U.S. patent application number 11/881385 was filed with the patent office on 2008-12-11 for sirna targeting proprotein convertase subtilisin/kexin type 9 (pcsk9).
This patent application is currently assigned to DHARMACON, INC.. Invention is credited to Anastasia Khvorova, Devin Leake, William Marshall, Steven Read, Angela Reynolds, Stephen Scaringe.
Application Number | 20080306015 11/881385 |
Document ID | / |
Family ID | 32329096 |
Filed Date | 2008-12-11 |
United States Patent
Application |
20080306015 |
Kind Code |
A1 |
Khvorova; Anastasia ; et
al. |
December 11, 2008 |
siRNA targeting proprotein convertase subtilisin/kexin type 9
(PCSK9)
Abstract
Efficient sequence specific gene silencing is possible through
the use of siRNA technology. By selecting particular siRNAs by
rational design, one can maximize the generation of an effective
gene silencing reagent, as well as methods for silencing genes.
Methods, compositions, and kits generated through rational design
of siRNAs are disclosed including those directed to PCSK9.
Inventors: |
Khvorova; Anastasia;
(Boulder, CO) ; Reynolds; Angela; (Conifer,
CO) ; Leake; Devin; (Denver, CO) ; Marshall;
William; (Boulder, CO) ; Read; Steven;
(Denver, CO) ; Scaringe; Stephen; (Lafayette,
CO) |
Correspondence
Address: |
KALOW & SPRINGUT LLP
488 MADISON AVENUE, 19TH FLOOR
NEW YORK
NY
10022
US
|
Assignee: |
DHARMACON, INC.
Lafayette
CO
|
Family ID: |
32329096 |
Appl. No.: |
11/881385 |
Filed: |
July 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10940892 |
Sep 14, 2004 |
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11881385 |
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PCT/US04/14885 |
May 12, 2004 |
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10940892 |
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10714333 |
Nov 14, 2003 |
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PCT/US04/14885 |
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60502050 |
Sep 10, 2003 |
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60426137 |
Nov 14, 2002 |
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Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
C12N 15/111 20130101;
C12N 2320/11 20130101; A61P 3/10 20180101; A61P 35/00 20180101;
C12Y 502/01008 20130101; A61P 21/00 20180101; C12Y 113/12007
20130101; A61P 35/02 20180101; C12N 15/113 20130101; A61P 37/02
20180101; C12N 15/1138 20130101; C12N 15/1136 20130101; A61K 31/713
20130101; C12N 2320/10 20130101; C12N 15/1135 20130101; A61P 25/28
20180101; C12N 15/1137 20130101; G16B 20/00 20190201; C12N 2310/14
20130101; A61P 13/12 20180101; C12N 15/1048 20130101 |
Class at
Publication: |
514/44 ;
536/24.5 |
International
Class: |
A61K 31/7105 20060101
A61K031/7105; C07H 21/02 20060101 C07H021/02 |
Claims
1. An siRNA comprising a sense region and an antisense region,
wherein said sense region and said antisense region together form a
duplex region, said antisense region and said sense region are each
18-30 nucleotides in length and said antisense region comprises a
sequence that is at least 90% complementary to a sequence selected
from the group consisting of SEQ. ID NOs. 438-565.
2. An siRNA comprising a sense region and an antisense region,
wherein said sense region and said antisense region together form a
duplex region and said sense region and said antisense region are
each 18-30 nucleotides in length, and said antisense region
comprises a sequence that is 100% complementary to a contiguous
stretch of at least 18 bases of a sequence selected from the group
consisting of SEQ. ID NOs. 438-565.
3. The siRNA of claim 2, wherein each of said antisense region and
said sense region are 19-30 nucleotides in length, and said
antisense region comprises a sequence that is 100% complementary to
said sequence selected from the group consisting of: SEQ. ID NOs.
438-565.
4. A pool of at least two siRNAs, wherein said pool comprises a
first siRNA and a second siRNA, said first siRNA comprises a first
antisense region and a first sense region that together form a
first duplex region and each of said first antisense region and
said first sense region are 18-30 nucleotides in length and said
first antisense region is at least 90% complementary to 18 bases of
a first sequence selected from the group consisting of: SEQ. ID
NOs. 438-565 and said second siRNA comprises a second antisense
region and a second sense region that together form a second duplex
region and each of said second antisense region and said second
sense region are 18-30 nucleotides in length and said second
antisense region is at least 90% complementary to 18 bases of a
second sequence selected from the group consisting of: SEQ. ID NOs.
438-565, wherein said first antisense region and said second
antisense region are not identical.
5. The pool of claim 4, wherein said first antisense region
comprises a sequence that is 100% complementary to at least 18
bases of said first sequence, and said second antisense region
comprises a sequence that is 100% complementary to at least 18
bases of said second sequence.
6. The pool of claim 4, wherein said first siRNA is 19-30
nucleotides in length and said first antisense region comprises a
sequence that is at least 90% complementary to said first sequence,
and second siRNA is 19-30 nucleotides in length and said second
antisense region comprises a sequence that is at least 90%
complementary to said second sequence.
7. The pool of claim 4, wherein said first antisense region is
19-30 nucleotides in length and said first antisense region
comprises a sequence that is 100% complementary to at least 18
bases of said first sequence, and said second antisense region is
19-30 nucleotides in length and said second antisense region
comprises a sequence that is 100% complementary to said second
sequence.
8. The siRNA of claim 1, wherein said antisense region and said
sense region are each 19-25 nucleotides in length.
9. The siRNA of claim 4, wherein said first antisense region, said
first sense region, said second sense region and said second
antisense region are each 19-25 nucleotides in length.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
10/714,333, filed Nov. 14, 2003, which claims the benefit of U.S.
Provisional Application No. 60/426,137, filed Nov. 14, 2002, and
also claims the benefit of U.S. Provisional Application No.
60/502,050, filed Sep. 10, 2003; this application is also a
continuation-in-part of U.S. Ser. No. 10/940,892, filed Sep. 14,
2004, which is a continuation of PCT Application No.
PCT/US04/14885, international filing date May 12, 2004. The
disclosures of the priority applications, including the sequence
listings and tables submitted in electronic form in lieu of paper,
are incorporated by reference into the instant specification.
SEQUENCE LISTING
[0002] The sequence listing for this application has been submitted
in accordance with 37 CFR .sctn.1.52(e) and 37 CFR .sctn.1.821 on
CD-ROM in lieu of paper on a disk containing the sequence listing
file entitled "DHARMA.sub.--2100-US64_CRF.txt" created Jun. 12,
2007, 112 kb. Applicants hereby incorporate by reference the
sequence listing provided on CD-ROM in lieu of paper into the
instant specification.
FIELD OF INVENTION
[0003] The present invention relates to RNA interference
("RNAi").
BACKGROUND OF THE INVENTION
[0004] Relatively recently, researchers observed that double
stranded RNA ("dsRNA") could be used to inhibit protein expression.
This ability to silence a gene has broad potential for treating
human diseases, and many researchers and commercial entities are
currently investing considerable resources in developing therapies
based on this technology.
[0005] Double stranded RNA induced gene silencing can occur on at
least three different levels: (i) transcription inactivation, which
refers to RNA guided DNA or histone methylation; (ii) siRNA induced
mRNA degradation; and (iii) mRNA induced transcriptional
attenuation.
[0006] It is generally considered that the major mechanism of RNA
induced silencing (RNA interference, or RNAi) in mammalian cells is
mRNA degradation. Initial attempts to use RNAi in mammalian cells
focused on the use of long strands of dsRNA. However, these
attempts to induce RNAi met with limited success, due in part to
the induction of the interferon response, which results in a
general, as opposed to a target-specific, inhibition of protein
synthesis. Thus, long dsRNA is not a viable option for RNAi in
mammalian systems.
[0007] More recently it has been shown that when short (18-30 bp)
RNA duplexes are introduced into mammalian cells in culture,
sequence-specific inhibition of target mRNA can be realized without
inducing an interferon response. Certain of these short dsRNAs,
referred to as small inhibitory RNAs ("siRNAs"), can act
catalytically at sub-molar concentrations to cleave greater than
95% of the target mRNA in the cell. A description of the mechanisms
for siRNA activity, as well as some of its applications are
described in Provost et al. (2002) Ribonuclease Activity and RNA
Binding of Recombinant Human Dicer, EMBO J. 21(21): 5864-5874;
Tabara et al. (2002) The dsRNA Binding Protein RDE-4 Interacts with
RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans,
Cell 109(7):861-71; Ketting et al. (2002) Dicer Functions in RNA
Interference and in Synthesis of Small RNA Involved in
Developmental Timing in C. elegans; Martinez et al.,
Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi,
Cell 110(5):563; Hutvagner & Zamore (2002) A microRNA in a
multiple-turnover RNAi enzyme complex, Science 297:2056.
[0008] From a mechanistic perspective, introduction of long double
stranded RNA into plants and invertebrate cells is broken down into
siRNA by a Type II endonuclease known as Dicer. Sharp, RNA
interference--2001, Genes Dev. 2001, 15:485. Dicer, a
ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base
pair short interfering RNAs with characteristic two base 3'
overhangs. Bernstein, Caudy, Hammond, & Hannon (2001) Role for
a bidentate ribonuclease in the initiation step of RNA
interference, Nature 409:363. The siRNAs are then incorporated into
an RNA-induced silencing complex (RISC) where one or more helicases
unwind the siRNA duplex, enabling the complementary antisense
strand to guide target recognition. Nykanen, Haley, & Zamore
(2001) ATP requirements and small interfering RNA structure in the
RNA interference pathway, Cell 107:309. Upon binding to the
appropriate target mRNA, one or more endonucleases within the RISC
cleaves the target to induce silencing. Elbashir, Lendeckel, &
Tuschl (2001) RNA interference is mediated by 21- and 22-nucleotide
RNAs, Genes Dev. 15:188, FIG. 1.
[0009] The interference effect can be long lasting and may be
detectable after many cell divisions. Moreover, RNAi exhibits
sequence specificity. Kisielow, M. et al., (2002) Isoform-specific
knockdown and expression of adaptor protein ShcA using small
interfering RNA, J. Biochem. 363: 1-5. Thus, the RNAi machinery can
specifically knock down one type of transcript, while not affecting
closely related mRNA. These properties make siRNA a potentially
valuable tool for inhibiting gene expression and studying gene
function and drug target validation. Moreover, siRNAs are
potentially useful as therapeutic agents against: (1) diseases that
are caused by over-expression or misexpression of genes; and (2)
diseases brought about by expression of genes that contain
mutations.
[0010] Successful siRNA-dependent gene silencing depends on a
number of factors. One of the most contentious issues in RNAi is
the question of the necessity of siRNA design, i.e., considering
the sequence of the siRNA used. Early work in C. elegans and plants
circumvented the issue of design by introducing long dsRNA (see,
for instance, Fire, A. et al. (1998) Nature 391:806-811). In this
primitive organism, long dsRNA molecules are cleaved into siRNA by
Dicer, thus generating a diverse population of duplexes that can
potentially cover the entire transcript. While some fraction of
these molecules are non-functional (i.e., induce little or no
silencing) one or more have the potential to be highly functional,
thereby silencing the gene of interest and alleviating the need for
siRNA design. Unfortunately, due to the interferon response, this
same approach is unavailable for mammalian systems. While this
effect can be circumvented by bypassing the Dicer cleavage step and
directly introducing siRNA, this tactic carries with it the risk
that the chosen siRNA sequence may be non-functional or
semi-functional.
[0011] A number of researches have expressed the view that siRNA
design is not a crucial element of RNAi. On the other hand, others
in the field have begun to explore the possibility that RNAi can be
made more efficient by paying attention to the design of the siRNA.
Unfortunately, none of the reported methods have provided a
satisfactory scheme for reliably selecting siRNA with acceptable
levels of functionality. Accordingly, there is a need to develop
rational criteria by which to select siRNA with an acceptable level
of functionality, and to identify siRNA that have this improved
level of functionality, as well as to identify siRNAs that are
hyperfunctional.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to increasing the
efficiency of RNAi, particularly in mammalian systems. Accordingly,
the present invention provides kits, siRNAs and methods for
increasing siRNA efficacy.
[0013] According to a first embodiment, the present invention
provides a kit for gene silencing, wherein said kit is comprised of
a pool of at least two siRNA duplexes, each of which is comprised
of a sequence that is complementary to a portion of the sequence of
one or more target messenger RNA, and each of which is selected
using non-target specific criteria.
[0014] According to a second embodiment, the present invention
provides a method for selecting an siRNA, said method comprising
applying selection criteria to a set of potential siRNA that
comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene; and
determining the relative functionality of the at least two
siRNAs.
[0015] According to a third embodiment, the present invention also
provides a method for selecting an siRNA wherein said selection
criteria are embodied in a formula comprising:
(-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-1-
0*C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-
9*U.sub.9-9*C.sub.18-8*G.sub.10-7*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.-
19+7*U.sub.17+8*A.sub.2+8*A.sub.4+8*A.sub.5+8*C.sub.4
+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.9+15*G.sub.1+18*A-
.sub.3+19*U.sub.10-Tm-3*(GC.sub.total)
-6*(GC.sub.15-19)-30*X; or Formula VIII
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+
(19)*A7+(6)*A8+(-4)*A9+(-5)*A10+(-2)*A11+(-5)*A12+(17)*A13+(-
3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(-13)*U1+(-
10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8+(-
5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-
10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-
9)*C5+(-20)*C6+(-18)*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-
5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-5)*C16+(-3)*C17+(-12)*C18+(-
18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-
4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-
24)*G13+(18)*G14+(11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+
6*(number of A+U in position 15-19)-3*(number of G+C in whole
siRNA), Formula X
wherein position numbering begins at the 5'-most position of a
sense strand, and A.sub.1=1 if A is the base at position 1 of the
sense strand, otherwise its value: is 0; A.sub.2=1 if A is the base
at position 2 of the sense strand, otherwise its value: is 0;
A.sub.3=1 if A is the base at position 3 of the sense strand,
otherwise its value: is 0; A.sub.4=1 if A is the base at position 4
of the sense strand, otherwise its value is 0; A.sub.5=1 if A is
the base at position 5 of the sense strand, otherwise its value is
0; A.sub.6=1 if A is the base at position 6 of the sense strand,
otherwise its value is 0; A.sub.7=1 if A is the base at position 7
of the sense strand, otherwise its value is 0; A.sub.10=1 if A is
the base at position 10 of the sense strand, otherwise its value is
0; A.sub.11=1 if A is the base at position 11 of the sense strand,
otherwise its value is 0; A.sub.13=1 if A is the base at position
13 of the sense strand, otherwise its value is 0; A.sub.19=1 if A
is the base at position 19 of the sense strand, otherwise if
another base is present or the sense strand is only 18 base pairs
in length, its value is 0; C.sub.3=1 if C is the base at position 3
of the sense strand, otherwise its value is 0; C.sub.4=1 if C is
the base at position 4 of the sense strand, otherwise its value is
0; C.sub.5=1 if C is the base at position 5 of the sense strand,
otherwise its value is 0; C.sub.6=1 if C is the base at position 6
of the sense strand, otherwise its value is 0; C.sub.7=1 if C is
the base at position 7 of the sense strand, otherwise its value: is
0; C.sub.9=1 if C is the base at position 9 of the sense strand,
otherwise its value is 0; C.sub.17=1 if C is the base at position
17 of the sense strand, otherwise its value is 0; C.sub.18=1 if C
is the base at position 18 of the sense strand, otherwise its value
is 0; C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0; G.sub.1=1 if G is the
base at position 1 on the sense strand, otherwise its value is 0;
G.sub.2=1 if G is the base at position 2 of the sense strand,
otherwise its value is 0; G.sub.8=1 if G is the base at position 8
on the sense strand, otherwise its value is 0; G.sub.10=1 if G is
the base at position 10 on the sense strand, otherwise its value is
0; G.sub.13=1 if G is the base at position 13 on the sense strand,
otherwise its value is 0; G.sub.19=1 if G is the base at position
19 of the sense strand, otherwise if another base is present or the
sense strand is only 18 base pairs in length, its value is 0;
U.sub.1=1 if U is the base at position 1 on the sense strand,
otherwise its value is 0; U.sub.2=1 if U is the base at position 2
on the sense strand, otherwise its value is 0; U.sub.3=1 if U is
the base at position 3 on the sense strand, otherwise its value is
0; U.sub.4=1 if U is the base at position 4 on the sense strand,
otherwise its value is 0; U.sub.7=1 if U is the base at position 7
on the sense strand, otherwise its value is 0; U.sub.9=1 if U is
the base at position 9 on the sense strand, otherwise its value is
0; U.sub.10=1 if U is the base at position 10 on the sense strand,
otherwise its value is 0; U.sub.15=1 if U is the base at position
15 on the sense strand, otherwise its value is 0; U.sub.16=1 if U
is the base at position 16 on the sense strand, otherwise its value
is 0; U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0; U.sub.18=1 if U is the base at
position 18 on the sense strand, otherwise its value is 0.
GC.sub.15-19=the number of G and C bases within positions 15-19 of
the sense strand, or within positions 15-18 if the sense strand is
only 18 base pairs in length; GC.sub.total=the number of G and C
bases in the sense strand; Tm=100 if the si RNA oligo has the
internal repeat longer then 4 base pairs, otherwise its value is 0;
and X=the number of times that the same nucleotide repeats four or
more times in a row.
[0016] According to a fourth embodiment, the invention provides a
method for developing an algorithm for selecting siRNA, said method
comprising: (a) selecting a set of siRNA; (b) measuring gene
silencing ability of each siRNA from said set; (c) determining
relative functionality of each siRNA; (d) determining improved
functionality by the presence or absence of at least one variable
selected from the group consisting of the presence or absence of a
particular nucleotide at a particular position, the total number of
As and Us in positions 15-19, the number of times that the same
nucleotide repeats within a given sequence, and the total number of
Gs and Cs; and (e) developing an algorithm using the information of
step (d).
[0017] According to a fifth embodiment, the present invention
provides a kit, wherein said kit is comprised of at least two
siRNAs, wherein said at least two siRNAs comprise a first optimized
siRNA and a second optimized siRNA, wherein said first optimized
siRNA and said second optimized siRNA are optimized according a
formula comprising Formula X.
[0018] The present invention also provides a method for identifying
a hyperfunctional siRNA, comprising applying selection criteria to
a set of potential siRNA that comprise 18-30 base pairs, wherein
said selection criteria are non-target specific criteria, and said
set comprises at least two siRNAs and each of said at least two
siRNAs contains a sequence that is at least substantially
complementary to a target gene; determining the relative
functionality of the at least two siRNAs and assigning each of the
at least two siRNAs a functionality score; and selecting siRNAs
from the at least two siRNAs that have a functionality score that
reflects greater than 80 percent silencing at a concentration in
the picomolar range, wherein said greater than 80 percent silencing
endures for greater than 120 hours.
[0019] According to a sixth embodiment, the present invention
provides a hyperfunctional siRNA that is capable of silencing
Bcl2.
[0020] According to a seventh embodiment, the present invention
provides a method for developing an siRNA algorithm for selecting
functional and hyperfunctional siRNAs for a given sequence. The
method comprises:
[0021] (a) selecting a set of siRNAs;
[0022] (b) measuring the gene silencing ability of each siRNA from
said set;
[0023] (c) determining the relative functionality of each
siRNA;
[0024] (d) determining the amount of improved functionality by the
presence or absence of at least one variable selected from the
group consisting of the total GC content, melting temperature of
the siRNA, GC content at positions 15-19, the presence or absence
of a particular nucleotide at a particular position, relative
thermodynamic stability at particular positions in a duplex, and
the number of times that the same nucleotide repeats within a given
sequence; and
[0025] (e) developing an algorithm using the information of step
(d).
[0026] According to this embodiment, preferably the set of siRNAs
comprises at least 90 siRNAs from at least one gene, more
preferably at least 180 siRNAs from at least two different genes,
and most preferably at least 270 and 360 siRNAs from at least three
and four different genes, respectively. Additionally, in step (d)
the determination is made with preferably at least two, more
preferably at least three, even more preferably at least four, and
most preferably all of the variables. The resulting algorithm is
not target sequence specific.
[0027] In another embodiment, the present invention provides
rationally designed siRNAs identified using the formulas above.
[0028] In yet another embodiment, the present invention is directed
to hyperfunctional siRNA.
[0029] The ability to use the above algorithms, which are not
sequence or species specific, allows for the cost-effective
selection of optimized siRNAs for specific target sequences.
Accordingly, there will be both greater efficiency and reliability
in the use of siRNA technologies.
[0030] In various embodiments, siRNAs that target proprotein
convertase subtilisin/kexin type 9 (PCSK9 also known as LOC255738)
are provided. In various embodiments, the siRNAs are rationally
designed. In various embodiments, the siRNAs are functional or
hyperfunctional.
[0031] In various embodiments, an siRNA that targets PCSK9 is
provided, wherein the siRNA is selected from the group consisting
of various siRNA sequences targeting PCSK9 that are disclosed
herein. In various embodiments, the siRNA sequence is selected from
the group consisting of SEQ ID NO. 438 to SEQ ID NO. 565.
[0032] In various embodiments, siRNA comprising a sense region and
an antisense region are provided, said sense region and said
antisense region together form a duplex region comprising 18-30
base pairs, and said sense region comprises a sequence that is at
least 90% similar to a sequence selected from the group consisting
of siRNA sequences targeting PCSK9 that are disclosed herein. In
various embodiments, the siRNA sequence is selected from the group
consisting of SEQ ID NO. 438 to SEQ ID NO. 565.
[0033] In various embodiments, an siRNA comprising a sense region
and an antisense region is provided, said sense region and said
antisense region together form a duplex region comprising 18-30
base pairs, and said sense region comprises a sequence that is
identical to a contiguous stretch of at least 18 bases of a
sequence selected from the group consisting of SEQ ID NO. 438 to
SEQ ID NO. 565. In various embodiments, the duplex region is 19-30
base pairs, and the sense region comprises a sequence that is
identical to a sequence selected from the group consisting of SEQ
ID NO. 438 to SEQ ID NO. 565.
[0034] In various embodiments, a pool of at least two siRNAs is
provided, wherein said pool comprises a first siRNA and a second
siRNA, said first siRNA comprising a duplex region of length 18-30
base pairs that has a first sense region that is at least 90%
similar to 18 bases of a first sequence selected from the group
consisting of SEQ ID NO. 438 to SEQ ID NO. 565, and said second
siRNA comprises a duplex region of length 18-30 base pairs that has
a second sense region that is at least 90% similar to 18 bases of a
second sequence selected from the group consisting of SEQ ID NO.
438 to SEQ ID NO. 565, wherein said first sense region and said
second sense region are not identical.
[0035] In various embodiments, the first sense region comprises a
sequence that is identical to at least 18 bases of a sequence
selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO.
565, and said second sense region comprises a sequence that is
identical to at least 18 bases of a sequence selected from the
group consisting of SEQ ID NO. 438 to SEQ ID NO. 565. In various
embodiments, the duplex of said first siRNA is 19-30 base pairs,
and said first sense region comprises a sequence that is at least
90% similar to a sequence selected from the group consisting of SEQ
ID NO. 438 to SEQ ID NO. 565, and said duplex of said second siRNA
is 19-30 base pairs and comprises a sequence that is at least 90%
similar to a sequence selected from the group consisting of SEQ ID
NO. 438 to SEQ ID NO. 565.
[0036] In various embodiments, the duplex of said first siRNA is
19-30 base pairs and said first sense region comprises a sequence
that is identical to at least 18 bases of a sequence selected from
the group consisting of SEQ ID NO. 438 to SEQ ID NO. 565, and said
duplex of said second siRNA is 19-30 base pairs and said second
region comprises a sequence that is identical to a sequence
selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO.
565.
[0037] 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 which is set forth in the appended
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 shows a model for siRNA-RISC interactions. RISC has
the ability to interact with either end of the siRNA or miRNA
molecule. Following binding, the duplex is unwound, and the
relevant target is identified, cleaved, and released.
[0039] FIG. 2 is a representation of the functionality of two
hundred and seventy siRNA duplexes that were generated to target
human cyclophilin, human diazepam-binding inhibitor (DB), and
firefly luciferase.
[0040] FIG. 3a is a representation of the silencing effect of 30
siRNAs in three different cells lines, HEK293, DU145, and Hela.
