2217-AS 2M17
acucucugacauacaumcmacdtdt 197 2217-AS 2M15
acucucugacauacmamucacdtdt 198 2217-AS 2M13
acucucugacaumamcaucacdtdt 199 2217-AS 2M11
acucucugacmamuacaucacdtdt 200 2217-AS 2M9 acucucugmamcauacaucacdtdt
201 2217-AS 2M7 acucucmumgacauacaucacdtdt 202 2217-AS 2M5
acucmumcugacauacaucacdtdt 203 2217-AS 2M3 acmumcucugacauacaucacdtdt
204 2217-AS 2M1 mamcucucugacauacaucacdtdt 205 2217-S 3D19
gugauguaugucagagdadgdudtdt 206 2217-S 3D16
gugauguaugucagadgdadgudtdt 207 2217-S 3D13
gugauguaugucdadgdagagudtdt 208 2217-S 3D10
gugauguaudgdudcagagagudtdt 209 2217-S 3D7
gugaugdudadugucagagagudtdt 210 2217-S 3D4
gugdadudguaugucagagagudtdt 211 2217-S 3D1
dgdudgauguaugucagagagudtdt 212 2217-S 2D19
gugauguaugucagagadgdudtdt 213 2217-S 2D17 gugauguaugucagagdadgudtdt
214 2217-S 2D15 gugauguaugucagdadgagudtdt 215 2217-S 2D13
gugauguaugucdadgagagudtdt 216 2217-S 2D11 gugauguaugdudcagagagudtdt
217 2217-S 2D9 gugauguadudgucagagagudtdt 218 2217-S 2D7
gugaugdudaugucagagagudtdt 219 2217-S 2D5 gugadudguaugucagagagudtdt
220 2217-S 2D3 gudgdauguaugucagagagudtdt 221 2217-S 2D1
dgdugauguaugucagagagudtdt 222 2217-S 3M19
gugauguaugucagagmamgmudtdt 223 2217-S 3M16
gugauguaugucagamgmamgudtdt 224 2217-S 3M13
gugauguaugucmamgmagagudtdt 225 2217-S 3M10
gugauguaumgmumcagagagudtdt 226 2217-S 3M7
gugaugmumamugucagagagudtdt 227 2217-S 3M4
gugmamumguaugucagagagudtdt 228 2217-S 3M1
mgmumgauguaugucagagagudtdt 229 2217-S 2M19
gugauguaugucagagamgmudtdt 230 2217-S 2M17 gugauguaugucagagmamgudtdt
231 2217-S 2M15 gugauguaugucagmamgagudtdt 232 2217-S 2M13
gugauguaugucmamgagagudtdt 233 2217-S 2M11 gugauguaugmumcagagagudtdt
234 2217-S 2M9 gugauguamumgucagagagudtdt 235 2217-S 2M7
gugaugmumaugucagagagudtdt 236 2217-S 2M5 gugamumguaugucagagagudtdt
237 2217-s 2M3 gumgmauguaugucagagagudtdt 238 2217-S 1M19
gugauguaugucagagagmudtdt 239 2217-S 1M18 gugauguaugucagagamgudtdt
240 2217-S 1M17 gugauguaugucagagmagudtdt 241 2217-S 1M16
gugauguaugucagamgagudtdt 242 2217-S 1M15 gugauguaugucagmagagudtdt
243 2217-S 1M14 gugauguaugucamgagagudtdt 244 2217-S 1M13
gugauguaugucmagagagudtdt 245 2217-S 1M12 gugauguaugumcagagagudtdt
246 2217-S 1M11 gugauguaugmucagagagudtdt 247 2217-S 1M10
gugauguaumgucagagagudtdt 248 2217-S 1M9 gugauguamugucagagagudtdt
249 2217-S 1M8 gugaugumaugucagagagudtdt 250 2217-S 1M7
gugaugmuaugucagagagudtdt 251 2217-S 1M6 gugaumguaugucagagagudtdt
252 2217-S 1M5 gugamuguaugucagagagudtdt 253 2217-S 1M4
gugmauguaugucagagagudtdt 254 2217-S 1M3 gumgauguaugucagagagudtdt
255 2217-S 1M2 gmugauguaugucagagagudtdt 256 2217-S 1M1
mgugauguaugucagagagudtdt 257 2217-AS 1M19 acucucugacauacaucamcdtdt
258 2217-AS 1M18 acucucugacauacaucmacdtdt 259 2217-AS 1M17
acucucugacauacaumcacdtdt 260 2217-AS 1M16 acucucugacauacamucacdtdt
261 2217-AS 1M15 acucucugacauacmaucacdtdt 262 2217-AS 1M14
acucucugacauamcaucacdtdt 263 2217-AS 1M13 acucucugacaumacaucacdtdt
264 2217-AS 1M12 acucucugacamuacaucacdtdt 265 2217-AS 1M11
acucucugacmauacaucacdtdt 266 2217-AS 1M10 acucucugamcauacaucacdtdt
267 2217-AS 1M9 acucucugmacauacaucacdtdt 268 2217-AS 1M8
acucucumgacauacaucacdtdt 269 2217-AS 1M7 acucucmugacauacaucacdtdt
270 2217-AS 1M6 acucumcugacauacaucacdtdt 271 2217-AS 1M5
acucmucugacauacaucacdtdt 272 2217-AS 1M4 acumcucugacauacaucacdtdt
273 2217-AS 1M3 acmucucugacauacaucacdtdt 274 2217-AS 1M2
amcucucugacauacaucacdtdt 275 2217-AS 1M1 macucucugacauacaucacdtdt
276 2217-S 1D19 gugauguaugucagagagdudtdt 277 2217-S 1D18
gugauguaugucagagadgudtdt 278 2217-S 1D17 gugauguaugucagagdagudtdt
279 2217-S 1D16 gugauguaugucagadgagudtdt 280 2217-S 1D15
gugauguaugucagdagagudtdt 281 2217-S 1D14 gugauguaugucadgagagudtdt
282 2217-S 1D13 gugauguaugucdagagagudtdt 283 2217-S 1D12
gugauguaugudcagagagudtdt 284 2217-S 1D11 gugauguaugducagagagudtdt
285 2217-S 1D10 gugauguaudgucagagagudtdt 286 2217-S 1D9
gugauguadugucagagagudtdt 287 2217-S 1D8 gugaugudaugucagagagudtdt
288 2217-S 1D7 gugaugduaugucagagagudtdt 289 2217-S 1D6
gugaudguaugucagagagudtdt 290 2217-S 1D5 gugaduguaugucagagagudtdt
291 2217-S 1D4 gugdauguaugucagagagudtdt 292 2217-S 1D3
gudgauguaugucagagagudtdt 293
2217-S 1D2 gdugauguaugucagagagudtdt 294 2217-S 1D1
dgugauguaugucagagagudtdt 295 2217-AS 1D19 acucucugacauacaucadcdtdt
296 2217-AS 1D18 acucucugacauacaucdacdtdt 297 2217-AS 1D17
acucucugacauacaudcacdtdt 298 2217-AS 1D16 acucucugacauacaducacdtdt
299 2217-AS 1D15 acucucugacauacdaucacdtdt 300 2217-AS 1D14
acucucugacauadcaucacdtdt 301 2217-AS 1D13 acucucugacaudacaucacdtdt
302 2217-AS 1D12 acucucugacaduacaucacdtdt 303 2217-AS 1D11
acucucugacdauacaucacdtdt 304 2217-AS 1D10 acucucugadcauacaucacdtdt
305 2217-AS 1D9 acucucugdacauacaucacdtdt 306 2217-AS 1D8
acucucudgacauacaucacdtdt 307 2217-AS 1D7 acucucdugacauacaucacdtdt
308 2217-AS 1D6 acucudcugacauacaucacdtdt 309 2217-AS 1D5
acucducugacauacaucacdtdt 310 2217-AS 1D4 acudcucugacauacaucacdtdt
311 2217-AS 1D3 acducucugacauacaucacdtdt 312 2217-AS 1D2
adcucucugacauacaucacdtdt 313 2217-AS 1D1 dacucucugacauacaucacdtdt
314 The letter "d" indicates that the nucleotide following the
letter "d" is deoxy at the 2' position. The letter "m" indicates
that the nucleotide following the letter "m" is modified with a
2'-O-methyl moiety.