FIG. 3b shows the frequency of different functional groups (>95%
silencing (black), >80% silencing (gray), >50% silencing
(dark gray), and <50% silencing (white)) based on GC content. In
cases where a given bar is absent from a particular GC percentage,
no siRNA were identified for that particular group. FIG. 3c shows
the frequency of different functional groups based on melting
temperature (Tm).
[0041] FIG. 4 is a representation of a statistical analysis that
revealed correlations between silencing and five sequence-related
properties of siRNA: (A) an A at position 19 of the sense strand,
(B) an A at position 3 of the sense strand, (C) a U at position 10
of the sense strand, (D) a base other than G at position 13 of the
sense strand, and (E) a base other than C at position 19 of the
sense strand. All variables were correlated with siRNA silencing of
firefly luciferase and human cyclophilin. siRNAs satisfying the
criterion are grouped on the left (Selected) while those that do
not, are grouped on the right (Eliminated). Y-axis is "% Silencing
of Control." Each position on the X-axis represents a unique
siRNA.
[0042] FIGS. 5A and 5B are representations of firefly luciferase
and cyclophilin siRNA panels sorted according to functionality and
predicted values using Formula VIII. The siRNA found within the
circle represent those that have Formula VIII values
(SMARTSCORES.TM., or siRNA rank) above zero. siRNA outside the
indicated area have calculated Formula VIII values that are below
zero. Y-axis is "Expression (% Control)." Each position on the
X-axis represents a unique siRNA.
[0043] FIG. 6A is a representation of the average internal
stability profile (AISP) derived from 270 siRNAs taken from three
separate genes (cyclophilin B, DBI and firefly luciferase). Graphs
represent AISP values of highly functional, functional, and
non-functional siRNA. FIG. 6B is a comparison between the AISP of
naturally derived GFP siRNA (filled squares) and the AISP of siRNA
from cyclophilin B, DBI, and luciferase having >90% silencing
properties (no fill) for the antisense strand. "DG" is the symbol
for .DELTA.G, free energy.
[0044] FIG. 7 is a histogram showing the differences in duplex
functionality upon introduction of base pair mismatches. The X-axis
shows the mismatch introduced in the siRNA and the position it is
introduced (e.g., 8C>A reveals that position 8 (which normally
has a C) has been changed to an A). The Y-axis is "% Silencing
(Normalized to Control)." The samples on the X-axis represent
siRNAs at 100 nM and are, reading from left to right: 1A to C, 1A
to G, 1A to U; 2A to C, 2A to G, 2A to U; 3A to C, 3A to G, 3A to
U; 4G to A, 4G to C; 4G to U; 5U to A, 5U to C, 5U to G; 6U to A,
6U to C, 6U to G; 7G to A, 7G to C, 7G to U; 8C to A, 8C to G, 8C
to U; 9G to A, 9G to C, 9G to U; 10C to A, 10C to G, 10C to U; 11G
to A, 11G to C, 11G to U; 12G to A, 12G to C, 12G to U; 13A to C,
13A to G, 13A to U; 14G to A, 14G to C, 14G to U; 15G to A, 15G to
C, 15G to U; 16A to C, 16A to G, 16A to U; 17G to A, 17G to C, 17G
to U; 18U to A, 18U to C, 18U to G; 19U to A, 19U to C, 19U to G;
20 wt; Control.
[0045] FIG. 8A is histogram that shows the effects of 5'sense and
antisense strand modification with 2'-O-methylation on
functionality. FIG. 8B is an expression profile showing a
comparison of sense strand off-target effects for IGFIR-3 and
2'-O-methyl IGFIR-3. Sense strand off-targets (lower box) are not
induced when the 5' end of the sense strand is modified with
2'-O-methyl groups (top box).
[0046] FIG. 9 shows a graph of SMARTSCORES.TM., or siRNA rank,
versus RNAi silencing values for more than 360 siRNA directed
against 30 different genes. SiRNA to the right of the vertical bar
represent those siRNA that have desirable SMARTSCORES.TM., or siRNA
rank.
[0047] FIGS. 10A-E compare the RNAi of five different genes (SEAP,
DBI, PLK, Firefly Luciferase, and Renilla Luciferase) by varying
numbers of randomly selected siRNA and four rationally designed
(SMART-selected) siRNA chosen using the algorithm described in
Formula VIII. In addition, RNAi induced by a pool of the four
SMART-selected siRNA is reported at two different concentrations
(100 and 400 nM). 10F is a comparison between a pool of randomly
selected EGFR siRNA (Pool 1) and a pool of SMART-selected EGFR
siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4 represent the
individual members that made up each respective pool. Note that
numbers for random siRNAs represent the position of the 5' end of
the sense strand of the duplex. The Y-axis represents the %
expression of the control(s). The X-axis is the percent expression
of the control.
[0048] FIG. 11 shows the Western blot results from cells treated
with siRNA directed against twelve different genes involved in the
clathrin-dependent endocytosis pathway (CHC, Dynil, CALM, CLCa,
CLCb, Eps15, Eps15R, Rab5a, Rab5b, Rab5c, .beta.2 subunit of AP-2
and EEA.1). siRNA were selected using Formula VIII. "Pool"
represents a mixture of duplexes 1-4. Total concentration of each
siRNA in the pool is 25 nM. Total concentration=4.times.25=100
nM.
[0049] FIG. 12 is a representation of the gene silencing
capabilities of rationally-selected siRNA directed against ten
different genes (human and mouse cyclophilin, C-myc, human lamin
A/C, QB (ubiquinol-cytochrome c reductase core protein 1), MEK1 and
MEK2, ATE1 (arginyl-tRNA protein transferase), GAPDH, and Eg5). The
Y-axis is the percent expression of the control. Numbers 1, 2, 3
and 4 represent individual rationally selected siRNA. "Pool"
represents a mixture of the four individual siRNA.
[0050] FIG. 13 is the sequence of the top ten Bcl2 siRNAs as
determined by Formula VIII. Sequences are listed 5' to 3'.
[0051] FIG. 14 is the knockdown by the top ten Bcl2 siRNAs at 100
nM concentrations. The Y-axis represents the amount of expression
relative to the non-specific (ns) and transfection mixture
control.
[0052] FIG. 15 represents a functional walk where siRNA beginning
on every other base pair of a region of the luciferase gene are
tested for the ability to silence the luciferase gene. The Y-axis
represents the percent expression relative to a control. The X-axis
represents the position of each individual siRNA. Reading from left
to right across the X-axis, the position designations are 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,
75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.
[0053] FIGS. 16A and 16B are histograms demonstrating the
inhibition of target gene expression by pools of 2 (16A) and 3
(16B) siRNA duplexes taken from the walk described in FIG. 15. The
Y-axis in each represents the percent expression relative to
control. The X-axis in each represents the position of the first
siRNA in paired pools, or trios of siRNAs. For instance, the first
paired pool contains siRNAs 1 and 3. The second paired pool
contains siRNAs 3 and 5. Pool 3 (of paired pools) contains siRNAs 5
and 7, and so on. For each of 16A and 16B, the X-axis from left to
right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,
65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and
Plasmid.
[0054] FIGS. 17A and 17B are histograms demonstrating the
inhibition of target gene expression by pools of 4 (17A) and 5
(17B) siRNA duplexes. The Y-axis in each represents the percent
expression relative to control. The X-axis in each represents the
position of the first siRNA in each pool. For each of 17A and 17B,
the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, and Plasmid.
[0055] FIGS. 18A and 18B are histograms demonstrating the
inhibition of target gene expression by siRNAs that are ten (18A)
and twenty (18B) base pairs base pairs apart. The Y-axis represents
the percent expression relative to a control. The X-axis represents
the position of the first siRNA in each pool. For each of 18A and
18B, the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,
85, 87, 89, and Plasmid.
[0056] FIG. 19 shows that pools of siRNAs (dark gray bar) work as
well (or better) than the best siRNA in the pool (light gray bar).
The Y-axis represents the percent expression relative to a control.
The X-axis represents the position of the first siRNA in each pool.
The X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, and Plasmid.
[0057] FIG. 20 shows that the combination of several semifunctional
siRNAs (dark gray) result in a significant improvement of gene
expression inhibition over individual (semi-functional; light gray)
siRNA. The Y-axis represents the percent expression relative to a
control.
[0058] FIGS. 21A, 21B and 21C show both pools (Library, Lib) and
individual siRNAs in inhibition of gene expression of
Beta-Galactosidase, Renilla Luciferase and SEAP (alkaline
phosphatase). Numbers on the X-axis indicate the position of the
5'-most nucleotide of the sense strand of the duplex. The Y-axis
represents the percent expression of each gene relative to a
control. Libraries contain 19 nucleotide long siRNAs (not including
overhangs) that begin at the following nucleotides: SEAP: Lib 1:
206, 766, 812, 923, Lib 2: 1117, 1280, 1300, 1487, Lib 3: 206, 766,
812, 923, 1117, 1280, 1300, 1487, Lib 4: 206, 812, 1117, 1300, Lib
5: 766, 923, 1280, 1487, Lib 6: 206, 1487; Bgal: Lib 1: 979, 1339,
2029, 2590, Lib 2: 1087, 1783, 2399, 3257, Lib 3: 979, 1783, 2590,
3257, Lib 4: 979, 1087, 1339, 1783, 2029, 2399, 2590, 3257, Lib 5:
979, 1087, 1339, 1783, Lib 6: 2029, 2399, 2590, 3257; Renilla: Lib
1: 174, 300, 432, 568, Lib 2: 592, 633, 729, 867, Lib 3: 174, 300,
432, 568, 592, 633, 729, 867, Lib 4: 174, 432, 592, 729, Lib 5:
300, 568, 633, 867, Lib 6: 592,568.
[0059] FIG. 22 shows the results of an EGFR and TfnR
internalization assay when single gene knockdowns are performed.
The Y-axis represents percent internalization relative to
control.
[0060] FIG. 23 shows the results of an EGFR and TfnR
internalization assay when multiple genes are knocked down (e.g.,
Rab5a, b, c). The Y-axis represents the percent internalization
relative to control.
[0061] FIG. 24 shows the simultaneous knockdown of four different
genes. siRNAs directed against G6PD, GAPDH, PLK, and UQC were
simultaneously introduced into cells. Twenty-four hours later,
cultures were harvested and assayed for mRNA target levels for each
of the four genes. A comparison is made between cells transfected
with individual siRNAs vs. a pool of siRNAs directed against all
four genes.
[0062] FIG. 25 shows the functionality of ten siRNAs at 0.3 nM
concentrations.
DETAILED DESCRIPTION
Definitions
[0063] Unless stated otherwise, the following terms and phrases
have the meanings provided below:
Complementary
[0064] 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. Complementary polynucleotide
strands 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 duplexes. As persons skilled in the art are aware, when using
RNA as opposed to DNA, uracil rather than thymine is the base that
is considered to be complementary to adenosine. However, when a U
is denoted in the context of the present invention, the ability to
substitute a T is implied, unless otherwise stated.
[0065] Perfect complementarity or 100% complementarity refers to
the situation in which each nucleotide unit of one polynucleotide
strand can hydrogen bond with a nucleotide unit of a second
polynucleotide strand. Less than perfect complementarity refers to
the situation in which some, but not all, nucleotide units of two
strands 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 exhibit 10%
complementarity. In the same example, if 18 base pairs on each
strand can hydrogen bond with each other, the polynucleotide
strands exhibit 90% complementarity.
Deoxynucleotide
[0066] The term "deoxynucleotide" refers to a nucleotide or
polynucleotide lacking a hydroxyl group (OH group) at the 2' and/or
3' position of a sugar moiety. Instead, it has a hydrogen bonded to
the 2' and/or 3' carbon. Within an RNA molecule that comprises one
or more deoxynucleotides, "deoxynucleotide" refers to the lack of
an OH group at the 2' position of the sugar moiety, having instead
a hydrogen bonded directly to the 2' carbon.
Deoxyribonucleotide
[0067] The terms "deoxyribonucleotide" and "DNA" refer to a
nucleotide or polynucleotide comprising at least one sugar moiety
that has an H, rather than an OH, at its 2' and/or 3'position.
Duplex Region
[0068] The phrase "duplex region" refers to the region in two
complementary or substantially complementary polynucleotides that
form base pairs with one another, either by Watson-Crick base
pairing or any other manner that allows for a stabilized duplex
between polynucleotide strands that are complementary or
substantially complementary. For example, a polynucleotide strand
having 21 nucleotide units can base pair with another
polynucleotide of 21 nucleotide units, yet only 19 bases on each
strand are complementary or substantially complementary, such that
the "duplex region" has 19 base pairs. The remaining bases may, for
example, exist as 5' and 3' overhangs. Further, within the duplex
region, 100% complementarity is not required; substantial
complementarity is allowable within a duplex region. Substantial
complementarity refers to 79% or greater complementarity. For
example, a mismatch in a duplex region consisting of 19 base pairs
results in 94.7% complementarity, rendering the duplex region
substantially complementary.
Filters
[0069] The term "filter" refers to one or more procedures that are
performed on sequences that are identified by the algorithm. In
some instances, filtering includes in silico procedures where
sequences identified by the algorithm can be screened to identify
duplexes carrying desirable or undesirable motifs. Sequences
carrying such motifs can be selected for, or selected against, to
obtain a final set with the preferred properties. In other
instances, filtering includes wet lab experiments. For instance,
sequences identified by one or more versions of the algorithm can
be screened using any one of a number of procedures to identify
duplexes that have hyperfunctional traits (e.g., they exhibit a
high degree of silencing at subnanomolar concentrations and/or
exhibit high degrees of silencing longevity).
Gene Silencing
[0070] The phrase "gene silencing" refers to a process by which the
expression of a specific gene product is lessened or attenuated.
Gene silencing can take place by a variety of pathways. Unless
specified otherwise, as used herein, gene silencing refers to
decreases in gene product expression that results from RNA
interference (RNAi), a defined, though partially characterized
pathway whereby small inhibitory RNA (siRNA) act in concert with
host proteins (e.g., the RNA induced silencing complex, RISC) to
degrade messenger RNA (mRNA) in a sequence-dependent fashion. The
level of gene silencing can be measured by a variety of means,
including, but not limited to, measurement of transcript levels by
Northern Blot Analysis, B-DNA techniques, transcription-sensitive
reporter constructs, expression profiling (e.g., DNA chips), and
related technologies. Alternatively, the level of silencing can be
measured by assessing the level of the protein encoded by a
specific gene. This can be accomplished by performing a number of
studies including Western Analysis, measuring the levels of
expression of a reporter protein that has e.g., fluorescent
properties (e.g., GFP) or enzymatic activity (e.g., alkaline
phosphatases), or several other procedures.
miRNA
[0071] The term "miRNA" refers to microRNA.
Nucleotide
[0072] 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.
[0073] 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. 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.
[0074] Modified bases refer to nucleotide bases such as, for
example, adenine, guanine, cytosine, thymine, 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, individually or in combination. More specific
examples 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.
[0075] 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. The term "nucleotide" is also meant to include the N3'
to P5' phosphoramidate, resulting from the substitution of a
ribosyl 3' oxygen with an amine group.
[0076] 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.
Off-Target Silencing and Off-Target Interference
[0077] The phrases "off-target silencing" and "off-target
interference" are defined as degradation of mRNA other than the
intended target mRNA due to overlapping and/or partial homology
with secondary mRNA messages.
Polynucleotide
[0078] 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/or irregularly
alternating deoxyribosyl moieties and ribosyl moieties (i.e.,
wherein alternate nucleotide units have an --OH, then and --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.
Polyribonucleotide
[0079] The term "polyribonucleotide" refers to a polynucleotide
comprising two or more modified or unmodified ribonucleotides
and/or their analogs. The term "polyribonucleotide" is used
interchangeably with the term "oligoribonucleotide."
Ribonucleotide and Ribonucleic Acid
[0080] 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 hydroxyl group attached to the 2'
position of a ribosyl moiety that has 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.
siRNA
[0081] The term "siRNA" refers to small inhibitory RNA duplexes
that induce the RNA interference (RNAi) pathway. These molecules
can vary in length (generally 18-30 base pairs) and contain varying
degrees of complementarity to their target mRNA in the antisense
strand. Some, but not all, siRNA have unpaired overhanging bases on
the 5' or 3' end of the sense strand and/or the antisense strand.
The term "siRNA" includes duplexes of two separate strands, as well
as single strands that can form hairpin structures comprising a
duplex region.
[0082] 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.
SMARTSCORE.TM., or siRNA Rank
[0083] The term "SMARTSCORE.TM.", or "siRNA rank" refers to a
number determined by applying any of the formulas to a given siRNA
sequence. The term "SMART-selected" or "rationally selected" or
"rational selection" refers to siRNA that have been selected on the
basis of their SMARTSCORES.TM., or siRNA ranking.
Substantially Similar
[0084] The phrase "substantially similar" refers to a similarity of
at least 90% with respect to the identity of the bases of the
sequence.
Target
[0085] The term "target" is used in a variety of different forms
throughout this document and is defined by the context in which it
is used. "Target mRNA" refers to a messenger RNA to which a given
siRNA can be directed against. "Target sequence" and "target site"
refer to a sequence within the mRNA to which the sense strand of an
siRNA shows varying degrees of homology and the antisense strand
exhibits varying degrees of complementarity. The phrase "siRNA
target" can refer to the gene, mRNA, or protein against which an
siRNA is directed. Similarly, "target silencing" can refer to the
state of a gene, or the corresponding mRNA or protein.
Transfection
[0086] The term "transfection" refers to a process by which agents
are introduced into a cell. The list of agents that can be
transfected is large and includes, but is not limited to, siRNA,
sense and/or anti-sense sequences, DNA encoding one or more genes
and organized into an expression plasmid, proteins, protein
fragments, and more. There are multiple methods for transfecting
agents into a cell including, but not limited to, electroporation,
calcium phosphate-based transfections, DEAE-dextran-based
transfections, lipid-based transfections, molecular conjugate-based
transfections (e.g., polylysine-DNA conjugates), microinjection and
others.
[0087] The present invention is directed to improving the
efficiency of gene silencing by siRNA. Through the inclusion of
multiple siRNA sequences that are targeted to a particular gene
and/or selecting an siRNA sequence based on certain defined
criteria, improved efficiency may be achieved.
[0088] 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.
[0089] Furthermore, this disclosure is not a primer on RNA
interference. Basic concepts known to persons skilled in the art
have not been set forth in detail.
[0090] The present invention is directed to increasing the
efficiency of RNAi, particularly in mammalian systems. Accordingly,
the present invention provides kits, siRNAs and methods for
increasing siRNA efficacy.
[0091] According to a first embodiment, the present invention
provides a kit for gene silencing, wherein said kit is comprised of
a pool of at least two siRNA duplexes, each of which is comprised
of a sequence that is complementary to a portion of the sequence of
one or more target messenger RNA, and each of which is selected
using non-target specific criteria. Each of the at least two siRNA
duplexes of the kit complementary to a portion of the sequence of
one or more target mRNAs is preferably selected using Formula
X.
[0092] According to a second embodiment, the present invention
provides a method for selecting an siRNA, said method comprising
applying selection criteria to a set of potential siRNA that
comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene; and
determining the relative functionality of the at least two
siRNAs.
[0093] In one embodiment, the present invention also provides a
method wherein said selection criteria are embodied in a formula
comprising:
(-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-1-
0*C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-
9*U.sub.9-9*C.sub.18-8*G.sub.10-7*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.-
19+7*U.sub.17+8*A.sub.2+8*A.sub.4+8*A.sub.5+8*C.sub.4
+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.9+15*G.sub.1+18*A-
.sub.3+19*U.sub.10-Tm-3*(GC.sub.total)
-6*(GC.sub.15-19)-30*X; or Formula VIII
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+
(19)*A7+(6)*A8+(-4)*A9+(-5)*A10+(-2)*A11+(-5)*A12+(17)*A13+(-
3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(-13)*U1+(-
10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8+(-
5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-
10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-
9)*C5+(-20)*C6+(-18)*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-
5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-5)*C16+(-3)*C17+(-12)*C18+(-
18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-
4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-
24)*G13+(18)*G14+(11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+
6*(number of A+U in position 15-19)-3*(number of G+C in whole
siRNA), Formula X
[0094] wherein position numbering begins at the 5'-most position of
a sense strand, and
[0095] A.sub.1=1 if A is the base at position 1 of the sense
strand, otherwise its value is 0;
[0096] A.sub.2=1 if A is the base at position 2 of the sense
strand, otherwise its value is 0;
[0097] A.sub.3=1 if A is the base at position 3 of the sense
strand, otherwise its value is 0;
[0098] A.sub.4=1 if A is the base at position 4 of the sense
strand, otherwise its value is 0;
[0099] A.sub.5=1 if A is the base at position 5 of the sense
strand, otherwise its value is 0;
[0100] A.sub.6=1 if A is the base at position 6 of the sense
strand, otherwise its value is 0;
[0101] A.sub.7=1 if A is the base at position 7 of the sense
strand, otherwise its value is 0;
[0102] A.sub.10=1 if A is the base at position 10 of the sense
strand, otherwise its value is 0;
[0103] A.sub.11=1 if A is the base at position 11 of the sense
strand, otherwise its value is 0;
[0104] A.sub.13=1 if A is the base at position 13 of the sense
strand, otherwise its value is 0;
[0105] A.sub.19=1 if A is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0106] C.sub.3=1 if C is the base at position 3 of the sense
strand, otherwise its value is 0;
[0107] C.sub.4=1 if C is the base at position 4 of the sense
strand, otherwise its value is 0;
[0108] C.sub.5=1 if C is the base at position 5 of the sense
strand, otherwise its value is 0;
[0109] C.sub.6=1 if C is the base at position 6 of the sense
strand, otherwise its value is 0;
[0110] C.sub.7=1 if C is the base at position 7 of the sense
strand, otherwise its value is 0;
[0111] C.sub.9=1 if C is the base at position 9 of the sense
strand, otherwise its value is 0;
[0112] C.sub.17=1 if C is the base at position 17 of the sense
strand, otherwise its value is 0;
[0113] C.sub.18=1 if C is the base at position 18 of the sense
strand, otherwise its value is 0;
[0114] C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0115] G.sub.1=1 if G is the base at position 1 on the sense
strand, otherwise its value is 0;
[0116] G.sub.2=1 if G is the base at position 2 of the sense
strand, otherwise its value is 0;
[0117] G.sub.8=1 if G is the base at position 8 on the sense
strand, otherwise its value is 0;
[0118] G.sub.10=1 if G is the base at position 10 on the sense
strand, otherwise its value is 0;
[0119] G.sub.13=1 if G is the base at position 13 on the sense
strand, otherwise its value is 0;
[0120] G.sub.19=1 if G is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0121] U.sub.1=1 if U is the base at position 1 on the sense
strand, otherwise its value is 0;
[0122] U.sub.2=1 if U is the base at position 2 on the sense
strand, otherwise its value is 0;
[0123] U.sub.3=1 if U is the base at position 3 on the sense
strand, otherwise its value is 0;
[0124] U.sub.4=1 if U is the base at position 4 on the sense
strand, otherwise its value is 0;
[0125] U.sub.7=1 if U is the base at position 7 on the sense
strand, otherwise its value is 0;
[0126] U.sub.9=1 if U is the base at position 9 on the sense
strand, otherwise its value is 0;
[0127] U.sub.10=1 if U is the base at position 10 on the sense
strand, otherwise its value is 0;
[0128] U.sub.15=1 if U is the base at position 15 on the sense
strand, otherwise its value is 0;
[0129] U.sub.16=1 if U is the base at position 16 on the sense
strand, otherwise its value is 0;
[0130] U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0;
[0131] U.sub.18=1 if U is the base at position 18 on the sense
strand, otherwise its value is 0.
[0132] GC.sub.15-19=the number of G and C bases within positions
15-19 of the sense strand, or within positions 15-18 if the sense
strand is only 18 base pairs in length;
[0133] GC.sub.total=the number of G and C bases in the sense
strand;
[0134] Tm=100 if the siRNA oligo has the internal repeat longer
then 4 base pairs, otherwise its value is 0; and
[0135] X=the number of times that the same nucleotide repeats four
or more times in a row.
[0136] Any of the methods of selecting siRNA in accordance with the
invention can further comprise comparing the internal stability
profiles of the siRNAs to be selected, and selecting those siRNAs
with the most favorable internal stability profiles. Any of the
methods of selecting siRNA can further comprise selecting either
for or against sequences that contain motifs that induce cellular
stress. Such motifs include, for example, toxicity motifs. Any of
the methods of selecting siRNA can further comprise either
selecting for or selecting against sequences that comprise
stability motifs.
[0137] In another embodiment, the present invention provides a
method of gene silencing, comprising introducing into a cell at
least one siRNA selected according to any of the methods of the
present invention. The siRNA can be introduced by allowing passive
uptake of siRNA, or through the use of a vector.
[0138] According to a third embodiment, the invention provides a
method for developing an algorithm for selecting siRNA, said method
comprising: (a) selecting a set of siRNA; (b) measuring gene
silencing ability of each siRNA from said set; (c) determining
relative functionality of each siRNA; (d) determining improved
functionality by the presence or absence of at least one variable
selected from the group consisting of the presence or absence of a
particular nucleotide at a particular position, the total number of
As and Us in positions 15-19, the number of times that the same
nucleotide repeats within a given sequence, and the total number of
Gs and Cs; and (e) developing an algorithm using the information of
step (d).
[0139] In another embodiment, the invention provides a method for
selecting an siRNA with improved functionality, comprising using
the above-mentioned algorithm to identify an siRNA of improved
functionality.
[0140] According to a fourth embodiment, the present invention
provides a kit, wherein said kit is comprised of at least two
siRNAs, wherein said at least two siRNAs comprise a first optimized
siRNA and a second optimized siRNA, wherein said first optimized
siRNA and said second optimized siRNA are optimized according a
formula comprising Formula X.
[0141] According to a fifth embodiment, the present invention
provides a method for identifying a hyperfunctional siRNA,
comprising applying selection criteria to a set of potential siRNA
that comprise 18-30 base pairs, wherein said selection criteria are
non-target specific criteria, and said set comprises at least two
siRNAs and each of said at least two siRNAs contains a sequence
that is at least substantially complementary to a target gene;
determining the relative functionality of the at least two siRNAs
and assigning each of the at least two siRNAs a functionality
score; and selecting siRNAs from the at least two siRNAs that have
a functionality score that reflects greater than 80 percent
silencing at a concentration in the picomolar range, wherein said
greater than 80 percent silencing endures for greater than 120
hours.
[0142] In other embodiments, the invention provides kits and/or
methods wherein the siRNA are comprised of two separate
polynucleotide strands; wherein the siRNA are comprised of a single
contiguous molecule such as, for example, a unimolecular siRNA
(comprising, for example, either a nucleotide or non-nucleotide
loop); wherein the siRNA are expressed from one or more vectors;
and wherein two or more genes are silenced by a single
administration of siRNA.
[0143] According to a sixth embodiment, the present invention
provides a hyperfunctional siRNA that is capable of silencing
Bcl2.
[0144] According to a seventh embodiment, the present invention
provides a method for developing an siRNA algorithm for selecting
functional and hyperfunctional siRNAs for a given sequence. The
method comprises:
[0145] (a) selecting a set of siRNAs;
[0146] (b) measuring the gene silencing ability of each siRNA from
said set;
[0147] (c) determining the relative functionality of each
siRNA;
[0148] (d) determining the amount of improved functionality by the
presence or absence of at least one variable selected from the
group consisting of the total GC content, melting temperature of
the siRNA, GC content at positions 15-19, the presence or absence
of a particular nucleotide at a particular position, relative
thermodynamic stability at particular positions in a duplex, and
the number of times that the same nucleotide repeats within a given
sequence; and
[0149] (e) developing an algorithm using the information of step
(d).
[0150] According to this embodiment, preferably the set of siRNAs
comprises at least 90 siRNAs from at least one gene, more
preferably at least 180 siRNAs from at least two different genes,
and most preferably at least 270 and 360 siRNAs from at least three
and four different genes, respectively. Additionally, in step (d)
the determination is made with preferably at least two, more
preferably at least three, even more preferably at least four, and
most preferably all of the variables. The resulting algorithm is
not target sequence specific.
[0151] In another embodiment, the present invention provides
rationally designed siRNAs identified using the formulas above.
[0152] In yet another embodiment, the present invention is directed
to hyperfunctional siRNA.
[0153] The ability to use the above algorithms, which are not
sequence or species specific, allows for the cost-effective
selection of optimized siRNAs for specific target sequences.
Accordingly, there will be both greater efficiency and reliability
in the use of siRNA technologies.
[0154] The methods disclosed herein can be used in conjunction with
comparing internal stability profiles of selected siRNAs, and
designing an siRNA with a desirable internal stability profile;
and/or in conjunction with a selection either for or against
sequences that contain motifs that induce cellular stress, for
example, cellular toxicity.
[0155] Any of the methods disclosed herein can be used to silence
one or more genes by introducing an siRNA selected, or designed, in
accordance with any of the methods disclosed herein. The siRNA(s)
can be introduced into the cell by any method known in the art,
including passive uptake or through the use of one or more
vectors.
[0156] Any of the methods and kits disclosed herein can employ
either unimolecular siRNAs, siRNAs comprised of two separate
polynucleotide strands, or combinations thereof. Any of the methods
disclosed herein can be used in gene silencing, where two or more
genes are silenced by a single administration of siRNA(s). The
siRNA(s) can be directed against two or more target genes, and
administered in a single dose or single transfection, as the case
may be.
Optimizing siRNA
[0157] According to one embodiment, the present invention provides
a method for improving the effectiveness of gene silencing for use
to silence a particular gene through the selection of an optimal
siRNA. An siRNA selected according to this method may be used
individually, or in conjunction with the first embodiment, i.e.,
with one or more other siRNAs, each of which may or may not be
selected by this criteria in order to maximize their
efficiency.
[0158] The degree to which it is possible to select an siRNA for a
given mRNA that maximizes these criteria will depend on the
sequence of the mRNA itself. However, the selection criteria will
be independent of the target sequence. According to this method, an
siRNA is selected for a given gene by using a rational design. That
said, rational design can be described in a variety of ways.
Rational design is, in simplest terms, the application of a proven
set of criteria that enhance the probability of identifying a
functional or hyperfunctional siRNA. In one method, rationally
designed siRNA can be identified by maximizing one or more of the
following criteria:
[0159] (1) A low GC content, preferably between about 30-52%.
[0160] (2) At least 2, preferably at least 3 A or U bases at
positions 15-19 of the siRNA on the sense strand.
[0161] (3) An A base at position 19 of the sense strand.
[0162] (4) An A base at position 3 of the sense strand.
[0163] (5) A U base at position 10 of the sense strand.
[0164] (6) An A base at position 14 of the sense strand.
[0165] (7) A base other than C at position 19 of the sense
strand.
[0166] (8) A base other than G at position 13 of the sense
strand.
[0167] (9) A Tm, which refers to the character of the internal
repeat that results in inter- or intramolecular structures for one
strand of the duplex, that is preferably not stable at greater than
50.degree. C., more preferably not stable at greater than
37.degree. C., even more preferably not stable at greater than
30.degree. C. and most preferably not stable at greater than
20.degree. C.
[0168] (10) A base other than U at position 5 of the sense
strand.
[0169] (11) A base other than A at position 11 of the sense
strand.
[0170] (12) A base other than an A at position 1 of the sense
strand.
[0171] (13) A base other than an A at position 2 of the sense
strand.
[0172] (14) An A base at position 4 of the sense strand.
[0173] (15) An A base at position 5 of the sense strand.
[0174] (16) An A base at position 6 of the sense strand.
[0175] (17) An A base at position 7 of the sense strand.
[0176] (18) An A base at position 8 of the sense strand.
[0177] (19) A base other than an A at position 9 of the sense
strand.
[0178] (20) A base other than an A at position 10 of the sense
strand.
[0179] (21) A base other than an A at position 11 of the sense
strand.
[0180] (22) A base other than an A at position 12 of the sense
strand.
[0181] (23) An A base at position 13 of the sense strand.
[0182] (24) A base other than an A at position 14 of the sense
strand.
[0183] (25) An A base at position 15 of the sense strand
[0184] (26) An A base at position 16 of the sense strand.
[0185] (27) An A base at position 17 of the sense strand.
[0186] (28) An A base at position 18 of the sense strand.
[0187] (29) A base other than a U at position 1 of the sense
strand.
[0188] (30) A base other than a U at position 2 of the sense
strand.
[0189] (31) A U base at position 3 of the sense strand.
[0190] (32) A base other than a U at position 4 of the sense
strand.
[0191] (33) A base other than a U at position 5 of the sense
strand.
[0192] (34) A U base at position 6 of the sense strand.
[0193] (35) A base other than a U at position 7 of the sense
strand.
[0194] (36) A base other than a U at position 8 of the sense
strand.
[0195] (37) A base other than a U at position 9 of the sense
strand.
[0196] (38) A base other than a U at position 1 of the sense
strand.
[0197] (39) A U base at position 13 of the sense strand.
[0198] (40) A base other than a U at position 14 of the sense
strand.
[0199] (41) A base other than a U at position 15 of the sense
strand.
[0200] (42) A base other than a U at position 16 of the sense
strand.
[0201] (43) A U base at position 17 of the sense strand.
[0202] (44) A U base at position 18 of the sense strand.
[0203] (45) A U base at position 19 of the sense strand.
[0204] (46) A C base at position 1 of the sense strand.
[0205] (47) A C base at position 2 of the sense strand.
[0206] (48) A base other than a C at position 3 of the sense
strand.
[0207] (49) A C base at position 4 of the sense strand.
[0208] (50) A base other than a C at position 5 of the sense
strand.
[0209] (51) A base other than a C at position 6 of the sense
strand.
[0210] (52) A base other than a C at position 7 of the sense
strand.
[0211] (53) A base other than a C at position 8 of the sense
strand.
[0212] (54) A C base at position 9 of the sense strand.
[0213] (55) A C base at position 10 of the sense strand.
[0214] (56) A C base at position 11 of the sense strand.
[0215] (57) A base other than a C at position 12 of the sense
strand.
[0216] (58) A base other than a C at position 13 of the sense
strand.
[0217] (59) A base other than a C at position 14 of the sense
strand.
[0218] (60) A base other than a C at position 15 of the sense
strand.
[0219] (61) A base other than a C at position 16 of the sense
strand.
[0220] (62) A base other than a C at position 17 of the sense
strand.
[0221] (63) A base other than a C at position 18 of the sense
strand.
[0222] (64) A G base at position 1 of the sense strand.
[0223] (65) A G base at position 2 of the sense strand.
[0224] (66) A G base at position 3 of the sense strand.
[0225] (67) A base other than a G at position 4 of the sense
strand.
[0226] (68) A base other than a G at position 5 of the sense
strand.
[0227] (69) A G base at position 6 of the sense strand.
[0228] (70) A G base at position 7 of the sense strand.
[0229] (71) A G base at position 8 of the sense strand.
[0230] (72) A G base at position 9 of the sense strand.
[0231] (73) A base other than a G at position 10 of the sense
strand.
[0232] (74) A G base at position 11 of the sense strand.
[0233] (75) A G base at position 12 of the sense strand.
[0234] (76) A G base at position 14 of the sense strand.
[0235] (77) A G base at position 15 of the sense strand.
[0236] (78) A G base at position 16 of the sense strand.
[0237] (79) A base other than a G at position 17 of the sense
strand.
[0238] (80) A base other than a G at position 18 of the sense
strand.
[0239] (81) A base other than a G at position 19 of the sense
strand.
[0240] The importance of various criteria can vary greatly. For
instance, a C base at position 10 of the sense strand makes a minor
contribution to duplex functionality. In contrast, the absence of a
C at position 3 of the sense strand is very important. Accordingly,
preferably an siRNA will satisfy as many of the aforementioned
criteria as possible.
[0241] With respect to the criteria, GC content, as well as a high
number of AU in positions 15-19 of the sense strand, may be
important for easement of the unwinding of double stranded siRNA
duplex. Duplex unwinding has been shown to be crucial for siRNA
functionality in vivo.
[0242] With respect to criterion 9, the internal structure is
measured in terms of the melting temperature of the single strand
of siRNA, which is the temperature at which 50% of the molecules
will become denatured. With respect to criteria 2-8 and 10-11, the
positions refer to sequence positions on the sense strand, which is
the strand that is identical to the mRNA.
[0243] In one preferred embodiment, at least criteria 1 and 8 are
satisfied. In another preferred embodiment, at least criteria 7 and
8 are satisfied. In still another preferred embodiment, at least
criteria 1, 8 and 9 are satisfied.
[0244] It should be noted that all of the aforementioned criteria
regarding sequence position specifics are with respect to the 5'
end of the sense strand. Reference is made to the sense strand,
because most databases contain information that describes the
information of the mRNA. Because according to the present invention
a chain can be from 18 to 30 bases in length, and the
aforementioned criteria assumes a chain 19 base pairs in length, it
is important to keep the aforementioned criteria applicable to the
correct bases.
[0245] When there are only 18 bases, the base pair that is not
present is the base pair that is located at the 3' of the sense
strand. When there are twenty to thirty bases present, then
additional bases are added at the 5' end of the sense chain and
occupy positions .sup.-1 to .sup.-11. Accordingly, with respect to
SEQ. ID NO. 0001 NNANANNNNUCNAANNNNA and SEQ. ID NO. 0028
GUCNNANANNNNUCNAANNNNA, both would have A at position 3, A at
position 5, U at position 10, C at position 11, A and position 13,
A and position 14 and A at position 19. However, SEQ. ID NO. 0028
would also have C at position -1, U at position -2 and G at
position -3.
[0246] For a 19 base pair siRNA, an optimal sequence of one of the
strands may be represented below, where N is any base, A, C, G, or
U:
TABLE-US-00001 SEQ. ID NO. 0001. NNANANNNNUCNAANNNNA SEQ. ID NO.
0002. NNANANNNNUGNAANNNNA SEQ. ID NO. 0003. NNANANNNNUUNAANNNNA
SEQ. ID NO. 0004. NNANANNNNUCNCANNNNA SEQ. ID NO. 0005.
NNANANNNNUGNCANNNNA SEQ. ID NO. 0006. NNANANNNNUUNCANNNNA SEQ. ID
NO. 0007. NNANANNNNUCNUANNNNA SEQ. ID NO. 0008. NNANANNNNUGNUANNNNA
SEQ. ID NO. 0009. NNANANNNNUUNUANNNNA SEQ. ID NO. 0010.
NNANCNNNNUCNAANNNNA SEQ. ID NO. 0011. NNANCNNNNUGNAANNNNA SEQ. ID
NO. 0012. NNANCNNNNUUNAANNNNA SEQ. ID NO. 0013. NNANCNNNNUCNCANNNNA
SEQ. ID NO. 0014. NNANCNNNNUGNCANNNNA SEQ. ID NO. 0015.
NNANCNNNNUUNCANNNNA SEQ. ID NO. 0016. NNANCNNNNUCNUANNNNA SEQ. ID
NO. 0017. NNANCNNNNUGNUANNNNA SEQ. ID NO. 0018. NNANCNNNNUUNUANNNNA
SEQ. ID NO. 0019. NNANGNNNNUCNAANNNNA SEQ. ID NO. 0020.
NNANGNNNNUGNAANNNNA SEQ. ID NO. 0021. NNANGNNNNUUNAANNNNA SEQ. ID
NO. 0022. NNANGNNNNUCNCANNNNA SEQ. ID NO. 0023. NNANGNNNNUGNCANNNNA
SEQ. ID NO. 0024. NNANGNNNNUUNCANNNNA SEQ. ID NO. 0025.
NNANGNNNNUCNUANNNNA SEQ. ID NO. 0026. NNANGNNNNUGNUANNNNA SEQ. ID
NO. 0027. NNANGNNNNNUNUANNNNA
[0247] In one embodiment, the sequence used as an siRNA is selected
by choosing the siRNA that score highest according to one of the
following seven algorithms that are represented by Formulas
I-VI:
Relative functionality of
siRNA=-(GC/3)+(AU.sub.15-19)-(Tm.sub.20.degree.C)*3-(G.sub.13)*3-(C.sub.1-
9)+(A.sub.19)*2+(A.sub.3)+(U.sub.10)+(A.sub.14)-(U.sub.5)-(A.sub.11)
Formula I
Relative functionality of
siRNA=-(GC/3)-(AU.sub.15-19)*3-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.su-
b.3) Formula II
Relative functionality of
siRNA=-(GC/3)+(AU.sub.15-19)-(Tm.sub.20.degree.C)*3 Formula III
Relative functionality of
siRNA=-GC/2+(AU.sub.15-19)/2-(Tm.sub.20.degree.C)*2-(G.sub.13)*3-(C.sub.1-
9)+(A.sub.19)*2+(A.sub.3)+(U.sub.10)+(A.sub.14)-(U.sub.5)-(A.sub.11)
Formula IV
Relative functionality of
siRNA=-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.sub.3)+(U.sub.10)+(A.sub.1-
4)-(U.sub.5)-(A.sub.11) Formula V
Relative functionality of
siRNA=-(G.sub.13)*3-(C.sub.19)+(A.sub.19)*2+(A.sub.3) Formula
VI
Relative functionality of
siRNA=-(GC/2)+(AU.sub.15-19)/2-(Tm.sub.20.degree.C)*1-(G.sub.13)*3-(C.sub-
.19)+(A.sub.19)*3+(A.sub.3)*3+(U.sub.10)/2+(A.sub.14)/2-(U.sub.5)/2-(A.sub-
.11)/2 Formula VII
[0248] In Formulas I-VII:
[0249] wherein A.sub.19=1 if A is the base at position 19 on the
sense strand, otherwise its value is 0,
[0250] AU.sub.15-19=0-5 depending on the number of A or U bases on
the sense strand at positions 15-19;
[0251] G.sub.13=1 if G is the base at position 13 on the sense
strand, otherwise its value is 0;
[0252] C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise its value is 0;
[0253] GC=the number of G and C bases in the entire sense
strand;
[0254] Tm.sub.20.degree.C=1 if the Tm is greater than 20.degree.
C.;
[0255] A.sub.3=1 if A is the base at position 3 on the sense
strand, otherwise its value is 0;
[0256] U.sub.10=1 if U is the base at position 10 on the sense
strand, otherwise its value is 0;
[0257] A.sub.14=1 if A is the base at position 14 on the sense
strand, otherwise its value is 0;
[0258] U.sub.5=1 if U is the base at position 5 on the sense
strand, otherwise its value is 0; and
[0259] A.sub.11=1 if A is the base at position 11 of the sense
strand, otherwise its value is 0.
[0260] Formulas I-VII provide relative information regarding
functionality. When the values for two sequences are compared for a
given formula, the relative functionality is ascertained; a higher
positive number indicates a greater functionality. For example, in
many applications a value of 5 or greater is beneficial.
[0261] Additionally, in many applications, more than one of these
formulas would provide useful information as to the relative
functionality of potential siRNA sequences. However, it is
beneficial to have more than one type of formula, because not every
formula will be able to help to differentiate among potential siRNA
sequences. For example, in particularly high GC mRNAs, formulas
that take that parameter into account would not be useful and
application of formulas that lack GC elements (e.g., formulas V and
VI) might provide greater insights into duplex functionality.
Similarly, formula II might by used in situations where hairpin
structures are not observed in duplexes, and formula IV might be
applicable for sequences that have higher AU content. Thus, one may
consider a particular sequence in light of more than one or even
all of these algorithms to obtain the best differentiation among
sequences. In some instances, application of a given algorithm may
identify an unusually large number of potential siRNA sequences,
and in those cases, it may be appropriate to re-analyze that
sequence with a second algorithm that is, for instance, more
stringent. Alternatively, it is conceivable that analysis of a
sequence with a given formula yields no acceptable siRNA sequences
(i.e. low SMARTSCORES.TM., or siRNA ranking). In this instance, it
may be appropriate to re-analyze that sequences with a second
algorithm that is, for instance, less stringent. In still other
instances, analysis of a single sequence with two separate formulas
may give rise to conflicting results (i.e. one formula generates a
set of siRNA with high SMARTSCORES.TM., or siRNA ranking, while the
other formula identifies a set of siRNA with low SMARTSCORES.TM.,
or siRNA ranking). In these instances, it may be necessary to
determine which weighted factor(s) (e.g. GC content) are
contributing to the discrepancy and assessing the sequence to
decide whether these factors should or should not be included.
Alternatively, the sequence could be analyzed by a third, fourth,
or fifth algorithm to identify a set of rationally designed
siRNA.
[0262] The above-referenced criteria are particularly advantageous
when used in combination with pooling techniques as depicted in
Table I:
TABLE-US-00002 TABLE I FUNCTIONAL PROBABILITY OLIGOS POOLS CRITERIA
>95% >80% <70% >95% >80% <70% CURRENT 33.0 50.0
23.0 79.5 97.3 0.3 NEW 50.0 88.5 8.0 93.8 99.98 0.005 (GC) 28.0
58.9 36.0 72.8 97.1 1.6
[0263] The term "current" used in Table I refers to Tuschl's
conventional siRNA parameters (Elbashir, S. M. et al. (2002)
"Analysis of gene function in somatic mammalian cells using small
interfering RNAs" Methods 26: 199-213). "New" refers to the design
parameters described in Formulas I-VII. "GC" refers to criteria
that select siRNA solely on the basis of GC content.
[0264] As Table I indicates, when more functional siRNA duplexes
are chosen, siRNAs that produce <70% silencing drops from 23% to
8% and the number of siRNA duplexes that produce >80% silencing
rises from 50% to 88.5%. Further, of the siRNA duplexes with
>80% silencing, a larger portion of these siRNAs actually
silence >95% of the target expression (the new criteria
increases the portion from 33% to 50%). Using this new criteria in
pooled siRNAs, shows that, with pooling, the amount of silencing
>95% increases from 79.5% to 93.8% and essentially eliminates
any siRNA pool from silencing less than 70%.
[0265] Table II similarly shows the particularly beneficial results
of pooling in combination with the aforementioned criteria.
However, Table II, which takes into account each of the
aforementioned variables, demonstrates even a greater degree of
improvement in functionality.
TABLE-US-00003 TABLE II FUNCTIONAL PROBABILITY OLIGOS POOLS NON-
NON- FUNCTIONAL AVERAGE FUNCTIONAL FUNCTIONAL AVERAGE FUNCTIONAL
RANDOM 20 40 50 67 97 3 CRITERIA 1 52 99 0.1 97 93 0.0040 CRITERIA
4 89 99 0.1 99 99 0.0000
[0266] The terms "functional," "Average," and "Non-functional" used
in Table II, refer to siRNA that exhibit >80%, >50%, and
<50% functionality, respectively. Criteria 1 and 4 refer to
specific criteria described above.
[0267] The above-described algorithms may be used with or without a
computer program that allows for the inputting of the sequence of
the mRNA and automatically outputs the optimal siRNA. The computer
program may, for example, be accessible from a local terminal or
personal computer, over an internal network or over the
Internet.