Example 11
Molecule 1 Modifications and Stability
[0524] For the purposes of Examples 11-18, the term "molecule 1
modifications" refers to molecules that contain 2'-O-methyl
modifications on positions 1 and 2 of the sense strand, 2'-O-methyl
modifications on all Cs and Us of the sense strand, 2'-Fluoro (Fl)
modification of all Cs and Us of the antisense strand, and a
phosphate modification on the 5' terminus of the antisense strand.
Similarly, the term "molecule 2 modifications" refers to molecules
that contain 2'-O-methyl modifications on positions 1 and 2 of the
sense strand, 2'-O-methyl modifications on all Cs and Us of the
sense strand, a Cy3 label on the 5' end of the sense strand,
2'-Fluoro (Fl) modifications on all Cs and Us of the antisense
strand, and a phosphate modification on the 5' terminus of the
antisense strand.
[0525] To assess the effects of molecule 1 modifications on siRNA
stability, four unique siRNA were synthesized in modified and
unmodified forms. Subsequently, these molecules were incubated in
100% serum at 37.degree. C. for varying periods of time and then
analyzed by PAGE to assess the intactness of the duplexes.
Visualization of sequences was accomplished by ethidium bromide
staining.
[0526] The results of these experiments are illustrated in FIG. 37
and show that duplexes carrying molecule 1 modifications are
drastically more stable than unmodified equivalents. Unmodified
molecules typically exhibit 50% degradation or greater within two
minutes of being exposed to serum at room temperature. In contrast,
the half-life for sequences carrying molecule 1 modifications
typically ran between 125 and 135 hours. Thus molecule 1
modifications significantly enhanced stability by approximately
500-fold.
Example 12
Molecule 1 Modifications and siRNA Silencing Potency
[0527] To assess the effects of molecule 1 modifications on siRNA
potency, two unique siRNA directed against human Cyclophilin B (U1
and U3) were synthesized in modified and unmodified forms using
2'-O-ACE chemistry and tested for functionality in a whole cell
assay. Briefly, modified and unmodified siRNA were transfected
(Lipofectamine 2000) into HeLa cells (10,000 cells/well, 96 well
plate) at concentrations between 0.01-200 nM and cultured for 24-48
hours. Subsequently, the level of expression of the intended target
was assessed using a branched DNA assay (Genospectra, Fremont,
Calif.).
[0528] Results of these experiments are illustrated in FIG. 38 and
show that duplexes carrying molecule 1 modifications perform
comparably with unmodified siRNA at all concentrations tested.
Thus, molecule 1 modifications do not appear to alter the potency
of siRNA.
Example 13
Molecule 1 Modifications and Silencing Longevity
[0529] To determine the effects of molecule 1 modifications on
siRNA silencing longevity, siRNA directed against the human
cyclophilin B gene were synthesized in the modified and unmodified
forms and transfected into HeLa cells (100 nM) as previously
described. Subsequently, the level of silencing was monitored over
the course of 7 days using a branched DNA assay.
[0530] An example of the results of these experiments are presented
in FIG. 39 and demonstrate that while the level of silencing
induced by unmodified molecules depreciates from roughly 70% to 0%
over the course of the seven day period, modified duplexes induce
>80% functionality throughout the course of the experiment.
Thus, siRNA modified with molecule 1 modifications enhance the
longevity of silencing induced by these duplexes.
Example 14
Molecule 1 Modifications and Toxicity
[0531] To determine the effects of molecule 1 modifications on
siRNA toxicity, 4 siRNA (U1-U4) directed against human cyclophilin
B were synthesized in the modified and unmodified forms and
transfected into HeLa cells at concentrations that ranged between
0.01-200 nM. At t=48 hours after transfection, Alamar Blue
viability assays were performed to assess the level of cell death
within the population. A side-by-side comparison between the
modified and unmodified duplexes showed no difference in the level
of cell death induced at any of the concentrations tested (FIG.
40).
Example 15
Molecule 1 Modifications and Off-Target Effects
[0532] To assess the effects of molecule 1 modifications on
off-target effects, four separate siRNA targeting human Cyclophilin
B (U1-U4) were synthesized in both the modified and unmodified
forms and transfected into HeLa cells at 100 nM concentrations.
Subsequently, total RNA from (1) mock-transfected, (2) transfected
(unmodified), and (3) transfected (modified) cells was purified
(Qiagen), converted into cDNA and labeled with Cy3
(mock-transfected) or Cy5 (transfected-unmodified,
transfected-modified) using Agilent's Low RNA Input Linear Amp Kit.
Labeled cDNA from mock-transfected and untransfected cells were
then mixed and hybridized to an Agilent Human 1A (V2) Oligo
Microarray containing over 21,000 probes. The number of off-targets
associated with modified and unmodified samples was assessed using
Agilent's Feature Extraction, Spotfire DecisionSite, and Spotfire
Functional Genomics, software (versions 7.2, 7.2, and 7.1,
respectively).
[0533] A summary of these off-target studies are shown in FIG. 41
and illustrate that modified and unmodified siRNA perform similarly
in terms of the numbers of off-targeted genes. Specifically, when
off-targeted genes were segregated based on the level of induction
or repression (compared to wild type gene expression), modified
siRNA performed similarly (or better than) unmodified
counterparts.