[0268] In addition to the formulas above, more detailed algorithms,
may be used for selecting siRNA. Preferably, at least one RNA
duplex of 18-30 base pairs is selected such that it is optimized
according a formula selected from:
(-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-1-
0*C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-
9*U.sub.9-9*C.sub.18-8*G.sub.10-7*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.-
19+7*U.sub.17+8*A.sub.2+8*A.sub.4+8*A.sub.5+8*C.sub.4
+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.9+15*G.sub.1+18*A-
.sub.3+19*U.sub.10-Tm-3*(GC.sub.total)
-6*(GC.sub.15-19)-30*X; and Formula VIII
(14.1)*A.sub.3+(14.9)*A.sub.6+(17.6)*A.sub.13+(24.7)*A.sub.19+(14.2)*U.s-
ub.10+(10.5)*
C.sub.9+(23.9)*G.sub.1+(16.3)*G.sub.2+(-12.3)*A.sub.11+(-19.3)*U.sub.1+(-
-12.1)*U.sub.2+(-
11)*U.sub.3+(-15.2)*U.sub.15+(-11.3)*U.sub.16+(-11.8)*C.sub.3+(-17.4)*C.-
sub.6+(-10.5)*C.sub.7+
(-13.7)*G.sub.13+(-25.9)*G.sub.19-Tm-3*(GC.sub.total)-6*(GC.sub.15-19)-3-
0*X; and Formula IX
(-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+
(19)*A7+(6)*A8+(-4)*A9+(-5)*A10+(-2)*A11+(-5)*A12+(17)*A13+(-
3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(-13)*U1+(-
10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8+(-
5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-
10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-
9)*C5+(-20)*C6+(-18)*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-
5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-5)*C16+(-3)*C17+(-12)*C18+(-
18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-
4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-
24)*G13+(18)*G14+(11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+
6*(number of A+U in position 15-19)-3*(number of G+C in whole
siRNA). Formula X
[0269] wherein
[0270] A.sub.1=1 if A is the base at position 1 of the sense
strand, otherwise its value is 0;
[0271] A.sub.2=1 if A is the base at position 2 of the sense
strand, otherwise its value is 0;
[0272] A.sub.3=1 if A is the base at position 3 of the sense
strand, otherwise its value is 0;
[0273] A.sub.4=1 if A is the base at position 4 of the sense
strand, otherwise its value is 0;
[0274] A.sub.5=1 if A is the base at position 5 of the sense
strand, otherwise its value is 0;
[0275] A.sub.6=1 if A is the base at position 6 of the sense
strand, otherwise its value is 0;
[0276] A.sub.7=1 if A is the base at position 7 of the sense
strand, otherwise its value is 0;
[0277] A.sub.10=1 if A is the base at position 10 of the sense
strand, otherwise its value is 0;
[0278] A.sub.11=1 if A is the base at position 11 of the sense
strand, otherwise its value is 0;
[0279] A.sub.13=1 if A is the base at position 13 of the sense
strand, otherwise its value is 0;
[0280] A.sub.19=1 if A is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0281] C.sub.3=1 if C is the base at position 3 of the sense
strand, otherwise its value is 0;
[0282] C.sub.4=1 if C is the base at position 4 of the sense
strand, otherwise its value is 0;
[0283] C.sub.5=1 if C is the base at position 5 of the sense
strand, otherwise its value is 0;
[0284] C.sub.6=1 if C is the base at position 6 of the sense
strand, otherwise its value is 0;
[0285] C.sub.7=1 if C is the base at position 7 of the sense
strand, otherwise its value is 0;
[0286] C.sub.9=1 if C is the base at position 9 of the sense
strand, otherwise its value is 0;
[0287] C.sub.17=1 if C is the base at position 17 of the sense
strand, otherwise its value is 0;
[0288] C.sub.18=1 if C is the base at position 18 of the sense
strand, otherwise its value is 0;
[0289] C.sub.19=1 if C is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0290] G.sub.1=1 if G is the base at position 1 on the sense
strand, otherwise its value is 0;
[0291] G.sub.2=1 if G is the base at position 2 of the sense
strand, otherwise its value is 0;
[0292] G.sub.8=1 if G is the base at position 8 on the sense
strand, otherwise its value is 0;
[0293] G.sub.10=1 if G is the base at position 10 on the sense
strand, otherwise its value is 0;
[0294] G.sub.13=1 if G is the base at position 13 on the sense
strand, otherwise its value is 0;
[0295] G.sub.19=1 if G is the base at position 19 of the sense
strand, otherwise if another base is present or the sense strand is
only 18 base pairs in length, its value is 0;
[0296] U.sub.1=1 if U is the base at position 1 on the sense
strand, otherwise its value is 0;
[0297] U.sub.2=1 if U is the base at position 2 on the sense
strand, otherwise its value is 0;
[0298] U.sub.3=1 if U is the base at position 3 on the sense
strand, otherwise its value is 0;
[0299] U.sub.4=1 if U is the base at position 4 on the sense
strand, otherwise its value is 0;
[0300] U.sub.7=1 if U is the base at position 7 on the sense
strand, otherwise its value is 0;
[0301] U.sub.9=1 if U is the base at position 9 on the sense
strand, otherwise its value is 0;
[0302] U.sub.10=1 if U is the base at position 10 on the sense
strand, otherwise its value is 0;
[0303] U.sub.15=1 if U is the base at position 15 on the sense
strand, otherwise its value is 0;
[0304] U.sub.16=1 if U is the base at position 16 on the sense
strand, otherwise its value is 0;
[0305] U.sub.17=1 if U is the base at position 17 on the sense
strand, otherwise its value is 0;
[0306] U.sub.18=1 if U is the base at position 18 on the sense
strand, otherwise its value is 0;
[0307] GC.sub.15-19=the number of G and C bases within positions
15-19 of the sense strand, or within positions 15-18 if the sense
strand is only 18 base pairs in length;
[0308] GC.sub.total=the number of G and C bases in the sense
strand;
[0309] Tm=100 if the siRNA oligo has the internal repeat longer
then 4 base pairs, otherwise its value is 0; and
[0310] X=the number of times that the same nucleotide repeats four
or more times in a row.
[0311] The above formulas VIII, IX, and X, as well as formulas
I-VII, provide methods for selecting siRNA in order to increase the
efficiency of gene silencing. A subset of variables of any of the
formulas may be used, though when fewer variables are used, the
optimization hierarchy becomes less reliable.
[0312] With respect to the variables of the above-referenced
formulas, a single letter of A or C or G or U followed by a
subscript refers to a binary condition. The binary condition is
that either the particular base is present at that particular
position (wherein the value is "1") or the base is not present
(wherein the value is "0"). Because position 19 is optional, i.e.,
there might be only 18 base pairs, when there are only 18 base
pairs, any base with a subscript of 19 in the formulas above would
have a zero value for that parameter. Before or after each variable
is a number followed by *, which indicates that the value of the
variable is to be multiplied or weighed by that number.
[0313] The numbers preceding the variables A, or G, or C, or U in
Formulas VIII, IX, and X (or after the variables in Formula I-VII)
were determined by comparing the difference in the frequency of
individual bases at different positions in functional siRNA and
total siRNA. Specifically, the frequency in which a given base was
observed at a particular position in functional groups was compared
with the frequency that that same base was observed in the total,
randomly selected siRNA set. If the absolute value of the
difference between the functional and total values was found to be
greater than 6%, that parameter was included in the equation. Thus,
for instance, if the frequency of finding a "G" at position 13
(G.sub.13) is found to be 6% in a given functional group, and the
frequency of G.sub.13 in the total population of siRNAs is 20%, the
difference between the two values is 6%-20%-14%. As the absolute
value is greater than six (6), this factor (-14) is included in the
equation. Thus, in Formula VIII, in cases where the siRNA under
study has a G in position 13, the accrued value is (-14)*(1)=-14.
In contrast, when a base other than G is found at position 13, the
accrued value is (-14)*(0)=0.
[0314] When developing a means to optimize siRNAs, the inventors
observed that a bias toward low internal thermodynamic stability of
the duplex at the 5'-antisense (AS) end is characteristic of
naturally occurring miRNA precursors. The inventors extended this
observation to siRNAs for which functionality had been assessed in
tissue culture.
[0315] With respect to the parameter GC.sub.15-19, a value of 0-5
will be ascribed depending on the number of G or C bases at
positions 15 to 19. If there are only 18 base pairs, the value is
between 0 and 4.
[0316] With respect to the criterion GC.sub.total content, a number
from 0-30 will be ascribed, which correlates to the total number of
G and C nucleotides on the sense strand, excluding overhangs.
Without wishing to be bound by any one theory, it is postulated
that the significance of the GC content (as well as AU content at
positions 15-19, which is a parameter for formulas III-VII) relates
to the easement of the unwinding of a double-stranded siRNA duplex.
Duplex unwinding is believed to be crucial for siRNA functionality
in vivo and overall low internal stability, especially low internal
stability of the first unwound base pair is believed to be
important to maintain sufficient processivity of RISC
complex-induced duplex unwinding. If the duplex has 19 base pairs,
those at positions 15-19 on the sense strand will unwind first if
the molecule exhibits a sufficiently low internal stability at that
position. As persons skilled in the art are aware, RISC is a
complex of approximately twelve proteins; Dicer is one, but not the
only, helicase within this complex. Accordingly, although the GC
parameters are believed to relate to activity with Dicer, they are
also important for activity with other RISC proteins.
[0317] The value of the parameter Tm is 0 when there are no
internal repeats longer than (or equal to) four base pairs present
in the siRNA duplex; otherwise the value is 1. Thus for example, if
the sequence ACGUACGU, or any other four nucleotide (or more)
palindrome exists within the structure, the value will be one (1).
Alternatively if the structure ACGGACG, or any other 3 nucleotide
(or less) palindrome exists, the value will be zero (0).
[0318] The variable "X" refers to the number of times that the same
nucleotide occurs contiguously in a stretch of four or more units.
If there are, for example, four contiguous As in one part of the
sequence and elsewhere in the sequence four contiguous Cs, X=2.
Further, if there are two separate contiguous stretches of four of
the same nucleotides or eight or more of the same nucleotides in a
row, then X=2. However, X does not increase for five, six or seven
contiguous nucleotides.
[0319] Again, when applying Formula VIII, Formula IX, or Formula X,
to a given mRNA, (the "target RNA" or "target molecule"), one may
use a computer program to evaluate the criteria for every sequence
of 18-30 base pairs or only sequences of a fixed length, e.g., 19
base pairs. Preferably the computer program is designed such that
it provides a report ranking of all of the potential siRNAs 18-30
base pairs, ranked according to which sequences generate the
highest value. A higher value refers to a more efficient siRNA for
a particular target gene. The computer program that may be used may
be developed in any computer language that is known to be useful
for scoring nucleotide sequences, or it may be developed with the
assistance of commercially available product such as Microsoft's
PRODUCT.NET. Additionally, rather than run every sequence through
one and/or another formula, one may compare a subset of the
sequences, which may be desirable if for example only a subset are
available. For instance, it may be desirable to first perform a
BLAST (Basic Local Alignment Search Tool) search and to identify
sequences that have no homology to other targets. Alternatively, it
may be desirable to scan the sequence and to identify regions of
moderate GC context, then perform relevant calculations using one
of the above-described formulas on these regions. These
calculations can be done manually or with the aid of a
computer.
[0320] As with Formulas I-VII, either Formula VIII, Formula IX, or
Formula X may be used for a given mRNA target sequence. However, it
is possible that according to one or the other formula more than
one siRNA will have the same value. Accordingly, it is beneficial
to have a second formula by which to differentiate sequences.
Formulas IX and X were derived in a similar fashion as Formula
VIII, yet used a larger data set and thus yields sequences with
higher statistical correlations to highly functional duplexes. The
sequence that has the highest value ascribed to it may be referred
to as a "first optimized duplex." The sequence that has the second
highest value ascribed to it may be referred to as a "second
optimized duplex." Similarly, the sequences that have the third and
fourth highest values ascribed to them may be referred to as a
third optimized duplex and a fourth optimized duplex, respectively.
When more than one sequence has the same value, each of them may,
for example, be referred to as first optimized duplex sequences or
co-first optimized duplexes. Formula X is similar to Formula IX,
yet uses a greater numbers of variables and for that reason,
identifies sequences on the basis of slightly different
criteria.
[0321] It should also be noted that the output of a particular
algorithm will depend on several of variables including: (1) the
size of the data base(s) being analyzed by the algorithm, and (2)
the number and stringency of the parameters being applied to screen
each sequence. Thus, for example, in U.S. patent application Ser.
No. 10/714,333, entitled "Functional and Hyperfunctional siRNA,"
filed Nov. 14, 2003, Formula VIII was applied to the known human
genome (NCBI REFSEQ database) through ENTREZ (EFETCH). As a result
of these procedures, roughly 1.6 million siRNA sequences were
identified. Application of Formula VIII to the same database in
March of 2004 yielded roughly 2.2 million sequences, a difference
of approximately 600,000 sequences resulting from the growth of the
database over the course of the months that span this period of
time. Application of other formulas (e.g., Formula X) that change
the emphasis of, include, or eliminate different variables can
yield unequal numbers of siRNAs. Alternatively, in cases where
application of one formula to one or more genes fails to yield
sufficient numbers of siRNAs with scores that would be indicative
of strong silencing, said genes can be reassessed with a second
algorithm that is, for instance, less stringent.
[0322] siRNA sequences identified using Formula VIII and Formula X
(minus sequences generated by Formula VIII) are contained within
the sequence listing. The data included in the sequence listing is
described more fully below. The sequences identified by Formula
VIII and Formula X that are disclosed in the sequence listing may
be used in gene silencing applications.
[0323] It should be noted that for Formulas VIII, IX, and X all of
the aforementioned criteria are identified as positions on the
sense strand when oriented in the 5' to 3' direction as they are
identified in connection with Formulas I-VII unless otherwise
specified.
[0324] Formulas I-X, may be used to select or to evaluate one, or
more than one, siRNA in order to optimize silencing. Preferably, at
least two optimized siRNAs that have been selected according to at
least one of these formulas are used to silence a gene, more
preferably at least three and most preferably at least four. The
siRNAs may be used individually or together in a pool or kit.
Further, they may be applied to a cell simultaneously or
separately. Preferably, the at least two siRNAs are applied
simultaneously. Pools are particularly beneficial for many research
applications. However, for therapeutics, it may be more desirable
to employ a single hyperfunctional siRNA as described elsewhere in
this application.
[0325] When planning to conduct gene silencing, and it is necessary
to choose between two or more siRNAs, one should do so by comparing
the relative values when the siRNA are subjected to one of the
formulas above. In general a higher scored siRNA should be
used.
[0326] Useful applications include, but are not limited to, target
validation, gene functional analysis, research and drug discovery,
gene therapy and therapeutics. Methods for using siRNA in these
applications are well known to persons of skill in the art.
[0327] Because the ability of siRNA to function is dependent on the
sequence of the RNA and not the species into which it is
introduced, the present invention is applicable across a broad
range of species, including but not limited to all mammalian
species, such as humans, dogs, horses, cats, cows, mice, hamsters,
chimpanzees and gorillas, as well as other species and organisms
such as bacteria, viruses, insects, plants and C. elegans.
[0328] The present invention is also applicable for use for
silencing a broad range of genes, including but not limited to the
roughly 45,000 genes of a human genome, and has particular
relevance in cases where those genes are associated with diseases
such as diabetes, Alzheimer's, cancer, as well as all genes in the
genomes of the aforementioned organisms.
[0329] The siRNA selected according to the aforementioned criteria
or one of the aforementioned algorithms are also, for example,
useful in the simultaneous screening and functional analysis of
multiple genes and gene families using high throughput strategies,
as well as in direct gene suppression or silencing.
Development of the Algorithms
[0330] To identify siRNA sequence features that promote
functionality and to quantify the importance of certain currently
accepted conventional factors--such as G/C content and target site
accessibility--the inventors synthesized an siRNA panel consisting
of 270 siRNAs targeting three genes, Human Cyclophilin, Firefly
Luciferase, and Human DBI. In all three cases, siRNAs were directed
against specific regions of each gene. For Human Cyclophilin and
Firefly Luciferase, ninety siRNAs were directed against a 199 bp
segment of each respective mRNA. For DBI, 90 siRNAs were directed
against a smaller, 109 base pair region of the mRNA. The sequences
to which the siRNAs were directed are provided below.
[0331] It should be noted that in certain sequences, "t" is
present. This is because many databases contain information in this
manner. However, the t denotes a uracil residue in in RNA and
siRNA. Any algorithm will, unless otherwise specified, process at
in a sequence as a u.
TABLE-US-00004 Human cyclophilin: 193-390, M60857 SEQ. ID NO. 29:
gttccaaaaa cagtggataa ttttgtggcc ttagctacag gagagaaagg atttggctac
aaaaacagca aattccatcg tgtaatcaag gacttcatga tccagggcgg agacttcacc
aggggagatg gcacaggagg aaagagcatc tacggtgagc gcttccccga tgagaacttc
aaactgaagc actacgggcc tggctggg Firefly luciferase: 1434-1631,
U47298 (pGL3, Promega) SEQ. ID NO. 30: tgaacttccc gccgccgttg
ttgttttgga gcacggaaag acgatgacgg aaaaagagat cgtggattac gtcgccagtc
aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa gtaccgaaag
gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat cctcataaag gccaagaagg
DRI, NM_020548 (202-310) (every position) SEQ. ID NO. 0031:
acgggcaagg ccaagtggga tgcctggaat gagctgaaag ggacttccaa ggaagatgcc
atgaaagctt acatcaacaa agtagaagag ctaaagaaaa aatacggg
[0332] A list of the siRNAs appears in Table III (see Examples
Section, Example II)
[0333] The set of duplexes was analyzed to identify correlations
between siRNA functionality and other biophysical or thermodynamic
properties. When the siRNA panel was analyzed in functional and
non-functional subgroups, certain nucleotides were much more
abundant at certain positions in functional or non-functional
groups. More specifically, the frequency of each nucleotide at each
position in highly functional siRNA duplexes was compared with that
of nonfunctional duplexes in order to assess the preference for or
against any given nucleotide at every position. These analyses were
used to determine important criteria to be included in the siRNA
algorithms (Formulas VIII, IX, and X).
[0334] The data set was also analyzed for distinguishing
biophysical properties of siRNAs in the functional group, such as
optimal percent of GC content, propensity for internal structures
and regional thermodynamic stability. Of the presented criteria,
several are involved in duplex recognition, RISC activation/duplex
unwinding, and target cleavage catalysis.
[0335] The original data set that was the source of the
statistically derived criteria is shown in FIG. 2. Additionally,
this figure shows that random selection yields siRNA duplexes with
unpredictable and widely varying silencing potencies as measured in
tissue culture using HEK293 cells. In the figure, duplexes are
plotted such that each x-axis tick-mark represents an individual
siRNA, with each subsequent siRNA differing in target position by
two nucleotides for Human Cyclophilin B and Firefly Luciferase, and
by one nucleotide for Human DBI. Furthermore, the y-axis denotes
the level of target expression remaining after transfection of the
duplex into cells and subsequent silencing of the target.
[0336] siRNA identified and optimized in this document work equally
well in a wide range of cell types. FIG. 3a shows the evaluation of
thirty siRNAs targeting the DBI gene in three cell lines derived
from different tissues. Each DBI siRNA displays very similar
functionality in HEK293 (ATCC, CRL-1573, human embryonic kidney),
HeLa (ATCC, CCL-2, cervical epithelial adenocarcinoma) and DU145
(HTB-81, prostate) cells as determined by the B-DNA assay. Thus,
siRNA functionality is determined by the primary sequence of the
siRNA and not by the intracellular environment. Additionally, it
should be noted that although the present invention provides for a
determination of the functionality of siRNA for a given target, the
same siRNA may silence more than one gene. For example, the
complementary sequence of the silencing siRNA may be present in
more than one gene. Accordingly, in these circumstances, it may be
desirable not to use the siRNA with highest SMARTSCORE.TM., or
siRNA ranking. In such circumstances, it may be desirable to use
the siRNA with the next highest SMARTSCORE.TM., or siRNA
ranking.
[0337] To determine the relevance of G/C content in siRNA function,
the G/C content of each duplex in the panel was calculated and the
functional classes of siRNAs (<F50, .gtoreq.F50, .gtoreq.F80,
.gtoreq.F95 where F refers to the percent gene silencing) were
sorted accordingly. The majority of the highly-functional siRNAs
(.gtoreq.F95) fell within the G/C content range of 36%-52% (FIG.
3B). Twice as many non-functional (<F50) duplexes fell within
the high G/C content groups (>57% GC content) compared to the
36%-52% group. The group with extremely low GC content (26% or
less) contained a higher proportion of non-functional siRNAs and no
highly-functional siRNAs. The G/C content range of 30%-52% was
therefore selected as Criterion I for siRNA functionality,
consistent with the observation that a G/C range 30%-70% promotes
efficient RNAi targeting. Application of this criterion alone
provided only a marginal increase in the probability of selecting
functional siRNAs from the panel: selection of F50 and F95 siRNAs
was improved by 3.6% and 2.2%, respectively. The siRNA panel
presented here permitted a more systematic analysis and
quantification of the importance of this criterion than that used
previously.
[0338] A relative measure of local internal stability is the A/U
base pair (bp) content; therefore, the frequency of A/U bp was
determined for each of the five terminal positions of the duplex
(5' sense (S)/5' antisense (AS)) of all siRNAs in the panel.
Duplexes were then categorized by the number of A/U bp in positions
1-5 and 15-19 of the sense strand. The thermodynamic flexibility of
the duplex 5'-end (positions 1-5; S) did not appear to correlate
appreciably with silencing potency, while that of the 3'-end
(positions 15-19; S) correlated with efficient silencing. No
duplexes lacking A/U bp in positions 15-19 were functional. The
presence of one A/U bp in this region conferred some degree of
functionality, but the presence of three or more A/Us was
preferable and therefore defined as Criterion II. When applied to
the test panel, only a marginal increase in the probability of
functional siRNA selection was achieved: a 1.8% and 2.3% increase
for F50 and F95 duplexes, respectively (Table IV).
[0339] The complementary strands of siRNAs that contain internal
repeats or palindromes may form internal fold-back structures.
These hairpin-like structures exist in equilibrium with the
duplexed form effectively reducing the concentration of functional
duplexes. The propensity to form internal hairpins and their
relative stability can be estimated by predicted melting
temperatures. High Tm reflects a tendency to form hairpin
structures. Lower Tm values indicate a lesser tendency to form
hairpins. When the functional classes of siRNAs were sorted by
T.sub.m (FIG. 3c), the following trends were identified: duplexes
lacking stable internal repeats were the most potent silencers (no
F95 duplex with predicted hairpin structure T.sub.m>60.degree.
C.). In contrast, about 60% of the duplexes in the groups having
internal hairpins with calculated T.sub.m values less than
20.degree. C. were F80. Thus, the stability of internal repeats is
inversely proportional to the silencing effect and defines
Criterion III (predicted hairpin structure
T.sub.m.ltoreq.20.degree. C.).
Sequence-Based Determinants of siRNA Functionality
[0340] When the siRNA panel was sorted into functional and
non-functional groups, the frequency of a specific nucleotide at
each position in a functional siRNA duplex was compared with that
of a nonfunctional duplex in order to assess the preference for or
against a certain nucleotide. FIG. 4 shows the results of these
queries and the subsequent resorting of the data set (from FIG. 2).
The data is separated into two sets: those duplexes that meet the
criteria, a specific nucleotide in a certain position--grouped on
the left (Selected) and those that do not--grouped on the right
(Eliminated). The duplexes are further sorted from most functional
to least functional with the y-axis of FIG. 4a-e representing the %
expression i.e., the amount of silencing that is elicited by the
duplex (Note: each position on the X-axis represents a different
duplex). Statistical analysis revealed correlations between
silencing and several sequence-related properties of siRNAs. FIG. 4
and Table IV show quantitative analysis for the following five
sequence-related properties of siRNA: (A) an A at position 19 of
the sense strand; (B) an A at position 3 of the sense strand; (C) a
U at position 10 of the sense strand; (D) a base other than G at
position 13 of the sense strand; and (E) a base other than C at
position 19 of the sense strand.
[0341] When the siRNAs in the panel were evaluated for the presence
of an A at position 19 of the sense strand, the percentage of
non-functional duplexes decreased from 20% to 11.8%, and the
percentage of F95 duplexes increased from 21.7% to 29.4% (Table
IV). Thus, the presence of an A in this position defined Criterion
IV.
[0342] Another sequence-related property correlated with silencing
was the presence of an A in position 3 of the sense strand (FIG.
4b). Of the siRNAs with A3, 34.4% were F95, compared with 21.7%
randomly selected siRNAs. The presence of a U base in position 10
of the sense strand exhibited an even greater impact (FIG. 4c). Of
the duplexes in this group, 41.7% were F95. These properties became
criteria V and VI, respectively.
[0343] Two negative sequence-related criteria that were identified
also appear on FIG. 4. The absence of a G at position 13 of the
sense strand, conferred a marginal increase in selecting functional
duplexes (FIG. 4d). Similarly, lack of a C at position 19 of the
sense strand also correlated with functionality (FIG. 4e). Thus,
among functional duplexes, position 19 was most likely occupied by
A, and rarely occupied by C. These rules were defined as criteria
VII and VIII, respectively.
[0344] Application of each criterion individually provided marginal
but statistically significant increases in the probability of
selecting a potent siRNA. Although the results were informative,
the inventors sought to maximize potency and therefore consider
multiple criteria or parameters. Optimization is particularly
important when developing therapeutics. Interestingly, the
probability of selecting a functional siRNA based on each
thermodynamic criteria was 2%-4% higher than random, but 4%-8%
higher for the sequence-related determinates. Presumably, these
sequence-related increases reflect the complexity of the RNAi
mechanism and the multitude of protein-RNA interactions that are
involved in RNAi-mediated silencing.
TABLE-US-00005 TABLE IV IMPROVEMENT PERCENT OVER CRITERION
FUNCTIONAL RANDOM (%) I. 30%-52% G/C Content <F50 16.4 -3.6
.gtoreq.F50 83.6 3.6 .gtoreq.F80 60.4 4.3 .gtoreq.F95 23.9 2.2 II.