Example 16
Molecule 2 Modifications and Silencing Potential
[0534] To test the functionality of siRNA containing molecule 2
modifications, Cyclo14 (5'GGCCTTAGCTACAGGAGAG, sense strand SEQ. ID
NO. 322), an siRNA directed against human cyclophilin B, was
synthesized with molecule 2 modifications and tested for the
ability to silence the intended target. Briefly, Cyclo14 was
synthesized with the appropriate modifications using 2'-O-ACE
chemistry. Subsequently, T482 HeLa cells (10,000 cells per well, 96
well plates) were plated, cultured overnight, and transfected
(Lipofectamine 2000) with the cyclo14 duplex at 100 nM
concentrations. Three days after transfection, the level of mRNA
silencing was assessed using a branched DNA assay (Genospectra,
Fremont, Calif.).
[0535] Results of these studies are presented in FIG. 42.
Unmodified Cyclo 14 duplexes typically induce 80-95% silencing.
Cyclo 14 duplexes modified with the Cy3 label alone induced roughly
70-80% silencing, and Cyclo14 siRNA carrying molecule 2
modifications induced 80% or better silencing. As similar
modification of non-specific sequences induced little or no
silencing, addition of molecule 2 modifications has little or no
effect on duplex functionality.
Example 17
The Effects of Molecule 2 Modifications on Trackability
[0536] To test the usefulness of molecule 2 modifications as a
means of assessing transfection efficiencies, Cyclo 14 siRNA were
prepared with the aforementioned modifications and visualized by
fluorescence microscopy. Specifically Cyclo14 was synthesized with
the appropriate modifications using 2'-O-ACE chemistry.
Subsequently, T482 HeLa cells (10,000 cells per well, 96 well
plates) were plated, cultured overnight, and transfected
(Lipofectamine 2000) with the appropriate duplex at 100 nM
concentrations. Forty-eight hours after transfection, cultures were
incubated with Hoechst 33342 (2 .mu.g/ml, 20 minutes, 37.degree.
C.) and then visualized on a Leica DM1L fluorescence microscope
using Dapi and Rhodamine filters.
[0537] A fluorescence micrograph (figure not included herein) of
HeLa cells transfected with Cyclo14 siRNA carrying molecule 2
modifications shows a strong perinuclear and nuclear stain. Cells
transfected with unmodified Cyclo14 siRNA show no equivalent
staining (data not shown), thus molecule 2 modifications provide an
excellent means of assessing the intracellular position of any
given siRNA and the success of transfection.
Example 18
The Effects of Molecule 2 Modifications on Stability
[0538] To test the whether molecule 2 modifications enhanced the
intracellular stability of duplexes, Cyclo 14 siRNA carrying
molecule 2 modifications were transfected into HeLa cells and
compared with Cy3 labeled Cyclo14 transfected cells over the course
of seven days. Addition of the aforementioned modifications
significantly enhanced siRNA stability over duplexes modified with
Cy3 alone. While both samples exhibit strong staining patterns on
Day 2, the Cy3-Cyclo14 transfected cells lose their stain by day 7.
In contrast, cells containing Cyclo14 siRNA carrying molecule 2
modifications retain a strong pattern of staining on day 7.
Moreover, unlike Cy3-labeled duplexes, siRNA carrying molecule 2
modifications also promote nuclear access to the duplex.
Example 19
Identification of Chemical Modifications that Modify Silencing
Activity
[0539] Using 2'-O-ACE chemistry as a platform for RNA synthesis, a
modification walk consisting of one, two, or three consecutively
modified nucleotides in sense (S) and antisense (AS) strands was
performed on SEAP-2217, an siRNA directed against human secreted
alkaline phosphatase (SEAP, SEAP-2217-sense strand 5'-G
UGAUGUAUGUCA GA G A G U dT dT (SEQ. ID NO. 326)). Subsequently, the
silencing efficiency of these modified siRNAs was evaluated by
cotransfecting each duplex with a SEAP expression vector (Clontech)
into HEK293 cells (100 nM siRNA, 50 ng/well SEAP expression vector,
Lipofectamine 2000) and assaying for a decrease in target protein
activity twenty-four hours after transfection. FIG. 43A and FIG.
43B show the relationship between modification and function for
2'-O-methylated SEAP-2217 siRNA. Unmodified duplexes targeting SEAP
induce >90% silencing of the SEAP gene. Single base
modifications of both S and AS strands induced little or no effect
on siRNA activity, suggesting that no single 2'-hydroxyl group on
either strand plays an indispensable role in RNAi. In contrast, a
walk of dual, side-by-side, modifications identified several key
positions where the introduction of modified bases interfered
significantly with silencing activity. The most profound
interference with function was observed when two consecutive bases
(positions 1 and 2) or three consecutive bases (positions 1, 2, and
3) of the 5' end of the AS strand were modified, thus hinting of a
cooperative effect between adjacent modified groups. As similar
modifications of the S strand failed to alter duplex functionality,
paired 2'-O-methyl modified bases enable a distinction of S and AS
strands.
[0540] An identical set of experiments was performed using 2'-deoxy
modifications. Studies shown in FIG. 44A and FIG. 44B demonstrate
that addition of three consecutive 2'-deoxy groups at different
positions (e.g., 1+2+3, 4+5+6, etc. . . . ) on either strand of the
duplex had no effect on duplex silencing activity. Similar results
were observed for walks that examined the effects of two and one
consecutive 2'-deoxy groups (data not shown).
[0541] To further test the effects of 2'-O-methyl modifications on
duplex functionality, a series of siRNA directed against the
luciferase gene (luc 8, 18, 56, 58, 63,and 81) were synthesized
using 2'-O-ACE chemistry and modified to contain O-methyl groups at
the 2' position of the ribose ring.
TABLE-US-00007 Luc 8 5'-GAAAAAUCAGAGAGAUCCU (SEQ. ID NO. 327) Luc
18 5'-UACCGGAAAACUCGACGCA (SEQ. ID NO. 328) Luc 56
5'-ACGUCGCCAGUCAAGUAAC (SEQ. ID NO. 329) Luc 58
5'-GAUUACGUCGCCAGUCAAG (SEQ. ID NO. 330) Luc 63
5'-AGAGAUCGUGGAUUACGUC (SEQ. ID NO. 331) Luc 81
5'-UGUUGUUUUGGAGCACGGA (SEQ. ID NO. 332)
(Sequences listed above are the sense strand.)
[0542] Specifically, siRNA containing 2'-O-methyl modifications on
the two 5'-most nucleotides of (1) the sense strand, (2) the
antisense strand, or (3) both strands, were co-transfected along
with a Luc-expression plasmid (pCMVLuc, 50 ng/well) into HEK293
cells. Subsequently, a side-by-side comparison of the silencing
ability of each duplex was performed to determine the effects of
this modification on target transcript degradation.
[0543] Results of these studies showed that addition of the
2'-O-methyl groups only to the AS strand dramatically diminished
the ability of the duplex to silence the target mRNA (see FIG.
45A-F). In contrast, duplexes carrying this modification on the
sense strand performed as well (luc 58, 63, 81) or better (luc 56,
8, 18) than equivalent, unmodified siRNA, suggesting that
modification of the sense strand biased strand selection by RISC
and (in some cases) increased the effective antisense strand
concentration. Enhanced silencing could be the result of a decrease
in the binding affinity of RISC to the 5' sense end of the molecule
(and therefore an increase in the availability of free RISC for
association to the opposing end), decreased ability of native
kinases to phosphorylate the sense strand (thus decreasing
competition between the sense and antisense strand for access to
RISC), or a decline in the ability of RISC to unravel the duplex
from the 5'-sense end. siRNA containing 2'-O-methyl modifications
on both strands exhibited decreased silencing abilities that were
between the values observed for molecules that contained
modifications on either single strand. One interpretation of these
results is that 2'-O-methyl modifications lowers the binding
affinity that RISC has for the modified strand. In cases where both
strands are modified, neither strand receives an advantage over its
complement, and a new equilibrium representing an average of the
functionality of both modified molecules is established.
[0544] To test whether the diminished level of silencing observed
in cells containing 2'-O-methylated S/AS siRNA was the result of a
debilitated capacity of cellular kinases to phosphorylate the
duplexes, siRNAs carrying the 2'-O-methyl modifications were
modified to carry a phosphate group on the 5' end of the AS strand.
Specifically, Luc siRNAs carrying 2'-O-methyl groups on either: (1)
positions 1 and 2 of the 5' end of the antisense strand; or (2)
positions 1 and 2 of the 5' end of both antisense and sense
strands, were 5'-phosphorylated on the AS strand during synthesis.
These duplexes were then introduced into HEK293 cells using
previously described procedures and tested for the ability to
silence the desired target. Results showed that in 83% of the cases
tested (10/12), 5' phosphorylation of the antisense strand improved
the silencing efficiency of the duplex over the equivalent
unphosphorylated molecule (FIG. 45A-F). In the remaining two cases,
silencing remained unchanged or was improved only marginally.
[0545] A more detailed examination of the effects of chemical
modifications on silencing using dose response curves support the
previous data. Five different siRNAs directed against the
luciferase gene (luc 8, 56, 58, 63, and 81) were synthesized using
the 2'-O-ACE chemistry in both 2'-O-methyl modified and unmodified
forms. These duplexes were then transfected into HeLa cells (along
with a luciferase expression vector) at concentrations that varied
between 0.01-200 nM and assayed (t=24-48 hours) for the level of
silencing of the luciferase protein. The results from these
experiments are summarized in FIG. 46 and lead to the following
conclusions: (1) addition of 2'-O-methyl groups to positions 1 and
2 of the 5' end of the antisense strand consistently disrupts
silencing activity; (2) addition of 2'-O-methyl groups to positions
1 and 2 of the 5' end of the sense strand of siRNA either improves
or has little or no effect on target specific silencing; (3)
addition of 2'-O-methyl groups to positions 1 and 2 of the 5' end
of both strands (sense and antisense) generates an intermediary
silencing effect that is between that observed with unmodified
duplexes and duplexes carrying a 2'-O-methyl modification on the 5'
end of the antisense strand; and (4) addition of a phosphate group
to the 5' end of the antisense strand of molecules that have
2'-O-methyl modifications on positions 1 and 2 of the 5' end of
both the sense and antisense strands, improved the silencing
activity of the molecule over duplexes that had 2'-O-methyl
modifications on the 5' end of both strands.
[0546] These results suggest that addition of the 2'-O-methyl
modification of positions 1 and 2 of the sense and antisense
strands of siRNA in addition to 5' phosphorylation of the first
terminal nucleotide of the antisense strand enhances the potency of
the duplex at a variety of concentrations. Moreover, the effects of
various modifications lead to the following conclusions: (1)
addition of 2'-O-methyl groups to the 5' end of the sense or
antisense strand severely reduces silencing by that strand; and (2)
addition of a 5' phosphate group restores, nearly completely,
silencing activity even in the presence of the 2'-O-methyl group.
Thus, while some fraction of the inhibitory effects of
AS-2'-O-methylation are likely the result of inhibition of duplex
phosphorylation, some portion of the effects are most probably due
to the effects of this form of modification on other steps in the
RNAi pathway such as RISC binding, siRNA unwinding, RISC mediated
siRNA-target association, or RISC mediated target cleavage.
Importantly, the potential effect of 2'-O-methylation on these
other steps led the authors to consider that the possibility that
said modifications might also alter the ability of RISC to
distinguish between intended targets that have 100% homology with
the antisense strand and off-targets that have lesser amounts of
homology.
Example 20
The Effects of 2'-O-methylation and 5' Phosphorylation on
Off-Targeting
[0547] The observation that 2'-O-methylation of the antisense
strand disrupts silencing while equivalent sense strand labeling
has no effect on target specific silencing suggests a strategy for
eliminating off-target silencing caused by the sense strand.
Furthermore, the observation that steps downstream of duplex
phosphorylation may be affected by 2'-O-methylation leads to the
prospect that siRNA containing this combination of modifications
(2'-O-methylation of positions 1 and 2 of the 5' end of the sense
and antisense strands, plus phosphorylation of the 5' end of the AS
strand) may have high potency and low sense/antisense off-targeting
effects.
[0548] To test the effects of 5'-phosphorylation of the antisense
strand in combination with 2'-O-methylation of: (1) the sense
strand; (2) the antisense strand; and (3) the sense and antisense
strands on off-targeting, two siRNA directed against four different
genes (8 siRNA total: MAPK14-193 and -153, MPHOSPH1-202 and -203,
IGF1R-73 and -74, and PTEN-213 and -214) were synthesized using the
2'-O-ACE chemistry and modified at the appropriate sites. When
experiments were conducted under conditions where MAPK14-153
contains only a phosphate group on the 5' end of the antisense
strand, extensive off-targeting is observed. When the sense strand
is further modified with 2'-O-methyl groups at positions 1 and 2,
off-targeting due to that strand is eliminated, but a novel set of
anti-sense strand off-targets are generated. This increase in
antisense strand off-targeting is likely due to increased AS
strand-RISC interactions in the absence of competition by the sense
strand. When the antisense strand contains a phosphate group on the
5' end and is modified with 2'-O-methyl groups at positions 1 and
2, off-targets due to the antisense strand are lost, but sense
strand off-targets are once again observed. Finally, when both the
sense strand and the antisense strand are modified with 2'-O-methyl
groups (on positions 1 and 2 of each strand) and a phosphate group
is attached to the 5' end of the antisense strand, a drastic
reduction in the level of off-targeting by both strands is
observed.