At least 3 A/U <F50 18.2 -1.8 bases at positions .gtoreq.F50
81.8 1.8 15-19 of the sense .gtoreq.F80 59.7 3.6 strand .gtoreq.F95
24.0 2.3 III. Absence of internal <F50 16.7 -3.3 repeats, as
measured .gtoreq.F50 83.3 3.3 by Tm of secondary .gtoreq.F80 61.1
5.0 structure .ltoreq.20.degree. C. .gtoreq.F95 24.6 2.9 IV. An A
base at <F50 11.8 -8.2 position 19 .gtoreq.F50 88.2 8.2 of the
sense strand .gtoreq.F80 75.0 18.9 .gtoreq.F95 29.4 7.7 V. An A
base at <F50 17.2 -2.8 position 3 of .gtoreq.F50 82.8 2.8 the
sense strand .gtoreq.F80 62.5 6.4 .gtoreq.F95 34.4 12.7 VI. A U
base at <F50 13.9 -6.1 position 10 of .gtoreq.F50 86.1 6.1 the
sense strand .gtoreq.F80 69.4 13.3 .gtoreq.F95 41.7 20 VII. A base
other than <F50 18.8 -1.2 C at position 19 .gtoreq.F50 81.2 1.2
of the sense strand .gtoreq.F80 59.7 3.6 .gtoreq.F95 24.2 2.5 VIII.
A base other than <F50 15.2 -4.8 G at position 13 .gtoreq.F50
84.8 4.8 of the sense strand .gtoreq.F80 61.4 5.3 .gtoreq.F95 26.5
4.8
The siRNA Selection Algorithm
[0345] In an effort to improve selection further, all identified
criteria, including but not limited to those listed in Table IV
were combined into the algorithms embodied in Formula VIII, Formula
IX, and Formula X. Each siRNA was then assigned a score (referred
to as a SMARTSCORE.TM., or siRNA ranking) according to the values
derived from the formulas. Duplexes that scored higher than 0 or
-20 (unadjusted), for Formulas VIII and IX, respectively,
effectively selected a set of functional siRNAs and excluded all
non-functional siRNAs. Conversely, all duplexes scoring lower than
0 and -20 (minus 20) according to formulas VIII and IX,
respectively, contained some functional siRNAs but included all
non-functional siRNAs. A graphical representation of this selection
is shown in FIG. 5. It should be noted that the scores derived from
the algorithm can also be provided as "adjusted" scores. To convert
Formula VIII unadjusted scores into adjusted scores it is necessary
to use the following equation:
(160+unadjusted score)/2.25
[0346] When this takes place, an unadjusted score of "0" (zero) is
converted to 75. Similarly, unadjusted scores for Formula X can be
converted to adjusted scores. In this instance, the following
equation is applied:
(228+unadjusted score)/3.56
[0347] When these manipulations take place, an unadjusted score of
38 is converted to an adjusted score of 75.
[0348] The methods for obtaining the seven criteria embodied in
Table IV are illustrative of the results of the process used to
develop the information for Formulas VIII, IX, and X. Thus similar
techniques were used to establish the other variables and their
multipliers. As described above, basic statistical methods were use
to determine the relative values for these multipliers.
[0349] To determine the value for "Improvement over Random" the
difference in the frequency of a given attribute (e.g., GC content,
base preference) at a particular position is determined between
individual functional groups (e.g., <F50) and the total siRNA
population studied (e.g., 270 siRNA molecules selected randomly).
Thus, for instance, in Criterion I (30%-52% GC content) members of
the <F50 group were observed to have GC contents between 30-52%
in 16.4% of the cases. In contrast, the total group of 270 siRNAs
had GC contents in this range, 20% of the time. Thus for this
particular attribute, there is a small negative correlation between
30%-52% GC content and this functional group (i.e.,
16.4%-20%=-3.6%). Similarly, for Criterion VI, (a "U" at position
10 of the sense strand), the >F95 group contained a "U" at this
position 41.7% of the time. In contrast, the total group of 270
siRNAs had a "U" at this position 21.7% of the time, thus the
improvement over random is calculated to be 20% (or
41.7%-21.7%).
Identifying the Average Internal Stability Profile of Strong
siRNA
[0350] In order to identify an internal stability profile that is
characteristic of strong siRNA, 270 different siRNAs derived from
the cyclophilin B, the diazepam binding inhibitor (DBI), and the
luciferase gene were individually transfected into HEK293 cells and
tested for their ability to induce RNAi of the respective gene.
Based on their performance in the in vivo assay, the sequences were
then subdivided into three groups, (i) >95% silencing; (ii)
80-95% silencing; and (iii) less than 50% silencing. Sequences
exhibiting 51-84% silencing were eliminated from further
consideration to reduce the difficulties in identifying relevant
thermodynamic patterns.
[0351] Following the division of siRNA into three groups, a
statistical analysis was performed on each member of each group to
determine the average internal stability profile (AISP) of the
siRNA. To accomplish this the Oligo 5.0 Primer Analysis Software
and other related statistical packages (e.g., Excel) were exploited
to determine the internal stability of pentamers using the nearest
neighbor method described by Freier et al., (1986) Improved
free-energy parameters for predictions of RNA duplex stability,
Proc Natl. Acad. Sci. USA 83(24): 9373-7. Values for each group at
each position were then averaged, and the resulting data were
graphed on a linear coordinate system with the Y-axis expressing
the .DELTA.G (free energy) values in kcal/mole and the X-axis
identifying the position of the base relative to the 5' end.
[0352] The results of the analysis identified multiple key regions
in siRNA molecules that were critical for successful gene
silencing. At the 3'-most end of the sense strand (5'antisense),
highly functional siRNA (>95% gene silencing, see FIG. 6a,
>F95) have a low internal stability (AISP of position
19=.about.-7.6 kcal/mol). In contrast low-efficiency siRNA (i.e.,
those exhibiting less than 50% silencing, <F50) display a
distinctly different profile, having high .DELTA.G values
(.about.-8.4 kcal/mol) for the same position. Moving in a 5' (sense
strand) direction, the internal stability of highly efficient siRNA
rises (position 12=.about.-8.3 kcal/mole) and then drops again
(position 7=.about.-7.7 kcal/mol) before leveling off at a value of
approximately -8.1 kcal/mol for the 5' terminus. siRNA with poor
silencing capabilities show a distinctly different profile. While
the AISP value at position 12 is nearly identical with that of
strong siRNAs, the values at positions 7 and 8 rise considerably,
peaking at a high of .about.-9.0 kcal/mol. In addition, at the 5'
end of the molecule the AISP profile of strong and weak siRNA
differ dramatically. Unlike the relatively strong values exhibited
by siRNA in the >95% silencing group, siRNAs that exhibit poor
silencing activity have weak AISP values (-7.6, -7.5, and -7.5
kcal/mol for positions 1, 2 and 3 respectively).
[0353] Overall the profiles of both strong and weak siRNAs form
distinct sinusoidal shapes that are roughly 180.degree.
out-of-phase with each other. While these thermodynamic
descriptions define the archetypal profile of a strong siRNA, it
will likely be the case that neither the .DELTA.G values given for
key positions in the profile or the absolute position of the
profile along the Y-axis (i.e., the .DELTA.G-axis) are absolutes.
Profiles that are shifted upward or downward (i.e., having on an
average, higher or lower values at every position) but retain the
relative shape and position of the profile along the X-axis can be
foreseen as being equally effective as the model profile described
here. Moreover, it is likely that siRNA that have strong or even
stronger gene-specific silencing effects might have exaggerated
.DELTA.G values (either higher or lower) at key positions. Thus,
for instance, it is possible that the 5'-most position of the sense
strand (position 19) could have .DELTA.G values of 7.4 kcal/mol or
lower and still be a strong siRNA if, for instance, a
G-C.fwdarw.G-T/U mismatch were substituted at position 19 and
altered duplex stability. Similarly, position 12 and position 7
could have values above 8.3 kcal/mol and below 7.7 kcal/mole,
respectively, without abating the silencing effectiveness of the
molecule. Thus, for instance, at position 12, a stabilizing
chemical modification (e.g., a chemical modification of the 2'
position of the sugar backbone) could be added that increases the
average internal stability at that position. Similarly, at position
7, mismatches similar to those described previously could be
introduced that would lower the .DELTA.G values at that
position.
[0354] Lastly, it is important to note that while functional and
non-functional siRNA were originally defined as those molecules
having specific silencing properties, both broader or more limiting
parameters can be used to define these molecules. As used herein,
unless otherwise specified, "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, and
"highly-functional siRNA" are molecules that induce great than 95%
gene silencing. These definitions are not intended to be rigid and
can vary depending upon the design and needs of the application.
For instance, it is possible that a researcher attempting to map a
gene to a chromosome using a functional assay, may identify an
siRNA that reduces gene activity by only 30%. While this level of
gene silencing may be "non-functional" for, e.g., therapeutic
needs, it is sufficient for gene mapping purposes and is, under
these uses and conditions, "functional." For these reasons,
functional siRNA can be defined as those molecules having greater
than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% silencing
capabilities at 100 nM transfection conditions. Similarly,
depending upon the needs of the study and/or application,
non-functional and semi-functional siRNA can be defined as having
different parameters. For instance, semi-functional siRNA can be
defined as being those molecules that induce 20%, 30%, 40%, 50%,
60%, or 70% silencing at 100 nM transfection conditions. Similarly,
non-functional siRNA can be defined as being those molecules that
silence gene expression by less than 70%, 60%, 50%, 40%, 30%, or
less. Nonetheless, unless otherwise stated, the descriptions stated
in the "Definitions" section of this text should be applied.
[0355] Functional attributes can be assigned to each of the key
positions in the AISP of strong siRNA. The low 5' (sense strand)
AISP values of strong siRNAs may be necessary for determining which
end of the molecule enters the RISC complex. In contrast, the high
and low AISP values observed in the central regions of the molecule
may be critical for siRNA-target mRNA interactions and product
release, respectively.
[0356] If the AISP values described above accurately define the
thermodynamic parameters of strong siRNA, it would be expected that
similar patterns would be observed in strong siRNA isolated from
nature. Natural siRNAs exist in a harsh, RNase-rich environment and
it can be hypothesized that only those siRNA that exhibit
heightened affinity for RISC (i.e., siRNA that exhibit an average
internal stability profile similar to those observed in strong
siRNA) would survive in an intracellular environment. This
hypothesis was tested using GFP-specific siRNA isolated from N.
benthamiana. Llave et al. (2002) Endogenous and
Silencing-Associated Small RNAs in Plants, The Plant Cell 14,
1605-1619, introduced long double-stranded GFP-encoding RNA into
plants and subsequently re-isolated GFP-specific siRNA from the
tissues. The AISP of fifty-nine of these GFP-siRNA were determined,
averaged, and subsequently plotted alongside the AISP profile
obtained from the cyclophilin B/DBI/luciferase siRNA having >90%
silencing properties (FIG. 6b). Comparison of the two groups show
that profiles are nearly identical. This finding validates the
information provided by the internal stability profiles and
demonstrates that: (1) the profile identified by analysis of the
cyclophilin B/DBI/luciferase siRNAs are not gene specific; and (2)
AISP values can be used to search for strong siRNAs in a variety of
species.
[0357] Both chemical modifications and base-pair mismatches can be
incorporated into siRNA to alter the duplex's AISP and
functionality. For instance, introduction of mismatches at
positions 1 or 2 of the sense strand destabilized the 5' end of the
sense strand and increases the functionality of the molecule (see
Luc, FIG. 7). Similarly, addition of 2'-O-methyl groups to
positions 1 and 2 of the sense strand can also alter the AISP and
(as a result) increase both the functionality of the molecule and
eliminate off-target effects that results from sense strand
homology with the unrelated targets (FIGS. 8a, 8b).
Rationale for Criteria in a Biological Context
[0358] The fate of siRNA in the RNAi pathway may be described in 5
major steps: (1) duplex recognition and pre-RISC complex formation;
(2) ATP-dependent duplex unwinding/strand selection and RISC
activation; (3) mRNA target identification; (4) mRNA cleavage, and
(5) product release (FIG. 1). Given the level of nucleic
acid-protein interactions at each step, siRNA functionality is
likely influenced by specific biophysical and molecular properties
that promote efficient interactions within the context of the
multi-component complexes. Indeed, the systematic analysis of the
siRNA test set identified multiple factors that correlate well with
functionality. When combined into a single algorithm, they proved
to be very effective in selecting active siRNAs.
[0359] The factors described here may also be predictive of key
functional associations important for each step in RNAi. For
example, the potential formation of internal hairpin structures
correlated negatively with siRNA functionality. Complementary
strands with stable internal repeats are more likely to exist as
stable hairpins thus decreasing the effective concentration of the
functional duplex form. This suggests that the duplex is the
preferred conformation for initial pre-RISC association. Indeed,
although single complementary strands can induce gene silencing,
the effective concentration required is at least two orders of
magnitude higher than that of the duplex form.
[0360] siRNA-pre-RISC complex formation is followed by an
ATP-dependent duplex unwinding step and "activation" of the RISC.
The siRNA functionality was shown to correlate with overall low
internal stability of the duplex and low internal stability of the
3' sense end (or differential internal stability of the 3' sense
compare to the 5' sense strand), which may reflect strand selection
and entry into the RISC. Overall duplex stability and low internal
stability at the 3' end of the sense strand were also correlated
with siRNA functionality. Interestingly, siRNAs with very high and
very low overall stability profiles correlate strongly with
non-functional duplexes. One interpretation is that high internal
stability prevents efficient unwinding while very low stability
reduces siRNA target affinity and subsequent mRNA cleavage by the
RISC.
[0361] Several criteria describe base preferences at specific
positions of the sense strand and are even more intriguing when
considering their potential mechanistic roles in target recognition
and mRNA cleavage. Base preferences for A at position 19 of the
sense strand but not C, are particularly interesting because they
reflect the same base preferences observed for naturally occurring
miRNA precursors. That is, among the reported miRNA precursor
sequences 75% contain a U at position 1 which corresponds to an A
in position 19 of the sense strand of siRNAs, while G was
under-represented in this same position for miRNA precursors. These
observations support the hypothesis that both miRNA precursors and
siRNA duplexes are processed by very similar if not identical
protein machinery. The functional interpretation of the
predominance of a U/A base pair is that it promotes flexibility at
the 5'antisense ends of both siRNA duplexes and miRNA precursors
and facilitates efficient unwinding and selective strand entrance
into an activated RISC.
[0362] Among the criteria associated with base preferences that are
likely to influence mRNA cleavage or possibly product release, the
preference for U at position 10 of the sense strand exhibited the
greatest impact, enhancing the probability of selecting an F80
sequence by 13.3%. Activated RISC preferentially cleaves target
mRNA between nucleotides 10 and 11 relative to the 5' end of the
complementary targeting strand. Therefore, it may be that U, the
preferred base for most endoribonucleases, at this position
supports more efficient cleavage. Alternatively, a U/A bp between
the targeting siRNA strand and its cognate target in RNA may create
an optimal conformation for the RISC-associated "slicing"
activity.
Post Algorithm Filters
[0363] According to another embodiment, the output of any one of
the formulas previously listed can be filtered to remove or select
for siRNAs containing undesirable or desirable motifs or
properties, respectively. In one example, sequences identified by
any of the formulas can be filtered to remove any and all sequences
that induce toxicity or cellular stress. Introduction of an siRNA
containing a toxic motif into a cell can induce cellular stress
and/or cell death (apoptosis) which in turn can mislead researchers
into associating a particular (e.g., nonessential) gene with, e.g.,
an essential function. Alternatively, sequences generated by any of
the before mentioned formulas can be filtered to identify and
retain duplexes that contain toxic motifs. Such duplexes may be
valuable from a variety of perspectives including, for instance,
uses as therapeutic molecules. A variety of toxic motifs exist and
can exert their influence on the cell through RNAi and non-RNAi
pathways. Examples of toxic motifs are explained more fully in
commonly assigned U.S. Provisional Patent Application Ser. No.
60/538,874, entitled "Identification of Toxic Sequences," filed
Jan. 23, 2004. Briefly, toxic: motifs include A/G UUU A/G/U, G/C
AAA G/C, and GCCA, or a complement of any of the foregoing.
[0364] In another instance, sequences identified by any of the
before mentioned formulas can be filtered to identify duplexes that
contain motifs (or general properties) that provide serum stability
or induce serum instability. In one envisioned application of siRNA
as therapeutic molecules, duplexes targeting disease-associated
genes will be introduced into patients intravenously. As the
half-life of single and double stranded RNA in serum is short,
post-algorithm filters designed to select molecules that contain
motifs that enhance duplex stability in the presence of serum
and/or (conversely) eliminate duplexes that contain motifs that
destabilize siRNA in the presence of serum, would be
beneficial.
[0365] In another instance, sequences identified by any of the
before mentioned formulas can be filtered to identify duplexes that
are hyperfunctional. Hyperfunctional sequences are defined as those
sequences 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. Filters that
identify hyperfunctional molecules can vary widely. In one example,
the top ten, twenty, thirty, or forty siRNA can be assessed for the
ability to silence a given target at, e.g., concentrations of 1 nM
and 0.5 nM to identify hyperfunctional molecules.
Pooling
[0366] According to another embodiment, the present invention
provides a pool of at least two siRNAs, preferably in the form of a
kit or therapeutic reagent, wherein one strand of each of the
siRNAs, the sense strand comprises a sequence that is substantially
similar to a sequence within a target mRNA. The opposite strand,
the antisense strand, will preferably comprise a sequence that is
substantially complementary to that of the target mRNA. More
preferably, one strand of each siRNA will comprise a sequence that
is identical to a sequence that is contained in the target mRNA.
Most preferably, each siRNA will be 19 base pairs in length, and
one strand of each of the siRNAs will be 100% complementary to a
portion of the target mRNA.
[0367] By increasing the number of siRNAs directed to a particular
target using a pool or kit, one is able both to increase the
likelihood that at least one siRNA with satisfactory functionality
will be included, as well as to benefit from additive or
synergistic effects. Further, when two or more siRNAs directed
against a single gene do not have satisfactory levels of
functionality alone, if combined, they may satisfactorily promote
degradation of the target messenger RNA and successfully inhibit
translation. By including multiple siRNAs in the system, not only
is the probability of silencing increased, but the economics of
operation are also improved when compared to adding different
siRNAs sequentially. This effect is contrary to the conventional
wisdom that the concurrent use of multiple siRNA will negatively
impact gene silencing (e.g., Holen, T. et al. (2003) Similar
behavior of single strand and double strand siRNAs suggests they
act through a common RNAi pathway. NAR 31: 2401-21407).
[0368] In fact, when two siRNAs were pooled together, 54% of the
pools of two siRNAs induced more than 95% gene silencing. Thus, a
2.5-fold increase in the percentage of functionality was achieved
by randomly combining two siRNAs. Further, over 84% of pools
containing two siRNAs induced more than 80% gene silencing.
[0369] More preferably, the kit is comprised of at least three
siRNAs, wherein one strand of each siRNA comprises a sequence that
is substantially similar to a sequence of the target mRNA and the
other strand comprises a sequence that is substantially
complementary to the region of the target mRNA. As with the kit
that comprises at least two siRNAs, more preferably one strand will
comprise a sequence that is identical to a sequence that is
contained in the mRNA and another strand that is 100% complementary
to a sequence that is contained in the mRNA. During experiments,
when three siRNAs were combined together, 60% of the pools induced
more than 95% gene silencing and 92% of the pools induced more than
80% gene silencing.
[0370] Further, even more preferably, the kit is comprised of at
least four siRNAs, wherein one strand of each siRNA comprises a
sequence that is substantially similar to a region of the sequence
of the target mRNA, and the other strand comprises a sequence that
is substantially complementary to the region of the target mRNA. As
with the kit or pool that comprises at least two siRNAs, more
preferably one strand of each of the siRNA duplexes will comprise a
sequence that is identical to a sequence that is contained in the
mRNA, and another strand that is 100% complementary to a sequence
that is contained in the mRNA.
[0371] Additionally, kits and pools with at least five, at least
six, and at least seven siRNAs may also be useful with the present
invention. For example, pools of five siRNA induced 95% gene
silencing with 77% probability and 80% silencing with 98.8%
probability. Thus, pooling of siRNAs together can result in the
creation of a target-specific silencing reagent with almost a 99%
probability of being functional. The fact that such high levels of
success are achievable using such pools of siRNA, enables one to
dispense with costly and time-consuming target-specific validation
procedures.
[0372] For this embodiment, as well as the other aforementioned
embodiments, each of the siRNAs within a pool will preferably
comprise 18-30 base pairs, more preferably 18-25 base pairs, and
most preferably 19 base pairs. Within each siRNA, preferably at
least 18 contiguous bases of the antisense strand will be 100%
complementary to the target mRNA. More preferably, at least 19
contiguous bases of the antisense strand will be 100% complementary
to the target mRNA. Additionally, there may be overhangs on either
the sense strand or the antisense strand, and these overhangs may
be at either the 5' end or the 3' end of either of the strands, for
example there may be one or more overhangs of 1-6 bases. When
overhangs are present, they are not included in the calculation of
the number of base pairs. The two nucleotide 3' overhangs mimic
natural siRNAs and are commonly used but are not essential.
Preferably, the overhangs should consist of two nucleotides, most
often dTdT or UU at the 3' end of the sense and antisense strand
that are not complementary to the target sequence. The siRNAs may
be produced by any method that is now known or that comes to be
known for synthesizing double stranded RNA that one skilled in the
art would appreciate would be useful in the present invention.
Preferably, the siRNAs will be produced by Dharmacon's proprietary
ACE.RTM. technology. However, other methods for synthesizing siRNAs
are well known to persons skilled in the art and include, but are
not limited to, any chemical synthesis of RNA oligonucleotides,
ligation of shorter oligonucleotides, in vitro transcription of RNA
oligonucleotides, the use of vectors for expression within cells,
recombinant Dicer products and PCR products.
[0373] The siRNA duplexes within the aforementioned pools of siRNAs
may correspond to overlapping sequences within a particular mRNA,
or non-overlapping sequences of the mRNA. However, preferably they
correspond to non-overlapping sequences. Further, each siRNA may be
selected randomly, or one or more of the siRNA may be selected
according to the criteria discussed above for maximizing the
effectiveness of siRNA.
[0374] Included in the definition of siRNAs are siRNAs that contain
substituted and/or labeled nucleotides that may, for example, be
labeled by radioactivity, fluorescence or mass. The most common
substitutions are at the 2' position of the ribose sugar, where
moieties such as H (hydrogen) F, NH.sub.3, OCH.sub.3 and other O--
alkyl, alkenyl, alkynyl, and orthoesters, may be substituted, or in
the phosphorous backbone, where sulfur, amines or hydrocarbons may
be substituted for the bridging of non-bridging atoms in the
phosphodiester bond. Examples of modified siRNAs are explained more
fully in commonly assigned U.S. patent application Ser. No.
10/613,077, filed Jul. 1, 2003.
[0375] Additionally, as noted above, the cell type into which the
siRNA is introduced may affect the ability of the siRNA to enter
the cell; however, it does not appear to affect the ability of the
siRNA to function once it enters the cell. Methods for introducing
double-stranded RNA into various cell types are well known to
persons skilled in the art.
[0376] As persons skilled in the art are aware, in certain species,
the presence of proteins such as RdRP, the RNA-dependent RNA
polymerase, may catalytically enhance the activity of the siRNA.
For example, RdRP propagates the RNAi effect in C. elegans and
other non-mammalian organisms. In fact, in organisms that contain
these proteins, the siRNA may be inherited. Two other proteins that
are well studied and known to be a part of the machinery are
members of the Argonaute family and Dicer, as well as their
homologues. There is also initial evidence that the RISC complex
might be associated with the ribosome so the more efficiently
translated mRNAs will be more susceptible to silencing than
others.
[0377] Another very important factor in the efficacy of siRNA is
mRNA localization. In general, only cytoplasmic in RNAs are
considered to be accessible to RNAi to any appreciable degree.
However, appropriately designed siRNAs, for example, siRNAs
modified with internucleotide linkages or 2'-O-methyl groups, may
be able to cause silencing by acting in the nucleus. Examples of
these types of modifications are described in commonly assigned
U.S. patent application Ser. Nos. 10/431,027 and 10/613,077.
[0378] As described above, even when one selects at least two
siRNAs at random, the effectiveness of the two may be greater than
one would predict based on the effectiveness of two individual
siRNAs. This additive or synergistic effect is particularly
noticeable as one increases to at least three siRNAs, and even more
noticeable as one moves to at least four siRNAs. Surprisingly, the
pooling of the non-functional and semi-functional siRNAs,
particularly more than five siRNAs, can lead to a silencing mixture
that is as effective if not more effective than any one particular
functional siRNA.