[0549] Similar results to those described above were obtained for
the remaining seven siRNAs directed against IGF1R, PTEN, MAPK14,
and MPHOSPH1. Thus, from these experiments it is possible to
conclude that: (1) different siRNA directed against the same target
induced varying levels of off-target effects; (2) 2'-O-methyl
modification of positions 1 and 2 of the 5'-end of the antisense
strand (in combination with 5' phosphorylation of the antisense
strand) eliminates off-target effects due to that strand but
frequently leads to additional sense strand off-targets; (3)
2'-O-methyl modification of positions 1 and 2 of the 5'-end of the
sense strand (in combination with 5' phosphorylation of the
antisense strand) eliminates off-target effects due to that strand
but frequently leads to additional antisense strand off-targets;
and (4) 2'-O-methyl modification of positions 1 and 2 of both sense
and antisense strands (in combination with 5' phosphorylation of
the antisense strand) drastically eliminates off-target effects
from both strands. Thus, the combination of three separate
modifications (5' phosphorylation of the antisense strand,
2'-O-methylation of positions 1 and 2 of the sense strand, and
2'-O-methylation of positions 1 and 2 of the antisense strand)
leads to drastic reduction in off-target effects that are generated
by both strands. In addition, in the three cases where specific
silencing activity was tested (MAPK14, PTEN, and MPHOSPH1), the
fully modified molecule (e.g., those that contain 5' phosphate on
the antisense strand, 2'-O-methylation of positions 1 and 2 of the
sense strand, and 2'-O-methylation of positions 1 and 2 of the
antisense strand) that has minimal off-target effects, silenced the
intended target as well or better than siRNA that contain only a
5'-phosphate group on the 5' end of the antisense strand.
[0550] The Effects of 2'-O-Methylation
[0551] The data described above clearly show that the addition of
2'-O-methyl groups to positions 1 and 2 of the 5' end of the AS
strand disrupts siRNA activity. How this modification disrupts
duplex function can be broken down into two distinct steps in the
RNAi pathway. In the first, 2'-O-methylation clearly disrupts the
ability of resident kinases to phosphorylate duplexes. Addition of
the 5' phosphate group synthetically alleviates the need for
cellular enzymes to fulfill this function and improves duplex
functionality. Thus, one of the effects of this chemical
modification is well-defined. While synthetic addition of phosphate
groups to the 5' end of the antisense strand of siRNA modified with
2'-O-methyl groups at nucleotide positions 1 and 2 on both strands
increases activity, full functionality is not recovered. This
result suggests that steps downstream of the phosphorylation event
(e.g., RISC binding, RISC mediated siRNA-target/off-target
interactions, and/or RISC mediated siRNA-target/off-target
cleavage) are also affected by 2'-O-methylation.
[0552] 2'-O-Methylation and Enhanced Duplex Functionality
[0553] The binding of siRNA to RISC and subsequent duplex unwinding
are critical parameters of functionality. Studies presented here
show that in some cases 2'-O-Me modification of the sense strand
alters the dose response curves in a fashion that is inversely
proportional to the original functionality of the molecule. One
interpretation of these findings is that RISC association with the
sense and antisense strands exists in an equilibrium that is
defined by "on" and "off" coefficients (e.g., k.sub.1, k.sub.1'',
k.sub.2, and k.sub.2', FIG. 47). Highly functional molecules
exhibit a K.sub.overall (k.sub.1 k.sub.2/k.sub.1' k.sub.2') that
reflects a bias toward association with the antisense strand (e.g.,
K.sub.overall=100). In contrast, non- or semi-functional molecules
(<F70), exhibit a K.sub.overall that is demonstrative of a more
impartial or balanced strand interaction (e.g., for <F70 siRNA,
K.sub.overall .about.1). Chemical modification of the sense strand
of non- or semi-functional molecules drives the equilibrium toward
RISC-AS interactions, thus dramatically changing the RISC-AS:
RISC-S ratio (K.sub.overall .about.5) and overall duplex
functionality. In contrast, in the case of highly functional
molecules, RISC-AS interactions are already preferred, thus
chemical modification does less to further enhance RISC-AS
association and functionality.
[0554] 2'-O-Methylation and Off-Target Silencing
[0555] While synthetic addition of phosphate groups to the 5' end
of the antisense strand of duplexes containing 2'-O-methyl
modifications at positions 1 and 2 on both strands leads to partial
rescue of the silencing phenotype, the absence of full recovery
suggests that this modification affects additional steps that are
downstream of duplex phosphorylation (e.g., RISC binding, RISC
mediated siRNA-target/off-target interactions, and/or RISC mediated
siRNA-target/off-target cleavage). This fact opens the door to the
possibility that these same modifications might also place greater
demands on the degree of siRNA-target complementarity necessary for
silencing. It is possible that in cases where the position of the
2'-O-methyl group overlaps the site of homology between an
off-target and a given siRNA (sense or antisense strand), the
addition of the chemical modification destabilizes siRNA-off-target
interactions, thus preventing a critical step in RNAi mediated down
regulation. Alternatively, in cases where the position of the
2'-O-methyl group overlaps the site of homology between an
off-target and a given siRNA, the additional chemical modification
may alter duplex flexibility in that region and thus inhibit RISC's
ability to cleave the target molecule. In yet another scenario, the
site of the 2'-O-methyl modification may not overlap the position
of homology with the off-target. In this case, it is possible that
the destabilizing effects of the modifications are transmitted down
the molecule, thus eliminating the ability of RISC to pair and/or
cleave targets that have less than 100% homology to the siRNA. To
that end, 2'-O-methyl modifications at positions other than the 5'
end of the S or AS sense strand may be used to eliminate off-target
effects.
Example 21
Modified Duplexes as Exaequo Agents
[0556] In the course of running dosage or microarray experiments
with siRNA, it is desirable to maintain the total siRNA
concentration at a constant during transfection. Unfortunately,
addition of a second siRNA, even one that is non-specific, can
lessen the degree of silencing induced by the target specific
siRNA, possibly due to competition between the two sequences for
access to RISC. For that reason it is important to develop duplexes
that can act as exaequo agents that do not disrupt gene
targeting.
[0557] To access the ability of modified duplexes to act as
non-competitive exaequo agents in, e.g., dosage experiments, the
effect of modified and unmodified non-specific siRNA on specific
siRNA silencing was tested. To accomplish this, an siRNA duplex
directed against human cyclophilin B gene (e.g., cyclo4) was first
transfected into cells at a variety of concentrations (i.e., a dose
response). The target specific silencing induced by cyclo4 alone
was then compared with the silencing induced by this molecule in
the presence of a) un-modified non specific sequence #4 (NS4), or
b) modified NS4. In this case, the modified version of NS4 was
either 19 or 17 base pairs in length, (SEQ. ID NO. 333:
UAGCGACUAAACACAUCAA and SEQ. ID NO. 334: UAGCGACUAAACACAUC,
respectively) and contained 2'-O-methyl groups on positions 1 and 2
of the sense and antisense strand, 2'-O-methyl groups on the Cs and
Us of the sense strand, and 2' Fl groups on the Cs and Us of the
antisense strand. No phosphate group is attached to the 5' end of
the sense or antisense strand.
[0558] Specifically, HeLa cells were plated into 96 well plates and
allowed to adhere overnight. Subsequently, cells were transfected
with one of the following: [0559] a) cyclo4 siRNA (1-100 nM), (SEQ.
ID NO. 318: GGA AAG ACU GUU CCA AAA A) [0560] b) cyclo4 siRNA
(1-100 nM) plus NS4 (19 base pairs), [0561] c) cyclo4 siRNA (1-100
nM), plus modified NS4 (17 or 19 base pairs), [0562] d) cyclo4
siRNA (1-100 nM) plus a modified NS4 (17 or 19 base pairs) that
also contained a Cy3 label on the 5' end of the sense strand, using
Lipofectamine 2000. The concentrations of the second siRNA were
matched with that of cyclo4 such that the total siRNA concentration
during the transfection remained constant at 100 nM. Subsequently
target specific silencing was measured (t=24 hours) using a
branched DNA assay (Genospectra, Fremont, Calif.).
[0563] The results of these experiments are illustrated in FIG. 48
and demonstrate the following: [0564] 1. Cyclo4 is a potent duplex.
Decreasing the concentration of cyclo4 from 100 nM to 1 nM does not
alter the silencing ability of this duplex (F95, See FIG. 48A-I).
[0565] 2. Addition of a 19 base pair unmodified NS4 siRNA to cyclo4
causes an appreciable concentration dependent decrease in cyclo4
induced gene silencing, suggesting that NS4 competes with cyclo4
for, e.g., access to the RISC complex (see FIG. 48B-I). [0566] 3.
NS4 duplexes (17 or 19 base pairs) containing 2'-O-methyl groups on
positions 1 and 2 of the sense and antisense strand, 2'-O-methyl
groups on the Cs and Us of the sense strand, and 2' Fl groups on
the Cs and Us of the antisense strand, fail to diminish target
specific silencing by cyclo4, suggesting these duplexes do not
compete with cyclo4 for, e.g., RISC (see FIG. 43BII and BIII).
[0567] 4. NS4 duplexes (17 or 19 base pairs) containing 2'-O-methyl
groups on positions 1 and 2 of the sense and antisense strand,
2'-O-methyl groups on the Cs and Us of the sense strand, 2' Fl
groups on the Cs and Us of the antisense strand, and a Cy3 group on
the 5' end of the sense strand fail to diminish silencing by
cyclo4, suggesting these duplexes do not compete with cyclo4 for,
e.g., RISC (see FIG. 48AII and AIII)
[0568] These results demonstrate that siRNA containing the before
mentioned modifications fail to compete with functional siRNA and
can be used as exaequo agents in siRNA studies, including
microarray studies.
[0569] It is conceivable that the reason why siRNAs carrying the
above modifications fail to compete with the target specific siRNA
is that the modified duplexes fail to enter the cell. To test this,
an siRNA carrying 2'-O-methyl groups on positions 1 and 2 of the
sense and antisense strand, 2'-O-methyl groups on the Cs and Us of
the sense strand, and 2' Fl groups on the Cs and Us of the
antisense strand (no 5' phosphate group on the antisense strand)
was labeled with a Cy3 moiety on the 5' end of the sense strand and
transfected into HeLa cells. Forty-eight hours post transfection,
cells were analyzed by fluorescence microscopy. Results of these
experiments show that aforementioned modifications do not alter the
ability of these duplexes to enter the cell by Lipofectamine 2000
transfection. Micrographs (not reproduced herein) of: (A) HeLa
cells stained with cyclo14 siRNA duplex modified with 2'-O-methyl
groups on positions 1 and 2 of the sense and antisense strand,
2'-O-methyl groups on the Cs and Us of the sense strand, 2' Fl
groups added to the Cs and Us of the antisense strand and a Cy3
group on the 5' end of the sense strand; and (B) stained as in (A)
and counterstained with Hoechst 33342 to identify the position of
the nucleus demonstrated nuclear and perinuclear stains of siRNA
containing these modifications.
[0570] To test whether addition of 2'-O-methyl groups on positions
1 and 2 of the sense and antisense strand was sufficient to
eliminate the ability of a duplex to compete with other,
co-transfected siRNA, an siRNA directed against GAPDH (GAPDH4) was
transfected into HeLa cells at varying concentrations (0.781-100
nM) along with (1) non-specific control #2 (NS2--SEQ ID. NO. 334:
UAAGGCUAUGAAGAGAUAC), (2) non-specific control #2 carrying
2'-O-methyl modifications on positions 1 and 2 of both the sense
and antisense strand, or (3) cyclo14 (SEQ. ID. NO. 335:
GGCCUUAGCUACAGGAGAG, a cyclophilin siRNA that does not compete with
GAPDH 4). The concentrations of the second siRNA were matched with
that of GAPDH4 such that the total siRNA concentration during the
transfection was 100 nM. Cells were then cultured for an additional
24 hours before branched DNA assays were performed to assess the
level of GAPDH transcripts. Results of these experiments are
illustrated in FIG. 49 and show the following: [0571] 1. GAPDH4 in
the presence of Cyclo14 (or by itself, data not shown) induces
roughly 90% silencing of the native transcript. [0572] 2. Addition
of unmodified NS2 led to a dramatic decrease in GAPDH silencing,
and [0573] 3. Modification of NS2 with 2'-O-methyl groups at
positions 1 and 2 on both the sense and antisense strands
eliminated interference effects observed with unmodified
molecules.
[0574] These results demonstrate that siRNA containing the before
mentioned modifications fail to compete with functional siRNA and
can be used as exaequo agents in a variety of siRNA applications
including microarray studies.
[0575] Although the invention has been described and has been
illustrated in connection with certain specific or preferred
inventive embodiments, it will be understood by those of skill in
the art that the invention is capable of many further
modifications. This application is intended to cover any and all
variations, uses, or adaptations of the invention that follow, in
general, the principles of the invention and include departures
from the disclosure that come within known or customary practice
within the art and as may be applied to the essential features
described in this application and in the scope of the appended
claims.