[0379] Within the kits of the present invention, preferably each
siRNA will be present in a concentration of between 0.001 and 200
.mu.M, more preferably between 0.01 and 200 nM, and most preferably
between 0.1 and 10 nM.
[0380] In addition to preferably comprising at least four or five
siRNAs, the kits of the present invention will also preferably
comprise a buffer to keep the siRNA duplex stable. Persons skilled
in the art are aware of buffers suitable for keeping siRNA stable.
For example, the buffer may be comprised of 100 mM KCl, 30 mM
HEPES-pH 7.5, and 1 mM MgCl.sub.2. Alternatively, kits might
contain complementary strands that contain any one of a number of
chemical modifications (e.g., a 2'-O-ACE) that protect the agents
from degradation by nucleases. In this instance, the user may (or
may not) remove the modifying protective group (e.g., deprotect)
before annealing the two complementary strands together.
[0381] By way of example, the kits may be organized such that pools
of siRNA duplexes are provided on an array or microarray of wells
or drops for a particular gene set or for unrelated genes. The
array may, for example, be in 96 wells, 384 wells or 1284 wells
arrayed in a plastic plate or on a glass slide using techniques now
known or that come to be known to persons skilled in the art.
Within an array, preferably there will be controls such as
functional anti-lamin A/C, cyclophilin and two siRNA duplexes that
are not specific to the gene of interest.
[0382] In order to ensure stability of the siRNA pools prior to
usage, they may be retained in lyophilized form at minus twenty
degrees (-20.degree. C.) until they are ready for use. Prior to
usage, they should be resuspended; however, even once resuspended,
for example, in the aforementioned buffer, they should be kept at
minus twenty degrees, (-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 transfections are well known to persons skilled in the
art and include for example, room temperature.
[0383] The kits may be applied either in vivo or in vitro.
Preferably, the siRNA of the pools or kits is applied to a cell
through transfection, employing standard transfection protocols.
These methods are well known to persons skilled in the art and
include the use of lipid-based carriers, electroporation, cationic
carriers, and microinjection. Further, one could apply the present
invention by synthesizing equivalent DNA sequences (either as two
separate, complementary strands, or as hairpin molecules) instead
of siRNA sequences and introducing them into cells through vectors.
Once in the cells, the cloned DNA could be transcribed, thereby
forcing the cells to generate the siRNA. Examples of vectors
suitable for use with the present application include but are not
limited to the standard transient expression vectors, adenoviruses,
retroviruses, lentivirus-based vectors, as well as other
traditional expression vectors. Any vector that has an adequate
siRNA expression and procession module may be used. Furthermore,
certain chemical modifications to siRNAs, including but not limited
to conjugations to other molecules, may be used to facilitate
delivery. For certain applications it may be preferable to deliver
molecules without transfection by simply formulating in a
physiological acceptable solution.
[0384] This embodiment may be used in connection with any of the
aforementioned embodiments. Accordingly, the sequences within any
pool may be selected by rational design.
Multigene Silencing
[0385] In addition to developing kits that contain multiple siRNA
directed against a single gene, another embodiment includes the use
of multiple siRNA targeting multiple genes. Multiple genes may be
targeted through the use of high- or hyper-functional siRNA. High-
or hyper-functional siRNA that exhibit increased potency, require
lower concentrations to induce desired phenotypic (and thus
therapeutic) effects. This circumvents RISC saturation. It
therefore reasons that if lower concentrations of a single siRNA
are needed for knockout or knockdown expression of one gene, then
the remaining (uncomplexed) RISC will be free and available to
interact with siRNA directed against two, three, four, or more,
genes. Thus in this embodiment, the authors describe the use of
highly functional or hyper-functional siRNA to knock out three
separate genes. More preferably, such reagents could be combined to
knockout four distinct genes. Even more preferably, highly
functional or hyperfunctional siRNA could be used to knock out five
distinct genes. Most preferably, siRNA of this type could be used
to knockout or knockdown the expression of six or more genes.
Hyperfunctional siRNA
[0386] The term hyperfunctional siRNA (hf-siRNA) describes a subset
of the siRNA population that induces RNAi in cells at low- or
sub-nanomolar concentrations for extended periods of time. These
traits, heightened potency and extended longevity of the RNAi
phenotype, are highly attractive from a therapeutic standpoint.
Agents having higher potency require lesser amounts of the molecule
to achieve the desired physiological response, thus reducing the
probability of side effects due to "off-target" interference. In
addition to the potential therapeutic benefits associated with
hyperfunctional siRNA, hf-siRNA are also desirable from an economic
perspective. Hyperfunctional siRNA may cost less on a per-treatment
basis, thus reducing overall expenditures to both the manufacturer
and the consumer.
[0387] Identification of hyperfunctional siRNA involves multiple
steps that are designed to examine an individual siRNA agent's
concentration- and/or longevity-profiles. In one non-limiting
example, a population of siRNA directed against a single gene are
first analyzed using the previously described algorithm (Formula
VIII). Individual siRNA are then introduced into a test cell line
and assessed for the ability to degrade the target mRNA. It is
important to note that when performing this step it is not
necessary to test all of the siRNA. Instead, it is sufficient to
test only those siRNA having the highest SMARTSCORES.TM., or siRNA
ranking (i.e., SMARTSCORES.TM.M, or siRNA ranking >-10).
Subsequently, the gene silencing data is plotted against the
SMARTSCORES.TM., or siRNA rankings (see FIG. 9). siRNA that (1)
induce a high degree of gene silencing (i.e., they induce greater
than 80% gene knockdown) and (2) have superior SMARTSCORES.TM.
(i.e., a SMARTSCORE.TM., or siRNA ranking, of >-10, suggesting a
desirable average internal stability profile) are selected for
further investigations designed to better understand the molecule's
potency and longevity. In one, non-limiting study dedicated to
understanding a molecule's potency, an siRNA is introduced into one
(or more) cell types in increasingly diminishing concentrations
(e.g., 3.0.fwdarw.0.3 nM). Subsequently, the level of gene
silencing induced by each concentration is examined and siRNA that
exhibit hyperfunctional potency (i.e., those that induce 80%
silencing or greater at, e.g., picomolar concentrations) are
identified. In a second study, the longevity profiles of siRNA
having high (>-10) SMARTSCORES.TM., or siRNA rankings and
greater than 80% silencing are examined. In one non-limiting
example of how this is achieved, siRNA are introduced into a test
cell line and the levels of RNAi are measured over an extended
period of time (e.g., 24-168 hrs). siRNAs that exhibit strong RNA
interference patterns (i.e., >80% interference) for periods of
time greater than, e.g., 120 hours, are thus identified. Studies
similar to those described above can be performed on any and all of
the >10.sup.6 siRNA included in this document to further define
the most functional molecule for any given gene. Molecules
possessing one or both properties (extended longevity and
heightened potency) are labeled "hyperfunctional siRNA," and
earmarked as candidates for future therapeutic studies.
[0388] While the example(s) given above describe one means by which
hyperfunctional siRNA can be isolated, neither the assays
themselves nor the selection parameters used are rigid and can vary
with each family of siRNA. Families of siRNA include siRNAs
directed against a single gene, or directed against a related
family of genes.
[0389] The highest quality siRNA achievable for any given gene may
vary considerably. Thus, for example, in the case of one gene (gene
X), rigorous studies such as those described above may enable the
identification of an siRNA that, at picomolar concentrations,
induces 99.sup.+% silencing for a period of 10 days. Yet identical
studies of a second gene (gene Y) may yield an siRNA that at high
nanomolar concentrations (e.g., 100 nM) induces only 75% silencing
for a period of 2 days. Both molecules represent the very optimum
siRNA for their respective gene targets and therefore are
designated "hyperfunctional." Yet due to a variety of factors
including but not limited to target concentration, siRNA stability,
cell type, off-target interference, and others, equivalent levels
of potency and longevity are not achievable. Thus, for these
reasons, the parameters described in the before mentioned assays
can vary. While the initial screen selected siRNA that had
SMARTSCORES.TM. above -10 and a gene silencing capability of
greater than 80%, selections that have stronger (or weaker)
parameters can be implemented. Similarly, in the subsequent studies
designed to identify molecules with high potency and longevity, the
desired cutoff criteria (i.e., the lowest concentration that
induces a desirable level of interference, or the longest period of
time that interference can be observed) can vary. The
experimentation subsequent to application of the rational criteria
of this application is significantly reduced where one is trying to
obtain a suitable hyperfunctional siRNA for, for example,
therapeutic use. When, for example, the additional experimentation
of the type described herein is applied by one skilled in the art
with this disclosure in hand, a hyperfunctional siRNA is readily
identified.
[0390] The siRNA may be introduced into a cell by any method that
is now known or that comes to be known and that from reading this
disclosure, persons skilled in the art would determine would be
useful in connection with the present invention in enabling siRNA
to cross the cellular membrane. These methods include, but are not
limited to, any manner of transfection, such as, for example,
transfection employing DEAE-Dextran, calcium phosphate, cationic
lipids/liposomes, micelles, manipulation of pressure,
microinjection, electroporation, immunoporation, use of vectors
such as viruses, plasmids, cosmids, bacteriophages, cell fusions,
and coupling of the polynucleotides to specific conjugates or
ligands such as antibodies, antigens, or receptors, passive
introduction, adding moieties to the siRNA that facilitate its
uptake, and the like.
[0391] 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.
EXAMPLES
General Techniques and Nomenclatures
[0392] siRNA nomenclature. All siRNA duplexes are referred to by
sense strand. The first nucleotide of the 5'-end of the sense
strand is position 1, which corresponds to position 19 of the
antisense strand for a 19-mer. In most cases, to compare results
from different experiments, silencing was determined by measuring
specific transcript mRNA levels or enzymatic activity associated
with specific transcript levels, 24 hours post-transfection, with
siRNA concentrations held constant at 100 nM. For all experiments,
unless otherwise specified, transfection efficiency was ensured to
be over 95%, and no detectable cellular toxicity was observed. The
following system of nomenclature was used to compare and report
siRNA-silencing functionality: "F" followed by the degree of
minimal knockdown. For example, F50 signifies at least 50%
knockdown, F80 means at least 80%, and so forth. For this study,
all sub-F50 siRNAs were considered non-functional.
[0393] Cell culture and transfection. 96-well plates are coated
with 50 .mu.l of 50 mg/ml poly-L-lysine (Sigma) for 1 hr, and then
washed 3.times. with distilled water before being dried for 20 min.
HEK293 cells or HEK293Lucs or any other cell type of interest are
released from their solid support by trypsinization, diluted to
3.5.times.10.sup.5 cells/ml, followed by the addition of 100 .mu.L
of cells/well. Plates are then incubated overnight at 37.degree.
C., 5% CO.sub.2. Transfection procedures can vary widely depending
on the cell type and transfection reagents. In one non-limiting
example, a transfection mixture consisting of 2 mL Opti-MEM 1
(Gibco-BRL), 80 .mu.l Lipofectamine 2000 (Invitrogen), 15 .mu.L
SUPERNasin at 20 U/.mu.l (Ambion), and 1.5 .mu.l of reporter gene
plasmid at 1 .mu.g/.mu.l is prepared in 5-ml polystyrene round
bottom tubes. One hundred .mu.l of transfection reagent is then
combined with 100 .mu.l of siRNAs in polystyrene deep-well titer
plates (Beckman) and incubated for 20 to 30 min at room
temperature. Five hundred and fifty microliters of Opti-MEM is then
added to each well to bring the final siRNA concentration to 100
nM. Plates are then sealed with parafilm and mixed. Media is
removed from HEK293 cells and replaced with 95 .mu.l of
transfection mixture. Cells are incubated overnight at 37.degree.
C., 5% CO.sub.2.
[0394] Quantification of gene knockdown. A variety of
quantification procedures can be used to measure the level of
silencing induced by siRNA or siRNA pools. In one non-limiting
example: to measure mRNA levels 24 hrs post-transfection,
QuantiGene branched-DNA (bDNA) kits (Bayer) (Wang, et al,
Regulation of insulin preRNA splicing by glucose. Proc. Natl. Acad.
Sci. USA 1997, 94:4360.) are used according to manufacturer
instructions. To measure luciferase activity, media is removed from
HEK293 cells 24 hrs post-transfection, and 50 .mu.l of Steady-GLO
reagent (Promega) is added. After 5 minutes, plates are analyzed on
a plate reader.
Example I
Sequences Used to Develop the Algorithm
[0395] Anti-Firefly and anti-Cyclophilin siRNAs panels (FIG. 5a, b)
sorted according to using Formula VIII predicted values. All siRNAs
scoring more than 0 (formula VIII) and more then 20 (formula IX)
are fully functional. All ninety sequences for each gene (and DBI)
appear below in Table III.
TABLE-US-00006 TABLE III Cyclo 1 SEQ. ID 0032 GUUCCAAAAACAGUGGAUA
Cyclo 2 SEQ. ID 0033 UCCAAAAACAGUGGAUAAU Cyclo 3 SEQ. ID 0034
CAAAAACAGUGGAUAAUUU Cyclo 4 SEQ. ID 0035 AAAACAGUGGAUAAUUUUG Cyclo
5 SEQ. ID 0036 AACAGUGGAUAAUUUUGUG Cyclo 6 SEQ. ID 0037
CAGUGGAUAAUUUUGUGGC Cyclo 7 SEQ. ID 0038 GUGGAUAAUUUUGUGGCCU Cyclo
8 SEQ. ID 0039 GGAUAAUUUUGUGGCCUUA Cyclo 9 SEQ. ID 0040
AUAAUUUUGUGGCCUUAGC Cyclo 10 SEQ. ID 0041 AAUUUUGUGGCCUUAGCUA Cyclo
11 SEQ. ID 0042 UUUUGUGGCCUUAGCUACA Cyclo 12 SEQ. ID 0043
UUGUGGCCUUAGCUACAGG Cyclo 13 SEQ. ID 0044 GUGGCCUUAGCUACAGGAG Cyclo
14 SEQ. ID 0045 GGCCUUAGCUACAGGAGAG Cyclo 15 SEQ. ID 0046
CCUUAGCUACAGGAGAGAA Cyclo 16 SEQ. ID 0047 UUAGCUACAGGAGAGAAAG Cyclo
17 SEQ. ID 0048 AGCUACAGGAGAGAAAGGA Cyclo 18 SEQ. ID 0049
CUACAGGAGAGAAAGGAUU Cyclo 19 SEQ. ID 0050 ACAGGAGAGAAAGGAUUUG Cyclo
20 SEQ. ID 0051 AGGAGAGAAAGGAUUUGGC Cyclo 21 SEQ. ID 0052
GAGAGAAAGGAUUUGGCUA Cyclo 22 SEQ. ID 0053 GAGAAAGGAUUUGGCUACA Cyclo
23 SEQ. ID 0054 GAAAGGAUUUGGCUACAAA Cyclo 24 SEQ. ID 0055
AAGGAUUUGGCUACAAAAA Cyclo 25 SEQ. ID 0056 GGAUUUGGCUACAAAAACA Cyclo
26 SEQ. ID 0057 AUUUGGCUACAAAAACAGC Cyclo 27 SEQ. ID 0058
UUGGCUACAAAAACAGCAA Cyclo 28 SEQ. ID 0059 GGCUACAAAAACAGCAAAU Cyclo
29 SEQ. ID 0060 CUACAAAAACAGCAAAUUC Cyclo 30 SEQ. ID 0061
ACAAAAACAGCAAAUUCCA Cyclo 31 SEQ. ID 0062 AAAAACAGCAAAUUCCAUC Cyclo
32 SEQ. ID 0063 AAACAGCAAAUUCCAUCGU Cyclo 33 SEQ. ID 0064
ACAGCAAAUUCCAUCGUGU Cyclo 34 SEQ. ID 0065 AGCAAAUUCCAUCGUGUAA Cyclo
35 SEQ. ID 0066 CAAAUUCCAUCGUGUAAUC Cyclo 36 SEQ. ID 0067
AAUUCCAUCGUGUAAUCAA Cyclo 37 SEQ. ID 0068 UUCCAUCGUGUAAUCAAGG Cyclo
38 SEQ. ID 0069 CCAUCGUGUAAUCAAGGAC Cyclo 39 SEQ. ID 0070
AUCGUGUAAUCAAGGACUU Cyclo 40 SEQ. ID 0071 CGUGUAAUCAAGGACUUCA Cyclo
41 SEQ. ID 0072 UGUAAUCAAGGACUUCAUG Cyclo 42 SEQ. ID 0073
UAAUCAAGGACUUCAUGAU Cyclo 43 SEQ. ID 0074 AUCAAGGACUUCAUGAUCC Cyclo
44 SEQ. ID 0075 CAAGGACUUCAUGAUCCAG Cyclo 45 SEQ. ID 0076
AGGACUUCAUGAUCCAGGG Cyclo 46 SEQ. ID 0077 GACUUCAUGAUCCAGGGCG Cyclo
47 SEQ. ID 0078 CUUCAUGAUCCAGGGCGGA Cyclo 48 SEQ. ID 0079
UCAUGAUCCAGGGCGGAGA Cyclo 49 SEQ. ID 0080 AUGAUCCAGGGCGGAGACU Cyclo
50 SEQ. ID 0081 GAUCCAGGGCGGAGACUUC Cyclo 51 SEQ. ID 0082
UCCAGGGCGGAGACUUCAC Cyclo 52 SEQ. ID 0083 CAGGGCGGAGACUUCACCA Cyclo
53 SEQ. ID 0084 GGGCGGAGACUUCACCAGG Cyclo 54 SEQ. ID 0085
GCGGAGACUUCACCAGGGG Cyclo 55 SEQ. ID 0086 GGAGACUUCACCAGGGGAG Cyclo
56 SEQ. ID 0087 ACACUUCACCAGGGGAGAU Cyclo 57 SEQ. ID 0088
ACUUCACCAGGGGAGAUGG Cyclo 58 SEQ. ID 0089 UUCACCAGGGGAGAUGGCA Cyclo
59 SEQ. ID 0090 CACCAGGGGAGAUGGCACA Cyclo 60 SEQ. ID 0091
CCAGGGGAGAUGGCACAGG Cyclo 61 SEQ. ID 0092 AGGGGAGAUGGCACAGGAG Cyclo
62 SEQ. ID 0093 GGGAGAUGGCACAGGAGGA Cyclo 63 SEQ. ID 0094
GAGAUGGCACAGGAGGAAA Cyclo 64 SEQ. ID 0095 GAUGGCACAGGAGGAAAGA Cyclo
65 SEQ. ID 0096 UGGCACAGGAGGAAAGAGC Cyclo 66 SEQ. ID 0097
GCACAGGAGGAAAGAGCAU Cyclo 67 SEQ. ID 0098 ACAGGAGGAAAGAGCAUCU Cyclo
68 SEQ. ID 0099 AGGAGGAAAGAGCAUCUAC Cyclo 69 SEQ. ID 0100
GAGGAAAGAGCAUCUACGG Cyclo 70 SEQ. ID 0101 GGAAAGAGCAUCUACGGUG Cyclo
71 SEQ. ID 0102 AAAGAGCAUCUACGGUGAG Cyclo 72 SEQ. ID 0103
AGAGCAUCUACGGUGAGCG Cyclo 73 SEQ. ID 0104 AGCAUCUACGGUGAGCGCU Cyclo
74 SEQ. ID 0105 CAUCUACGGUGAGCGCUUC Cyclo 75 SEQ. ID 0106
UCUACGGUGAGCGCUUCCC Cyclo 76 SEQ. ID 0107 UACGGUGAGCGCUUCCCCG Cyclo
77 SEQ. ID 0108 CGGUGAGCGCUUCCCCGAU Cyclo 78 SEQ. ID 0109
GUGAGCGCUUCCCCGAUGA Cyclo 79 SEQ. ID 0110 GAGCGCUUCCCCGAUGAGA Cyclo
80 SEQ. ID 0111 GCGCUUCCCCGAUGAGAAC Cyclo 81 SEQ. ID 0112
GCUUCCCCGAUGAGAACUU Cyclo 82 SEQ. ID 0113 UUCCCCGAUGAGAACUUCA Cyclo
83 SEQ. ID 0114 CCCCGAUGAGAACUUCAAA Cyclo 84 SEQ. ID 0115
CCGAUGAGAACUUCAAACU Cyclo 85 SEQ. ID 0116 GAUGAGAACUUCAAACUGA Cyclo
86 SEQ. ID 0117 UGAGAACUUCAAACUGAAG Cyclo 87 SEQ. ID 0118
AGAACUUCAAACUGAAGCA Cyclo 88 SEQ. ID 0119 AACUUCAAACUGAAGCACU Cyclo
89 SEQ. ID 0120 CUUCAAACUGAAGCACUAC Cyclo 90 SEQ. ID 0121
UCAAACUGAAGCACUACGG DB 1 SEQ. ID 0122 ACGGGCAAGGCCAAGUGGG DB 2 SEQ.
ID 0123 CGGGCAAGGCCAAGUGGGA DB 3 SEQ. ID 0124 GGGCAAGGCCAAGUGGGAU
DB 4 SEQ. ID 0125 GGCAAGGCCAAGUGGGAUG DB 5 SEQ. ID 0126
GCAAGGCCAAGUGGGAUGC DB 6 SEQ. ID 0127 CAAGGCCAAGUGGGAUGCC DB 7 SEQ.
ID 0128 AAGGCCAAGUGGGAUGCCU DB 8 SEQ. ID 0129 AGGCCAAGUGGGAUGCCUG
DB 9 SEQ. ID 0130 GGCCAAGUGGGAUGCCUGG DB 10 SEQ. ID 0131
GCCAAGUGGGAUGCCUGGA DB 11 SEQ. ID 0132 CCAAGUGGGAUGCCUGGAA DB 12
SEQ. ID 0133 CAAGUGGGAUGCCUGGAAU DB 13 SEQ. ID 0134
AAGUGGGAUGCCUGGAAUG DB 14 SEQ. ID 0135 AGUGGGAUGCCUGGAAUGA DB 15
SEQ. ID 0136 GUGGGAUGCCUGGAAUGAG DB 16 SEQ. ID 0137
UGGGAUGCCUGGAAUGAGC DB 17 SEQ. ID 0138 GGGAUGCCUGGAAUGAGCU DB 18
SEQ. ID 0139 GGAUGCCUGGAAUGAGCUG DB 19 SEQ. ID 0140
GAUGCCUGGAAUGAGCUGA DB 20 SEQ. ID 0141 AUGCCUGGAAUGAGCUGAA DB 21
SEQ. ID 0142 UGCCUGGAAUGAGCUGAAA DB 22 SEQ. ID 0143
GCCUGGAAUGAGCUGAAAG DB 23 SEQ. ID 0144 CCUGGAAUGAGCUGAAAGG DB 24
SEQ. ID 0145 CUGGAAUGAGCUGAAAGGG DB 25 SEQ. ID 0146
UGGAAUGAGCUGAAAGGGA DB 26 SEQ. ID 0147 GGAAUGAGCUGAAAGGGAC DB 27
SEQ. ID 0148 GAAUGAGCUGAAAGGGACU DB 28 SEQ. ID 0149
AAUGAGCUGAAAGGGACUU DB 29 SEQ. ID 0150 AUGAGCUGAAAGGGACUUC DB 30
SEQ. ID 0151 UGAGCUGAAAGGGACUUCC DB 31 SEQ. ID 0152
GAGCUGAAAGGGACUUCCA DB 32 SEQ. ID 0153 AGCUGAAAGGGACUUCCAA DB 33
SEQ. ID 0154 GCUGAAAGGGACUUCCAAG DB 34 SEQ. ID 0155
CUGAAAGGGACUUCCAAGG
DB 35 SEQ. ID 0156 UGAAAGGGACUUCCAAGGA DB 36 SEQ. ID 0157
GAAAGGGACUUCCAAGGAA DB 37 SEQ. ID 0158 AAAGGGACUUCCAAGGAAG DB 38
SEQ. ID 0159 AAGGGACUUCCAAGGAAGA DB 39 SEQ. ID 0160
AGGGACUUCCAAGGAAGAU DB 40 SEQ. ID 0161 GGGACUUCCAAGGAAGAUG DB 41
SEQ. ID 0162 GGACUUCCAAGGAAGAUGC DB 42 SEQ. ID 0163
GACUUCCAAGGAAGAUGCC DB 43 SEQ. ID 0164 ACUUCCAAGGAAGAUGCCA DB 44
SEQ. ID 0165 CUUCCAAGGAAGAUGCCAU DB 45 SEQ. ID 0166
UUCCAAGGAAGAUGCCAUG DB 46 SEQ. ID 0167 UCCAAGGAAGAUGCCAUGA DB 47
SEQ. ID 0168 CCAAGGAAGAUGCCAUGAA DB 48 SEQ. ID 0169
CAAGGAAGAUGCCAUGAAA DB 49 SEQ. ID 0170 AAGGAAGAUGCCAUGAAAG DB 50
SEQ. ID 0171 AGGAAGAUGCCAUGAAAGC DB 51 SEQ. ID 0172
GGAAGAUGCCAUGAAAGCU DB 52 SEQ. ID 0173 GAAGAUGCCAUGAAAGCUU DB 53
SEQ. ID 0174 AAGAUGCCAUGAAAGCUUA DB 54 SEQ. ID 0175
AGAUGCCAUGAAAGCUUAC DB 55 SEQ. ID 0176 GAUGCCAUGAAAGCUUACA DB 56
SEQ. ID 0177 AUGCCAUGAAAGCUUACAU DB 57 SEQ. ID 0178
UGCCAUGAAAGCUUACAUC DB 58 SEQ. ID 0179 GCCAUGAAAGCUUACAUCA DB 59
SEQ. ID 0180 CCAUGAAAGCUUACAUCAA DB 60 SEQ. ID 0181
CAUGAAAGCUUACAUCAAC DB 61 SEQ. ID 0182 AUGAAAGCUUACAUCAACA DB 62
SEQ. ID 0183 UGAAAGCUUACAUCAACAA DB 63 SEQ. ID 0184
GAAAGCUUACAUCAACAAA DB 64 SEQ. ID 0185 AAAGCUUACAUCAACAAAG DB 65
SEQ. ID 0186 AAGCUUACAUCAACAAAGU DB 66 SEQ. ID 0187
AGCUUACAUCAACAAAGUA DB 67 SEQ. ID 0188 GCUUACAUCAACAAAGUAG DB 68
SEQ. ID 0189 CUUACAUCAACAAAGUAGA DB 69 SEQ. ID 0190
UUACAUCAACAAAGUAGAA DB 70 SEQ. ID 0191 UACAUCAACAAAGUAGAAG DB 71
SEQ. ID 0192 ACAUCAACAAAGUAGAAGA DB 72 SEQ. ID 0193
CAUCAACAAAGUAGAAGAG DB 73 SEQ. ID 0194 AUCAACAAAGUAGAAGAGC DB 74
SEQ. ID 0195 UCAACAAAGUAGAAGAGCU DB 75 SEQ. ID 0196
CAACAAAGUAGAAGAGCUA DB 76 SEQ. ID 0197 AACAAAGUAGAAGAGCUAA DB 77
SEQ. ID 0198 ACAAAGUAGAAGAGCUAAA DB 78 SEQ. ID 0199
CAAAGUAGAAGAGCUAAAG DB 79 SEQ. ID 0200 AAAGUAGAAGAGCUAAAGA DB 80
SEQ. ID 0201 AAGUAGAAGAGCUAAAGAA DB 81 SEQ. ID 0202
AGUAGAAGAGCUAAAGAAA DB 82 SEQ. ID 0203 GUAGAAGAGCUAAAGAAAA DB 83
SEQ. ID 0204 UAGAAGAGCUAAAGAAAAA DB 84 SEQ. ID 0205
AGAAGAGCUAAAGAAAAAA DB 85 SEQ. ID 0206 GAAGAGCUAAAGAAAAAAU DB 86
SEQ. ID 0207 AAGAGCUAAAGAAAAAAUA DB 87 SEQ. ID 0208
AGAGCUAAAGAAAAAAUAC DB 88 SEQ. ID 0209 GAGCUAAAGAAAAAAUACG DB 89
SEQ. ID 0210 AGCUAAAGAAAAAAUACGG DB 90 SEQ. ID 0211
GCUAAAGAAAAAAUACGGG Luc 1 SEQ. ID 0212 AUCCUCAUAAAGGCCAAGA Luc 2
SEQ. ID 0213 AGAUCCUCAUAAAGGCCAA Luc 3 SEQ. ID 0214
AGAGAUCCUCAUAAAGGCC Luc 4 SEQ. ID 0215 AGAGAGAUCCUCAUAAAGG Luc 5
SEQ. ID 0216 UCAGAGAGAUCCUCAUAAA Luc 6 SEQ. ID 0217
AAUCAGAGAGAUCCUCAUA Luc 7 SEQ. ID 0218 AAAAUCAGAGAGAUCCUCA Luc 8
SEQ. ID 0219 GAAAAAUCAGAGAGAUCCU Luc 9 SEQ. ID 0220
AAGAAAAAUCAGAGAGAUC Luc 10 SEQ. ID 0221 GCAAGAAAAAUCAGAGAGA Luc 11
SEQ. ID 0222 ACGCAAGAAAAAUCAGAGA Luc 12 SEQ. ID 0223
CGACGCAAGAAAAAUCAGA Luc 13 SEQ. ID 0224 CUCGACGCAAGAAAAAUCA Luc 14
SEQ. ID 0225 AACUCGACGCAAGAAAAAU Luc 15 SEQ. ID 0226
AAAACUCGACGCAAGAAAA Luc 16 SEQ. ID 0227 GGAAAACUCGACGCAAGAA Luc 17
SEQ. ID 0228 CCGGAAAACUCGACGCAAG Luc 18 SEQ. ID 0229
UACCGGAAAACUCGACGCA Luc 19 SEQ. ID 0230 CUUACCGGAAAACUCGACG Luc 20
SEQ. ID 0231 GUCUUACCGGAAAACUCGA Luc 21 SEQ. ID 0232
AGGUCUUACCGGAAAACUC Luc 22 SEQ. ID 0233 AAAGGUCUUACCGGAAAAC Luc 23
SEQ. ID 0234 CGAAAGGUCUUACCGGAAA Luc 24 SEQ. ID 0235
ACCGAAAGGUCUUACCGGA Luc 25 SEQ. ID 0236 GUACCGAAAGGUCUUACCG Luc 26
SEQ. ID 0237 AAGUACCGAAAGGUCUUAC Luc 27 SEQ. ID 0238
CGAAGUACCGAAAGGUCUU Luc 28 SEQ. ID 0239 GACGAAGUACCGAAAGGUC Luc 29
SEQ. ID 0240 UGGACGAAGUACCGAAAGG Luc 30 SEQ. ID 0241
UGUGGACGAAGUACCGAAA Luc 31 SEQ. ID 0242 UUUGUGGACGAAGUACCGA Luc 32
SEQ. ID 0243 UGUUUGUGGACGAAGUACC Luc 33 SEQ. ID 0244
UGUGUUUGUGGACGAAGUA Luc 34 SEQ. ID 0245 GUUGUGUUUGUGGACGAAG Luc 35
SEQ. ID 0246 GAGUUGUGUUUGUGGACGA Luc 36 SEQ. ID 0247
AGGAGUUGUGUUUGUGGAC Luc 37 SEQ. ID 0248 GGAGGAGUUGUGUUUGUGG Luc 38
SEQ. ID 0249 GCGGAGGAGUUGUGUUUGU Luc 39 SEQ. ID 0250
GCGCGGAGGAGUUGUGUUU Luc 40 SEQ. ID 0251 UUGCGCGGAGGAGUUGUGU Luc 41
SEQ. ID 0252 AGUUGCGCGGAGGAGUUGU Luc 42 SEQ. ID 0253
AAAGUUGCGCGGAGGAGUU Luc 43 SEQ. ID 0254 AAAAAGUUGCGCGGAGGAG Luc 44
SEQ. ID 0255 CGAAAAAGUUGCGCGGAGG Luc 45 SEQ. ID 0256
CGCGAAAAAGUUGCGCGGA Luc 46 SEQ. ID 0257 ACCGCGAAAAAGUUGCGCG Luc 47
SEQ. ID 0258 CAACCGCGAAAAAGUUGCG Luc 48 SEQ. ID 0259
AACAACCGCGAAAAAGUUG Luc 49 SEQ. ID 0260 GUAACAACCGCGAAAAAGU Luc 50
SEQ. ID 0261 AAGUAACAACCGCGAAAAA Luc 51 SEQ. ID 0262
UCAAGUAACAACCGCGAAA Luc 52 SEQ. ID 0263 AGUCAAGUAACAACCGCGA Luc 53
SEQ. ID 0264 CCAGUCAAGUAACAACCGC Luc 54 SEQ. ID 0265
CGCCAGUCAAGUAACAACC Luc 55 SEQ. ID 0266 GUCGCCAGUCAAGUAACAA Luc 56
SEQ. ID 0267 ACGUCGCCAGUCAAGUAAC Luc 57 SEQ. ID 0268
UUACGUCGCCAGUCAAGUA Luc 58 SEQ. ID 0269 GAUUACGUCGCCAGUCAAG Luc 59
SEQ. ID 0270 UGGAUUACGUCGCCAGUCA Luc 60 SEQ. ID 0271
CGUGGAUUACGUCGCCAGU Luc 61 SEQ. ID 0272 AUCGUGGAUUACGUCGCCA Luc 62
SEQ. ID 0273 AGAUCGUGGAUUACGUCGC Luc 63 SEQ. ID 0274
AGAGAUCGUGGAUUACGUC Luc 64 SEQ. ID 0275 AAAGAGAUCGUGGAUUACG Luc 65
SEQ. ID 0276 AAAAAGAGAUCGUGGAUUA Luc 66 SEQ. ID 0277
GGAAAAAGAGAUCGUGGAU Luc 67 SEQ. ID 0278 ACGGAAAAAGAGAUCGUGG Luc 68
SEQ. ID 0279 UGACGGAAAAAGAGAUCGU Luc 69 SEQ. ID 0280
GAUGACGGAAAAAGAGAUC Luc 70 SEQ. ID 0281 ACGAUGACGGAAAAAGAGA
Luc 71 SEQ. ID 0282 AGACGAUGACGGAAAAAGA Luc 72 SEQ. ID 0283
AAAGACGAUGACGGAAAAA Luc 73 SEQ. ID 0284 GGAAAGACGAUGACGGAAA Luc 74
SEQ. ID 0285 ACGGAAAGACGAUGACGGA Luc 75 SEQ. ID 0286
GCACGGAAAGACGAUGACG Luc 76 SEQ. ID 0287 GAGCACGGAAAGACGAUGA Luc 77
SEQ. ID 0288 UGGAGCACGGAAAGACGAU Luc 78 SEQ. ID 0289
UUUGGAGCACGGAAAGACG Luc 79 SEQ. ID 0290 GUUUUGGAGCACGGAAAGA Luc 80
SEQ. ID 0291 UUGUUUUGGAGCACGGAAA Luc 81 SEQ. ID 0292
UGUUGUUUUGGAGCACGGA Luc 82 SEQ. ID 0293 GUUGUUGUUUUGGAGCACG Luc 83
SEQ. ID 0294 CCGUUGUUGUUUUGGAGCA Luc 84 SEQ. ID 0295
CGCCGUUGUUGUUUUGGAG Luc 85 SEQ. ID 0296 GCCGCCGUUGUUGUUUUGG Luc 86
SEQ. ID 0297 CCGCCGCCGUUGUUGUUUU Luc 87 SEQ. ID 0298
UCCCGCCGCCGUUGUUGUU Luc 88 SEQ. ID 0299 CUUCCCGCCGCCGUUGUUG Luc 89
SEQ. ID 0300 AACUUCCCGCCGCCGUUGU Luc 90 SEQ. ID 0301
UGAACUUCCCGCCGCCGUU
Example II
Validation of the Algorithm Using DBI, Luciferase, PLK, EGFR, and
SEAP
[0396] The algorithm (Formula VIII) identified siRNAs for five
genes, human DBI, firefly luciferase (fLuc), renilla luciferase
(rLuc), human PLK, and human secreted alkaline phosphatase (SEAP).
Four individual siRNAs were selected on the basis of their
SMARTSCORES.TM. derived by analysis of their sequence using Formula
VIII (all of the siRNAs would be selected with Formula IX as well)
and analyzed for their ability to silence their targets expression.
In addition to the scoring, a BLAST search was conducted for each
siRNA. To minimize the potential for off-target silencing effects,
only those target sequences with more than three mismatches against
un-related sequences were selected. Semizarov, et al. (2003)
Specificity of short interfering RNA determined through gene
expression signatures, Proc. Natl. Acad. Sci. USA, 100:6347. These
duplexes were analyzed individually and in pools of 4 and compared
with several siRNAs that were randomly selected. The functionality
was measured as a percentage of targeted gene knockdown as compared
to controls. All siRNAs were transfected as described by the
methods above at 100 nM concentration into HEK293 using
Lipofectamine 2000. The level of the targeted gene expression was
evaluated by B-DNA as described above and normalized to the
non-specific control. FIG. 10 shows that the siRNAs selected by the
algorithm disclosed herein were significantly more potent than
randomly selected siRNAs. The algorithm increased the chances of
identifying an F50 siRNA from 48% to 91%, and an F80 siRNA from 13%
to 57%. In addition, pools of SMART siRNA silence the selected
target better than randomly selected pools (see FIG. 10F).
Example III
Validation of the Algorithm Using Genes Involved in
Clathrin-Dependent Endocytosis
[0397] Components of clathrin-mediated endocytosis pathway are key
to modulating intracellular signaling and play important roles in
disease. Chromosomal rearrangements that result in fusion
transcripts between the Mixed-Lineage Leukemia gene (MLL) and CALM
(clathrin assembly lymphoid myeloid leukemia gene) are believed to
play a role in leukemogenesis. Similarly, disruptions in Rab7 and
Rab9, as well as HIP1 (Huntingtin-interacting protein), genes that
are believed to be involved in endocytosis, are potentially
responsible for ailments resulting in lipid storage, and neuronal
diseases, respectively. For these reasons, siRNA directed against
clathrin and other genes involved in the clathrin-mediated
endocytotic pathway are potentially important research and
therapeutic tools.
[0398] siRNAs directed against genes involved in the
clathrin-mediated endocytosis pathways were selected using Formula
VIII. The targeted genes were clathrin heavy chain (CHC, accession
# NM.sub.--004859), clathrin light chain A (CLCa. NM.sub.--001833),
clathrin light chain B (CLCb, NM.sub.--001834), CALM (U45976),
.beta.2 subunit of AP-2 (.beta.2, NM.sub.--001282), Eps15
(NM.sub.--001981), Eps15R (NM.sub.--021235), dynamin II (DYNII,
NM.sub.--004945), Rab5a (BC001267), Rab5b (NM.sub.--002868), Rab5c
(AF141304), and EEA.1 (XM.sub.--018197).
[0399] For each gene, four siRNAs duplexes with the highest scores
were selected and a BLAST search was conducted for each of them
using the Human EST database. In order to minimize the potential
for off-target silencing effects, only those sequences with more
than three mismatches against un-related sequences were used. All
duplexes were synthesized at Dharmacon, Inc. as 21-mers with 3'-UU
overhangs using a modified method of 2'-ACE chemistry, Scaringe
(2000) Advanced 5'-silyl-2'-orthoester approach to RNA
oligonucleotide synthesis, Methods Enzymol. 317:3, and the
antisense strand was chemically phosphorylated to insure maximized
activity.
[0400] HeLa cells were grown in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum, antibiotics and
glutamine. siRNA duplexes were resuspended in 1.times.siRNA
Universal buffer (Dharmacon, Inc.) to 20 .mu.M prior to
transfection. HeLa cells in 12-well plates were transfected twice
with 4 .mu.l of 20 .mu.M siRNA duplex in 3 .mu.l Lipofectamine 2000
reagent (Invitrogen, Carlsbad, Calif., USA) at 24-hour intervals.
For the transfections in which 2 or 3 siRNA duplexes were included,
the amount of each duplex was decreased, so that the total amount
was the same as in transfections with single siRNAs. Cells were
plated into normal culture medium 12 hours prior to experiments,
and protein levels were measured 2 or 4 days after the first
transfection.
[0401] Equal amounts of lysates were resolved by electrophoresis,
blotted, and stained with the antibody specific to targeted
protein, as well as antibodies specific to unrelated proteins, PP1
phosphatase and Tsg101 (not shown). The cells were lysed in Triton
X-100/glycerol solubilization buffer as described previously.
Tebar, Bohlander, & Sorkin (1999) Clathrin Assembly Lymphoid
Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated
Pits, Interactions with Clathrin, and the Impact of Overexpression
on Clathrin-mediated Traffic, Mol. Biol. Cell, 10:2687. Cell
lysates were electrophoresed, transferred to nitrocellulose
membranes, and Western blotting was performed with several
antibodies followed by detection using enhanced chemiluminescence
system (Pierce, Inc). Several x-ray films were analyzed to
determine the linear range of the chemiluminescence signals, and
the quantifications were performed using densitometry and
AlphaImager v5.5 software (Alpha Innotech Corporation). In
experiments with Eps15R-targeted siRNAs, cell lysates were
subjected to immunoprecipitation with Ab860, and Eps15R was
detected in immunoprecipitates by Western blotting as described
above.
[0402] The antibodies to assess the levels of each protein by
Western blot were obtained from the following sources: monoclonal
antibody to clathrin heavy chain (TD.1) was obtained from American
Type Culture Collection (Rockville, Md., USA); polyclonal antibody
to dynamin II was obtained from Affinity Bioreagents, Inc. (Golden,
Colo., USA); monoclonal antibodies to EEA.1 and Rab5a were
purchased from BD Transduction Laboratories (Los Angeles, Calif.,
USA); the monoclonal antibody to Tsg101 was purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA); the monoclonal
antibody to GFP was from ZYMED Laboratories Inc. (South San
Francisco, Calif., USA); the rabbit polyclonal antibodies Ab32
specific to .alpha.-adaptins and Ab20 to CALM were described
previously (Sorkin et al. (1995) Stoichiometric Interaction of the
Epidermal Growth Factor Receptor with the Clathrin-associated
Protein Complex AP-2, J. Biol. Chem., 270:619), the polyclonal
antibodies to clathrin light chains A and B were kindly provided by
Dr. F. Brodsky (UCSF); monoclonal antibodies to PP1 (BD
Transduction Laboratories) and .alpha.-Actinin (Chemicon) were
kindly provided by Dr. M. Dell'Acqua (University of Colorado);
Eps15 Ab577 and Eps15R Ab860 were kindly provided by Dr. P. P. Di
Fiore (European Cancer Institute).
[0403] FIG. 11 demonstrates the in vivo functionality of 48
individual siRNAs, selected using Formula VIII (most of them will
meet the criteria incorporated by Formula IX as well) targeting 12
genes. Various cell lines were transfected with siRNA duplexes
(Dup1-4) or pools of siRNA duplexes (Pool), and the cells were
lysed 3 days after transfection with the exception of CALM (2 days)
and .beta.2 (4 days).
[0404] Note a .beta.1-adaptin band (part of AP-1 Golgi adaptor
complex) that runs slightly slower than .beta.2 adaptin. CALM has
two splice variants, 66 and 72 kD. The full-length Eps15R (a
doublet of 130 kD) and several truncated spliced forms of
.about.100 kD and .about.70 kD were detected in Eps15R
immunoprecipitates (shown by arrows). The cells were lysed 3 days
after transfection. Equal amounts of lysates were resolved by
electrophoresis and blotted with the antibody specific to a
targeted protein (GFP antibody for YFP fusion proteins) and the
antibody specific to unrelated proteins PP1 phosphatase or
.alpha.-actinin, and TSG101. The amount of protein in each specific
band was normalized to the amount of non-specific proteins in each
lane of the gel. Nearly all of them appear to be functional, which
establishes that Formula VIII and IX can be used to predict siRNAs'
functionality in general in a genome wide manner.
[0405] To generate the fusion of yellow fluorescent protein (YFP)
with Rab5b or Rab5c (YFP-Rab5b or YFP-Rab5c), a DNA fragment
encoding the full-length human Rab5b or Rab5c was obtained by PCR
using Pfu polymerase (Stratagene) with a SacI restriction site
introduced into the 5' end and a KpnI site into the 3' end and
cloned into pEYFP-C1 vector (CLONTECH, Palo Alto, Calif., USA).
GFP-CALM and YFP-Rab5a were described previously (Tebar, Bohlander,
& Sorkin (1999) Clathrin Assembly Lymphoid Myeloid Leukemia
(CALM) Protein: Localization in Endocytic-coated Pits, Interactions
with Clathrin, and the Impact of Overexpression on
Clathrin-mediated Traffic, Mol. Biol. Cell 10:2687).
Example IV
Validation of the Algorithm Using Eg5, GADPH, ATE1, MEK2, MEK1, QB,
Lamina/C, C-MYC, Human Cyclophilin, and Mouse Cyclophilin
[0406] A number of genes have been identified as playing
potentially important roles in disease etiology. Expression
profiles of normal and diseased kidneys has implicated Edg5 in
immunoglobulin A neuropathy, a common renal glomerular disease.
Myc1, MEK1/2 and other related kinases have been associated with
one or more cancers, while lamins have been implicated in muscular
dystrophy and other diseases. For these reasons, siRNA directed
against the genes encoding these classes of molecules would be
important research and therapeutic tools.
[0407] FIG. 12 illustrates four siRNAs targeting 10 different genes
(Table V for sequence and accession number information) that were
selected according to the Formula VIII and assayed as individuals
and pools in HEK293 cells. The level of siRNA induced silencing was
measured using the B-DNA assay. These studies demonstrated that
thirty-six out of the forty individual SMART-selected siRNA tested
are functional (90%) and all 10 pools are fully functional.
Example V
Validation of the Algorithm Using Bcl2
[0408] Bcl-2 is a .about.25 kD, 205-239 amino acid, anti-apoptotic
protein that contains considerable homology with other members of
the BCL family including BCLX, MCL1, BAX, BAD, and BIK. The protein
exists in at least two forms (Bcl2a, which has a hydrophobic tail
for membrane anchorage, and Bcl2b, which lacks the hydrophobic
tail) and is predominantly localized to the mitochondrial membrane.
While Bcl2 expression is widely distributed, particular interest
has focused on the expression of this molecule in B and T cells.
Bcl2 expression is down-regulated in normal germinal center B cells
yet in a high percentage of follicular lymphomas, Bcl2 expression
has been observed to be elevated. Cytological studies have
identified a common translocation ((14;18)(q32;q32)) amongst a high
percentage (>70%) of these lymphomas. This genetic lesion places
the Bcl2 gene in juxtaposition to immunoglobulin heavy chain gene
(IgH) encoding sequences and is believed to enforce inappropriate
levels of gene expression, and resistance to programmed cell death
in the follicle center B cells. In other cases, hypomethylation of
the Bcl2 promoter leads to enhanced expression and again,
inhibition of apoptosis. In addition to cancer, dysregulated
expression of Bcl-2 has been correlated with multiple sclerosis and
various neurological diseases.
[0409] The correlation between Bcl-2 translocation and cancer makes
this gene an attractive target for RNAi. Identification of siRNA
directed against the bcl2 transcript (or Bcl2-IgH fusions) would
further our understanding Bcl2 gene function and possibly provide a
future therapeutic agent to battle diseases that result from
altered expression or function of this gene.
In Silico Identification of Functional siRNA
[0410] To identify functional and hyperfunctional siRNA against the
Bcl2 gene, the sequence for Bcl-2 was downloaded from the NCBI
Unigene database and analyzed using the Formula VIII algorithm. As
a result of these procedures, both the sequence and
SMARTSCORES.TM., or siRNA rankings of the Bcl2 siRNA were obtained
and ranked according to their functionality. Subsequently, these
sequences were BLAST'ed (database) to insure that the selected
sequences were specific and contained minimal overlap with
unrelated genes. The SMARTSCORES.TM., or siRNA rankings for the top
10 Bcl-2 siRNA are identified in FIG. 13.
In Vivo Testing of Bcl-2 SiRNA
[0411] Bcl-2 siRNAs having the top ten SMARTSCORES.TM., or siRNA
rankings were selected and tested in a functional assay to
determine silencing efficiency. To accomplish this, each of the ten
duplexes were synthesized using 2'-O-ACE chemistry and transfected
at 100 nM concentrations into cells. Twenty-four hours later assays
were performed on cell extracts to assess the degree of target
silencing. Controls used in these experiments included mock
transfected cells, and cells that were transfected with a
non-specific siRNA duplex.