Sequence CWU 1
1
335121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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21921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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211921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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212921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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213921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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214921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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215021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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215121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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215221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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215321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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215421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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215521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 55uuuaugagga ucucucugat t
215621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 56uuuaugagga ucucucugat t
215721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 57uuuaugagga ucucucugat t
215821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 58uuuaugagga ucucucugat t
215921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
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216021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 60uuuaugagga ucucucugat t
216121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 61uuuaugagga ucucucugat t
216221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 62uuuaugagga ucucucugat t
216321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 63ucagagagau ccucauaaat t
216421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 64ucagagagau ccucauaaat t
216521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 65ucagagagau ccucauaaat t
216621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 66ucagagagau ccucauaaat t
216721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 67ucagagagau ccucauaaat t
216821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 68ucagagagau ccucauaaat t
216921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 69ucagagagau ccucauaaat t
217021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 70ucagagagau ccucauaaat t
217121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 71ucagagagau ccucauaaat t
217221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 72ucagagagau ccucauaaat t
217321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 73ucagagagau ccucauaaat t
217421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 74ucagagagau ccucauaaat t
217521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 75ucagagagau ccucauaaat t
217621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 76ucagagagau ccucauaaat t
217721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 77ucagagagau ccucauaaat t
217821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 78ucagagagau ccucauaaat t
217921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 79ucagagagau ccucauaaat t
218021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 80ucagagagau ccucauaaat t
218121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 81ucagagagau ccucauaaat t
218221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 82ucagagagau ccucauaaat t
218321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 83ucagagagau ccucauaaat t
218421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 84ucagagagau ccucauaaat t
218521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 85ucagagagau ccucauaaat t
218621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 86ucagagagau ccucauaaat t
218721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 87ucagagagau ccucauaaat t
218821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 88ucagagagau ccucauaaat t
218921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 89ucagagagau ccucauaaat t
219021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 90ucagagagau ccucauaaat t
219121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 91ucagagagau ccucauaaat t
219221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 92ucagagagau ccucauaaat t
219321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 93ucagagagau ccucauaaat t
219421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 94ucagagagau ccucauaaat t
219521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 95ucagagagau ccucauaaat t
219621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 96ucagagagau ccucauaaat t
219721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 97ucagagagau ccucauaaat t
219821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 98ucagagagau ccucauaaat t
219921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 99uuuaugagga ucucucugat t
2110021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 100uuuaugagga ucucucugat t
2110121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 101uuuaugagga ucucucugat t
2110221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 102uuuaugagga ucucucugat t
2110321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 103uuuaugagga ucucucugat t
2110421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 104uuuaugagga ucucucugat t
2110521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 105uuuaugagga ucucucugat t
2110621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 106uuuaugagga ucucucugat t
2110721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 107uuuaugagga ucucucugat t
2110821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 108uuuaugagga ucucucugat t
2110921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 109uuuaugagga ucucucugat t
2111021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 110uuuaugagga ucucucugat t
2111121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 111uuuaugagga ucucucugat t
2111221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 112uuuaugagga ucucucugat t
2111321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 113uuuaugagga ucucucugat t
2111421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 114uuuaugagga ucucucugat t
2111521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 115uuuaugagga ucucucugat t
2111621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 116uuuaugagga ucucucugat t
2111721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 117uuuaugagga ucucucugat t
2111821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 118uuuaugagga ucucucugat t
2111921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 119uuuaugagga ucucucugat t
2112021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 120uuuaugagga ucucucugat t
2112121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 121uuuaugagga ucucucugat t
2112221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 122uuuaugagga ucucucugat t
2112321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 123uuuaugagga ucucucugat t
2112421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 124uuuaugagga ucucucugat t
2112521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 125uuuaugagga ucucucugat t
2112621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 126uuuaugagga ucucucugat t
2112721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 127uuuaugagga ucucucugat t
2112821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 128uuuaugagga ucucucugat t
2112921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 129uuuaugagga ucucucugat t
2113021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 130uuuaugagga ucucucugat t
2113121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 131uuuaugagga ucucucugat t
2113221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 132uuuaugagga ucucucugat t
2113321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 133uuuaugagga ucucucugat t
2113421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 134uuuaugagga ucucucugat t
2113521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 135ucagagagau ccucauaaat t
2113621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 136ucagagagau ccucauaaat t
2113721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 137ucagagagau ccucauaaat t
2113821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 138ucagagagau ccucauaaat t
2113921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 139ucagagagau ccucauaaat t
2114021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 140ucagagagau ccucauaaat t
2114121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 141ucagagagau ccucauaaat t
2114221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 142ucagagagau ccucauaaat t
2114321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 143ucagagagau ccucauaaat t
2114421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 144ucagagagau ccucauaaat t
2114521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 145ucagagagau ccucauaaat t
2114621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 146ucagagagau ccucauaaat t
2114721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 147ucagagagau ccucauaaat t
2114821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 148ucagagagau ccucauaaat t
2114921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 149ucagagagau ccucauaaat t
2115021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 150ucagagagau ccucauaaat t
2115121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 151ucagagagau ccucauaaat t
2115221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 152ucagagagau ccucauaaat t
2115321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 153ucagagagau ccucauaaat t
2115421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 154ucagagagau ccucauaaat t
2115521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 155ucagagagau ccucauaaat t
2115621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 156ucagagagau ccucauaaat t
2115721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 157ucagagagau ccucauaaat t
2115821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 158ucagagagau ccucauaaat t
2115921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 159ucagagagau ccucauaaat t
2116021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 160ucagagagau ccucauaaat t
2116121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 161ucagagagau ccucauaaat t
2116221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 162ucagagagau ccucauaaat t
2116321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 163ucagagagau ccucauaaat t
2116421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 164ucagagagau ccucauaaat t
2116521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 165ucagagagau ccucauaaat t
2116621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 166ucagagagau ccucauaaat t
2116721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 167ucagagagau ccucauaaat t
2116821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 168ucagagagau ccucauaaat t
2116921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 169ucagagagau ccucauaaat t
2117021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 170ucagagagau ccucauaaat t
2117121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 171gugauguaug ucagagagut t
2117221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 172acucucugac auacaucact t
2117321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 173acucucugac auacaucact t
2117421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 174acucucugac auacaucact t
2117521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 175acucucugac auacaucact t
2117621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 176acucucugac auacaucact t
2117721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 177acucucugac auacaucact t
2117821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 178acucucugac auacaucact t
2117921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 179acucucugac auacaucact t
2118021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 180acucucugac auacaucact t
2118121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 181acucucugac auacaucact t
2118221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 182acucucugac auacaucact t
2118321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 183acucucugac auacaucact t
2118421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 184acucucugac auacaucact t
2118521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 185acucucugac auacaucact t
2118621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 186acucucugac auacaucact t
2118721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 187acucucugac auacaucact t
2118821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 188acucucugac auacaucact t
2118921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 189acucucugac auacaucact t
2119021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 190acucucugac auacaucact t
2119121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 191acucucugac auacaucact t
2119221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 192acucucugac auacaucact t
2119321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 193acucucugac auacaucact t
2119421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 194acucucugac auacaucact t
2119521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 195acucucugac auacaucact t
2119621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 196acucucugac auacaucact t
2119721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 197acucucugac auacaucact t
2119821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 198acucucugac auacaucact t
2119921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 199acucucugac auacaucact t
2120021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 200acucucugac auacaucact t
2120121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 201acucucugac auacaucact t
2120221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 202acucucugac auacaucact t
2120321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 203acucucugac auacaucact t
2120421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 204acucucugac auacaucact t
2120521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 205acucucugac auacaucact t
2120621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 206gugauguaug ucagagagut t
2120721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 207gugauguaug ucagagagut t
2120821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 208gugauguaug ucagagagut t
2120921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 209gugauguaug ucagagagut t
2121021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 210gugauguaug ucagagagut t
2121121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 211gugauguaug ucagagagut t
2121221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 212gugauguaug ucagagagut t
2121321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 213gugauguaug ucagagagut t
2121421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 214gugauguaug ucagagagut t
2121521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 215gugauguaug ucagagagut t
2121621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 216gugauguaug ucagagagut t
2121721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 217gugauguaug ucagagagut t
2121821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 218gugauguaug ucagagagut t
2121921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 219gugauguaug ucagagagut t
2122021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 220gugauguaug ucagagagut t
2122121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 221gugauguaug ucagagagut t
2122221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 222gugauguaug ucagagagut t
2122321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 223gugauguaug ucagagagut t
2122421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 224gugauguaug ucagagagut t
2122521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 225gugauguaug ucagagagut t
2122621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 226gugauguaug ucagagagut t
2122721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 227gugauguaug ucagagagut t
2122821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 228gugauguaug ucagagagut t
2122921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 229gugauguaug ucagagagut t
2123021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 230gugauguaug ucagagagut t
2123121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 231gugauguaug ucagagagut t
2123221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 232gugauguaug ucagagagut t
2123321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 233gugauguaug ucagagagut t
2123421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 234gugauguaug ucagagagut t
2123521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 235gugauguaug ucagagagut t
2123621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 236gugauguaug ucagagagut t
2123721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 237gugauguaug ucagagagut t
2123821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 238gugauguaug ucagagagut t
2123921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 239gugauguaug ucagagagut t
2124021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 240gugauguaug ucagagagut t
2124121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 241gugauguaug ucagagagut t
2124221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 242gugauguaug ucagagagut t
2124321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 243gugauguaug ucagagagut t
2124421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 244gugauguaug ucagagagut t
2124521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 245gugauguaug ucagagagut t
2124621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 246gugauguaug ucagagagut t
2124721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 247gugauguaug ucagagagut t
2124821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 248gugauguaug ucagagagut t
2124921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 249gugauguaug ucagagagut t
2125021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 250gugauguaug ucagagagut t
2125121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 251gugauguaug ucagagagut t
2125221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 252gugauguaug ucagagagut t
2125321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 253gugauguaug ucagagagut t
2125421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 254gugauguaug ucagagagut t
2125521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 255gugauguaug ucagagagut t
2125621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 256gugauguaug ucagagagut t
2125721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 257gugauguaug ucagagagut t
2125821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 258acucucugac auacaucact t
2125921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 259acucucugac auacaucact t
2126021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 260acucucugac auacaucact t
2126121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 261acucucugac auacaucact t
2126221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 262acucucugac auacaucact t
2126321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 263acucucugac auacaucact t
2126421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 264acucucugac auacaucact t
2126521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 265acucucugac auacaucact t
2126621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 266acucucugac auacaucact t
2126721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 267acucucugac auacaucact t
2126821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 268acucucugac auacaucact t
2126921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 269acucucugac auacaucact t
2127021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 270acucucugac auacaucact t
2127121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 271acucucugac auacaucact t
2127221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 272acucucugac auacaucact t
2127321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 273acucucugac auacaucact t
2127421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 274acucucugac auacaucact t
2127521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 275acucucugac auacaucact t
2127621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 276acucucugac auacaucact t
2127721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 277gugauguaug ucagagagut t
2127821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 278gugauguaug ucagagagut t
2127921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 279gugauguaug ucagagagut t
2128021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 280gugauguaug ucagagagut t
2128121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 281gugauguaug ucagagagut t
2128221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 282gugauguaug ucagagagut t
2128321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 283gugauguaug ucagagagut t
2128421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 284gugauguaug ucagagagut t
2128521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 285gugauguaug ucagagagut t
2128621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 286gugauguaug ucagagagut t
2128721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 287gugauguaug ucagagagut t
2128821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 288gugauguaug ucagagagut t
2128921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 289gugauguaug ucagagagut t
2129021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 290gugauguaug ucagagagut t
2129121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 291gugauguaug ucagagagut t
2129221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 292gugauguaug ucagagagut t
2129321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 293gugauguaug ucagagagut t
2129421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 294gugauguaug ucagagagut t
2129521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 295gugauguaug ucagagagut t
2129621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 296acucucugac auacaucact t
2129721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 297acucucugac auacaucact t
2129821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 298acucucugac auacaucact t
2129921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 299acucucugac auacaucact t
2130021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 300acucucugac auacaucact t
2130121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 301acucucugac auacaucact t
2130221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 302acucucugac auacaucact t
2130321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 303acucucugac auacaucact t
2130421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 304acucucugac auacaucact t
2130521DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 305acucucugac auacaucact t
2130621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 306acucucugac auacaucact t
2130721DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 307acucucugac auacaucact t
2130821DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 308acucucugac auacaucact t
2130921DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 309acucucugac auacaucact t
2131021DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 310acucucugac auacaucact t
2131121DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 311acucucugac auacaucact t
2131221DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 312acucucugac auacaucact t
2131321DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 313acucucugac auacaucact t
2131421DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' end 314gaaagagcau cuacggugat t
2131519DNAArtificial Sequence2'-O-Methyl 315gaaaggauuu ggcuacaaa
1931619DNAArtificial Sequence2'-O-Methyl 316acagcaaauu ccaucgugu
1931719DNAArtificial Sequence2'-O-Methyl 317acagcaaauu ccaucgugu
1931819DNAArtificial Sequence2'-O-Methyl 318ggaaagacug uuccaaaaa
193194DNAArtificial Sequencenucleotide loop 319uucg
43209DNAArtificial Sequencenucleotide loop 320uuuguguag
932110DNAArtificial Sequencenucleotide loop 321cuuccuguca
1032219DNAArtificial Sequence2'-O-Methyl 322ggccttagct acaggagag
193234DNAArtificial Sequencenucleotide loop 323uucg
43249DNAArtificial Sequencenucleotide loop 324uuuguguag
932510DNAArtificial Sequencenucleotide loop 325cuuccuguca
1032621DNAArtificial SequenceRNA/DNA, synthetic, RNA with
2'deoxythymidines at 3' 326gugauguaug ucagagagut t
2132719DNAArtificial Sequence2'-O-methyl 327gaaaaaucag agagauccu
1932819DNAArtificial Sequence2'-O-methyl 328uaccggaaaa cucgacgca
1932919DNAArtificial Sequence2'-O-methyl 329acgucgccag ucaaguaac
1933019DNAArtificial Sequence2'-O-methyl 330gauuacgucg ccagucaag
1933119DNAArtificial Sequence2'-O-methyl 331agagaucgug gauuacguc
1933219DNAArtificial Sequence2'-O-methyl 332uguuguuuug gagcacgga
1933319DNAArtificial Sequence2'-O-methyl 333uagcgacuaa acacaucaa
1933417DNAArtificial Sequence2'-O-methyl 334uagcgacuaa acacauc
1733519DNAArtificial Sequencesynthetic human cyclophilin siRNA
sense strand 335ggccuuagcu acaggagag 19
* * * * *