[0412] The results of these experiments are presented below (and in
FIG. 14) and show that all ten of the selected siRNA induce 80% or
better silencing of the Bcl2 message at 100 nM concentrations.
These data verify that the algorithm successfully identified
functional Bcl2 siRNA and provide a set of functional agents that
can be used in experimental and therapeutic environments.
TABLE-US-00007 siRNA 1 GGGAGAUAGUGAUGAAGUA SEQ. ID NO. 302 siRNA 2
GAAGUACAUCCAUUAUAAG SEQ. ID NO. 303 siRNA 3 GUACGACAACCGGGAGAUA
SEQ. ID NO. 304 siRNA 4 AGAUAGUGAUGAAGUACAU SEQ. ID NO. 305 siRNA 5
UGAAGACUCUGCUCAGUUU SEQ. ID NO. 306 siRNA 6 GCAUGCGGCCUCUGUUUGA
SEQ. ID NO. 307 siRNA 7 UGCGGCCUCUGUUUGAUUU SEQ. ID NO. 308 siRNA 8
GAGAUAGUGAUGAAGUACA SEQ. ID NO. 309 siRNA 9 GGAGAUAGUGAUGAAGUAC
SEQ. ID NO. 310 siRNA 10 GAAGACUCUGCUCAGUUUG SEQ. ID NO. 311 Bcl2
siRNA: Sense Strand, 5'.fwdarw.3'
Example VI
Sequences Selected by the Algorithm
[0413] Sequences of the siRNAs selected using Formulas (Algorithms)
VIII and IX with their corresponding ranking, which have been
evaluated for the silencing activity in vivo in the present study
(Formula VIII and IX, respectively) are shown in Table V. It should
be noted that the "t" residues in Table V, and elsewhere, when
referring to siRNA, should be replaced by "u" residues.
TABLE-US-00008 TABLE V FORMULA FORMULA GENE Name SEQ. ID No.
FTLLSEQTENCE VIII IX CLTC NM_004859 0312 GAAAGAATCTGTAGAGAAA 76
94.2 CLTC NM_004859 0313 GCAATGAGCTGTTTGAAGA 65 39.9 CLTC NM_004859
0314 TGACAAAGGTGGATAAATT 57 38.2 CLTC NM_004859 0315
GGAAATGGATCTCTTTGAA 54 49.4 CLTA NM_001833 0316 GGAAACTAATGGTCCAACA
22 55.5 CLTA NM_001833 0317 AGACAGTTATGCAGCTATT 4 22.9 CLTA
NM_001833 0318 CCAATTCTCGGAAGCAAGA 1 17 CLTA NM_001833 0319
GAAAGTAATGGTCCAACAG -1 -13 CLTB NM_001834 0320 GCGCCAGAGTGAACAAGTA
17 57.5 CLTB NM_001834 0321 GAAGGTGGCCCAGCTATGT 15 -8.6 CLTB
NM_001834 0322 GGAACCAGCGCCAGAGTGA 13 40.5 CLTB NM_001834 0323
GAGCGAGATTGCAGGCATA 20 61.7 CALM U45976 0324 GTTACTATCTGATCACTTG 36
-34.6 CALM U45976 0325 GAAATGGAACCACTAAGAA 33 46.1 CALM U45976 0326
GGAAATGGAACCACTAAGA 30 61.2 CALM U45976 0327 CAACTACACTTTCCAATGC 28
6.8 EPS15 NM_001981 0328 CCACCAAGATTTCATGATA 48 25.2 EPS15
NM_001981 0329 GATCGGAACTCCAACAAGA 43 49.3 EPS15 NM_001981 0330
AAACGGAGCTACAGATTAT 39 11.5 EPS15 NM_001981 0331
CCACACAGCATTCTTGTAA 33 -23.6 EPS15R NM_021235 0332
GAAGTTACCTTGAGCAATC 48 33 EPS15R NM_021235 0333 GGACTTGGCCGATCCAGAA
27 33 EPS15R NM_021235 0334 GCACTTGGATCGAGATGAG 20 1.3 EPS15R
NM_021235 0335 CAAAGACCAATTCGCGTTA 17 27.7 DNM2 NM_004945 0336
CCGAATCAATCGCATCTTC 6 -29.6 DNM2 NM_004945 0337 GACATGATCCTGCAGTTCA
5 -14 DNM2 NM_004945 0338 GAGCGAATCGTCACCACTT 5 24 DNM2 NM_004945
0339 CCTCCGAGCTGGCGTCTAC -4 -63.6 ARF6 AF93885 0340
TCACATGGTTAACCTCTAA 27 -21.1 ARF6 AF93885 0341 GATGAGGGACGCCATAATC
7 -38.4 ARF6 AF93885 0342 CCTCTAACTACAAATCTTA 4 16.9 ARF6 AF93885
0343 GGAAGGTGCTATCCAAAAT 4 11.5 RAB5A BC001267 0344
GCAAGCAAGTCCTAACATT 40 25.1 RAB5A BC001267 0345 GGAAGAGGAGTAGACCTTA
17 50.1 RAB5A BC001267 0346 AGGAATCAGTGTTGTAGTA 16 11.5 RAB5A
BC001267 0347 GAAGAGGAGTAGACCTTAC 12 7 RAB5B NM_002868 0348
GAAAGTCAAGCCTGGTATT 14 18.1 RAB5B NM_002868 0349
AAAGTCAAGCCTGGTATTA 6 -17.8 RAB5B NM_002868 0350
GCTATGAACGTGAATGATC 3 -21.1 RAB5B NM_002868 0351
CAAGCCTGGTATTACGTTT -7 -37.5 RAB5C AF141304 0352
GGAACAAGATCTGTCAATT 38 51.9 RAB5C AF141304 0353 GCAATGAACGTGAACGAAA
29 43.7 RAB5C AF141304 0354 CAATGAACGTGAACGAAAT 18 43.3 RAB5C
AF141304 0355 GGACAGGAGCGGTATCACA 6 18.2 EEA1 XM_018197 0356
AGACAGAGCTTGAGAATAA 67 64.1 EEA1 XM_018197 0357 GAGAAGATCTTTATGCAAA
60 48.7 EEA1 XM_018197 0358 GAAGAGAAATCAGCAGATA 58 45.7 EEA1
XM_018197 0359 GCAAGTAACTCAACTAACA 56 72.3 AP2B1 NM_001282 0360
GAGCTAATCTGCCACATTG 49 -12.4 AP2B1 NM_001282 0361
GCAGATGAGTTACTAGAAA 44 48.9 AP2B1 NM_001282 0362
CAACTTAATTGTCCAGAAA 41 28.2 AP2B1 NM_001282 0363
CAACACAGGATTCTGATAA 33 -5.8 PLK NM_005030 0364 AGATTGTGCCTAAGTCTCT
-35 -3.4 PLK NM_005030 0365 ATGAAGATCTGGAGGTGAA 0 -4.3 PLK
NM_005030 0366 TTTGAGACTTCTTGCCTAA -5 -27.7 PLK NM_005030 0367
AGATCACCCTCCTTAAATA 15 72.3 GAPDH NM_002046 0368
CAACGGATTTGGTCGTATT 27 -2.8 GAPDH NM_002046 0369
GAAATCCCATCACCATCTT 24 3.9 GAPDH NM_002046 0370 GACCTCAACTACATGGTTT
22 -22.9 GAPDH NM_002046 0371 TGGTTTACATGTTCCAATA 9 9.8 c-Myc 0372
GAAGAAATCGATGTTGTTT 31 -11.7 c-Myc 0373 ACACAAACTTGAACAGCTA 22 51.3
c-Myc 0374 GGAAGAAATCGATGTTGTT 18 26 c-Myc 0375 GAAACGACGAGAACAGTTG
18 -8.9 MAP2K1 NM_002755 0376 GCACATGGATGGAGGTTCT 26 16 MAP2K1
NM_002755 0377 GCAGAGAGAGCAGATTTGA 16 0.4 MAP2K1 NM_002755 0378
GAGGTTCTCTGGATCAAGT 14 15.5 MAP2K1 NM_002755 0379
GAGCAGATTTGAAGCAACT 14 18.5 MAP2K2 NM_030662 0380
CAAAGACGATGACTTCGAA 37 26.4 MAP2K2 NM_030662 0381
GATCAGCATTTGCATGGAA 24 -0.7 MAP2K2 NM_030662 0382
TCCAGGAGTTTGTCAATAA 17 -4.5 MAP2K2 NM_030662 0383
GGAAGCTGATCCACCTTGA 16 59.2 KNSL1(EG5) NM_004523 0384
GCAGAAATCTAAGGATATA 53 35.8 KNSL1(EG5) NM_004523 0385
CAACAAGGATGAAGTCTAT 50 18.3 KNSL1(EC5) NM_004523 0386
CAGCAGAAATCTAAGGATA 41 32.7 KNSL1(EG5) NM_004523 0387
CTAGATGGCTTTCTCAGTA 39 3.9 CyclophilinA NM_021130 0388
AGACAAGGTCCCAAAGACA -16 58.1 CyclophilinA NM_021130 0389
GGAATGGCAAGACCAGCAA -6 36 CyclophilinA NM_021130 0390
AGAATTATTCCAGGGTTTA -3 16.1 CyclophilinA NM_021130 0391
GCAGACAAGGTCCCAAAGA 8 8.9 LAMIN A/C NM_170707 0392
AGAAGCAGCTTCAGGATGA 31 38.8 LAMIN A/C NM_170707 0393
GAGCTTGACTTCCAGAAGA 33 22.4 LAMIN A/C NM_170707 0394
CCACCGAAGTTCACCCTAA 21 27.5 LAMIN A/C NM_170707 0395
GAGAAGAGCTCCTCCATCA 55 30.1 CyclophilinB M60857 0396
GAAAGAGCATCTACGGTGA 41 83.9 CyclophilinB M60857 0397
GAAAGGATTTGGCTACAAA 53 59.1 CyclophilinB M60857 0398
ACAGCAAATTCCATCGTGT -20 28.8 CyclophilinB M60857 0399
GGAAAGACTGTTCCAAAAA 2 27 DBI1 NM_020548 0400 CAACACGCCTCATCCTCTA 27
-7.6 DBI2 NM_020548 0401 CATGAAAGCTTACATCAAC 25 -30.8 DBI3
NM_020548 0402 AAGATGCCATGAAAGCTTA 17 22 DBI4 NM_020548 0403
GCACATACCGCCTGAGTCT 15 3.9 rLUC1 0404 GATCAAATCTGAAGAAGGA 57 49.2
rLUC2 0405 GCCAAGAAGTTTCCTAATA 50 13.7 rLUC3 0406
CAGCATATCTTGAACCATT 41 -2.2 rLUC4 0407 CAACAAAGGAAACGGATGA 39 29.2
SeAP1 NM_031313 0408 CGGAAACGGTCCAGGCTAT 6 26.9 SeAP2 NM_031313
0409 GCTTCGAGCAGACATGATA 4 -11.2 SeAP3 NM_031313 0410
CCTACACGGTCCTCCTATA 4 4.9 SeAP4 NM_031313 0411 GCCAAGAACCTCATCATCT
1 -9.9 fLUC1 0412 GATATGGGCTGAATACAAA 54 40.4 fLUC2 0413
GCACTCTGATTGACAAATA 47 54.7 fLUC3 0414 TGAAGTCTCTGATTAAGTA 46 34.5
fLUC4 0415 TCAGAGAGATCCTCATAAA 40 11.4 mCyclo_1 NM_008907 0416
GCAAGAAGATCACCATTTC 52 46.4 mCyclo_2 NM_008907 0417
GAGAGAAATTTGAGGATGA 36 70.7 mCyclo_3 NM_008907 0418
GAAAGGATTTGGCTATAAG 35 -1.5 mCyclo_4 NM_008907 0419
GAAAGAAGGCATGAACATT 27 10.3 BCL2_1 NM_000633 0420
GGGAGATAGTGATGAAGTA 21 72 BCL2_2 NM_000633 0421 GAAGTACATCCATTATAAG
1 3.3 BCL2_3 NM_000633 0422 CTACGACAACCGGGAGATA 1 35.9 BCL2_4
NM_000633 0423 AGATAGTGATGAAGTACAT -12 22.1 BCL2_5 NM_000633 0424
TGAAGACTCTGCTCAGTTT 36 19.1 BCL2_6 NM_000633 0425
GCATGCGGCCTCTGTTTGA 5 -9.7 QB1 NM_003365.1 0426 GCACACAGCUUACUACAUC
52 -4.8 QB2 NM_003365.1 0427 GAAAUGCCCUGGUAUCUCA 49 22.1 QB3
NM_003365.1 0428 GAAGGAACGUGAUGUGAUC 34 22.9 QB4 NM_003365.1 0429
GCACUACUCCUGUGUGUGA 28 20.4 ATE1-1 NM_007041 0430
GAACCCAGCUGGAGAACUU 45 15.5 ATE1-2 NM_007041 0431
GAUAUACAGUGUGAUCUUA 40 12.2 ATE1-3 NM_007041 0432
GUACUACGAUCCUGAUUAU 37 32.9 ATE1-4 NM_007041 0433
GUGCCGACCUUUACAAUUU 35 18.2
EGFR-1 NM_005228 0434 GAAGGAAACTGAATTCAAA 68 79.4 EGFR-1 NM_005228
0435 GGAAATATGTACTACGAAA 49 49.5 EGFR-1 NM_005228 0436
CCACAAAGCAGTGAATTTA 41 7.6 EGFR-1 NM_005228 0437
GTAACAAGCTCACGCAGTT 40 25.9
[0414] Many of the genes to which the described siRNA are directed
play critical roles in disease etiology. For this reason, the
siRNAs listed in the sequence listing may potentially act as
therapeutic agents. A number of prophetic examples follow and
should be understood in view of the siRNA that are identified in
the sequence listing. To isolate these siRNAs, the appropriate
message sequence for each gene is analyzed using one of the before
mentioned formulas (preferably formula VIII) to identify potential
siRNA targets. Subsequently these targets are BLAST'ed to eliminate
homology with potential off-targets.
Example VII
Evidence for the Benefits of Pooling
[0415] Evidence for the benefits of pooling have been demonstrated
using the reporter gene, luciferase. Ninety siRNA duplexes were
synthesized using Dharmacon proprietary ACE.RTM. chemistry against
one of the standard reporter genes: firefly luciferase. The
duplexes were designed to start two base pairs apart and to cover
approximately 180 base pairs of the luciferase gene (see sequences
in Table III). Subsequently, the siRNA duplexes were co-transfected
with a luciferase expression reporter plasmid into HEK293 cells
using standard transfection protocols and luciferase activity was
assayed at 24 and 48 hours.
[0416] Transfection of individual siRNAs showed standard
distribution of inhibitory effect. Some duplexes were active, while
others were not. FIG. 15 represents a typical screen of ninety
siRNA duplexes (SEQ. ID NO. 0032-0120) positioned two base pairs
apart. As the figure suggests, the functionality of the siRNA
duplex is determined more by a particular sequence of the
oligonucleotide than by the relative oligonucleotide position
within a gene or excessively sensitive part of the mRNA, which is
important for traditional anti-sense technology.
[0417] When two continuous oligonucleotides were pooled together, a
significant increase in gene silencing activity was observed (see
FIGS. 16A and B). A gradual increase in efficacy and the frequency
of pools functionality was observed when the number of siRNAs
increased to 3 and 4 (FIGS. 16A, 16B, 17A, and 17B). Further, the
relative positioning of the oligonucleotides within a pool did not
determine whether a particular pool was functional (see FIGS. 18A
and 18B, in which 100% of pools of oligonucleotides distanced by 2,
10 and 20 base pairs were functional).
[0418] However, relative positioning may nonetheless have an
impact. An increased functionality may exist when the siRNA are
positioned continuously head to toe (5' end of one directly
adjacent to the 3' end of the others).
[0419] Additionally, siRNA pools that were tested performed at
least as well as the best oligonucleotide in the pool, under the
experimental conditions whose results are depicted in FIG. 19.
Moreover, when previously identified non-functional and marginally
(semi) functional siRNA duplexes were pooled together in groups of
five at a time, a significant functional cooperative action was
observed (see FIG. 20). In fact, pools of semi-active
oligonucleotides were 5 to 25 times more functional than the most
potent oligonucleotide in the pool. Therefore, pooling several
siRNA duplexes together does not interfere with the functionality
of the most potent siRNAs within a pool, and pooling provides an
unexpected significant increase in overall functionality
Example VIII
Additional Evidence of the Benefits of Pooling
[0420] Experiments were performed on the following genes:
.beta.-galactosidase, Renilla luciferase, and Secreted alkaline
phosphatase, which demonstrates the benefits of pooling. (see FIGS.
21A, 21B and 21C). Individual and pools of siRNA (described in
Figure legends 21A-C) were transfected into cells and tested for
silencing efficiency. Approximately 50% of individual siRNAs
designed to silence the above-specified genes were functional,
while 100% of the pools that contain the same siRNA duplexes were
functional.
Example IX
Highly Functional siRNA
[0421] Pools of five siRNAs in which each two siRNAs overlap to
10-90% resulted in 98% functional entities (>80% silencing).
Pools of siRNAs distributed throughout the mRNA that were evenly
spaced, covering an approximate 20-2000 base pair range, were also
functional. When the pools of siRNA were positioned continuously
head to tail relative to in RNA sequences and mimicked the natural
products of Dicer cleaved long double stranded RNA, 98% of the
pools evidenced highly functional activity (>95% silencing).
Example X
Human Cyclophilin B
[0422] Table III above lists the siRNA sequences for the human
cyclophilin B protein. A particularly functional siRNA may be
selected by applying these sequences to any of Formula I to VII
above.
[0423] Alternatively, one could pool 2, 3, 4, 5 or more of these
sequences to create a kit for silencing a gene. Preferably, within
the kit there would be at least one sequence that has a relatively
high predicted functionality when any of Formulas I-VII is
applied.
Example XI
Sample Pools of siRNAs and their Application to Human Disease
[0424] The genetic basis behind human disease is well documented
and siRNA may be used as both research or diagnostic tools and
therapeutic agents, either individually or in pools. Genes involved
in signal transduction, the immune response, apoptosis, DNA repair,
cell cycle control, and a variety of other physiological functions
have clinical relevance and therapeutic agents that can modulate
expression of these genes may alleviate some or all of the
associated symptoms. In some instances, these genes can be
described as a member of a family or class of genes and siRNA
(randomly, conventionally, or rationally designed) can be directed
against one or multiple members of the family to induce a desired
result.
[0425] To identify rationally designed siRNA to each gene, the
sequence was analyzed using Formula VIII or Formula X to identify
rationally designed siRNA. To confirm the activity of these
sequences, the siRNA are introduced into a cell type of choice
(e.g., HeLa cells, HEK293 cells) and the levels of the appropriate
message are analyzed using one of several art proven techniques.
siRNA having heightened levels of potency can be identified by
testing each of the before mentioned duplexes at increasingly
limiting concentrations. Similarly, siRNA having increased levels
of longevity can be identified by introducing each duplex into
cells and testing functionality at 24, 48, 72, 96, 120, 144, 168,
and 192 hours after transfection. Agents that induce >95%
silencing at sub-nanomolar concentrations and/or induce functional
levels of silencing for >96 hours are considered
hyperfunctional.
Example XII
Validation of Multigene Knockout Using Rab5 and Eps
[0426] Two or more genes having similar, overlapping functions
often leads to genetic redundancy. Mutations that knockout only one
of, e.g., a pair of such genes (also referred to as homologs)
results in little or no phenotype due to the fact that the
remaining intact gene is capable of fulfilling the role of the
disrupted counterpart. To fully understand the function of such
genes in cellular physiology, it is often necessary to knockout or
knockdown both homologs simultaneously. Unfortunately, concomitant
knockdown of two or more genes is frequently difficult to achieve
in higher organisms (e.g., mice) thus it is necessary to introduce
new technologies dissect gene function. One such approach to
knocking down multiple genes simultaneously is by using siRNA. For
example, FIG. 11 showed that rationally designed siRNA directed
against a number of genes involved in the clathrin-mediated
endocytosis pathway resulted in significant levels of protein
reduction (e.g., >80%). To determine the effects of gene
knockdown on clathrin-related endocytosis, internalization assays
were performed using epidermal growth factor and transferrin.
Specifically, mouse receptor-grade EGF (Collaborative Research
Inc.) and iron-saturated human transferrin (Sigma) were iodinated
as described previously (Jiang, X., Huang, F., Marusyk, A. &
Sorkin, A. (2003) Mol Biol Cell 14, 858-70). HeLa cells grown in
12-well dishes were incubated with .sup.125I-EGF (1 ng/ml) or
.sup.125I-transferrin (1 .mu.g/ml) in binding medium (DM EM, 0.1%
bovine serum albumin) at 37.degree. C., and the ratio of
internalized and surface radioactivity was determined during 5-min
time course to calculate specific internalization rate constant
k.sub.e as described previously (Jiang, X et al.). The measurements
of the uptakes of radiolabeled transferrin and EGF were performed
using short time-course assays to avoid influence of the recycling
on the uptake kinetics, and using low ligand concentration to avoid
saturation of the clathrin-dependent pathway (for EGF Lund, K. A.,
Opresko, L. K., Strarbuck, C., Walsh, B. J. & Wiley, H. S.
(1990) J. Biol. Chem. 265, 15713-13723).
[0427] The effects of knocking down Rab5a, 5b, 5c, Eps, or Eps 15R
(individually) are shown in FIG. 22 and demonstrate that disruption
of single genes has little or no effect on EGF or Tfn
internalization. In contrast, simultaneous knock down of Rab5a, 5b,
and 5c, or Eps and Eps 15R, leads to a distinct phenotype (note:
total concentration of siRNA in these experiments remained constant
with that in experiments in which a single siRNA was introduced,
see FIG. 23). These experiments demonstrate the effectiveness of
using rationally designed siRNA to knockdown multiple genes and
validates the utility of these reagents to override genetic
redundancy.
Example XIII
Validation of Multigene Targeting Using G6PD, GAPDH, PLK, and
UQC
[0428] Further demonstration of the ability to knock down
expression of multiple genes using rationally designed siRNA was
performed using pools of siRNA directed against four separate
genes. To achieve this, siRNA were transfected into cells (total
siRNA concentration of 100 nM) and assayed twenty-four hours later
by B-DNA. Results shown in FIG. 24 show that pools of rationally
designed molecules are capable of simultaneously silencing four
different genes.
Example XIV
Validation of Multigene Knockouts as Demonstrated by Gene
Expression Profiling, a Prophetic Example
[0429] To further demonstrate the ability to concomitantly
knockdown the expression of multiple gene targets, single siRNA or
siRNA pools directed against a collection of genes (e.g., 4, 8, 16,
or 23 different targets) are simultaneously transfected into cells
and cultured for twenty-four hours. Subsequently, mRNA is harvested
from treated (and untreated) cells and labeled with one of two
fluorescent probes dyes (e.g., a red fluorescent probe for the
treated cells, a green fluorescent probe for the control cells.).
Equivalent amounts of labeled RNA from each sample is then mixed
together and hybridized to sequences that have been linked to a
solid support (e.g., a slide, "DNA CHIP"). Following hybridization,
the slides are washed and analyzed to assess changes in the levels
of target genes induced by siRNA.
Example XV
Identifying Hyperfunctional siRNA
[0430] Identification of Hyperfunctional Bcl-2 siRNA
[0431] The ten rationally designed Bcl2 siRNA (identified in FIG.
13, 14) were tested to identify hyperpotent reagents. To accomplish
this, each of the ten Bcl-2 siRNA were individually transfected
into cells at a 300 pM (0.3 nM) concentrations. Twenty-four hours
later, transcript levels were assessed by B-DNA assays and compared
with relevant controls. As shown in FIG. 25, while the majority of
Bcl-2 siRNA failed to induce functional levels of silencing at this
concentration, siRNA 1 and 8 induced >80% silencing, and siRNA 6
exhibited greater than 90% silencing at this subnanomolar
concentration.
By way of prophetic examples, similar assays could be performed
with any of the groups of rationally designed genes described in
the Examples. Thus for instance, rationally designed siRNA
sequences directed against a gene of interest could be introduced
into cells at increasingly limiting concentrations to determine
whether any of the duplexes are hyperfunctional.
Example XVI
Gene Silencing
Prophetic Example
[0432] Below is an example of how one might transfect a cell.
[0433] Select a cell line. The selection of a cell line is usually
determined by the desired application. The most important feature
to RNAi is the level of expression of the gene of interest. It is
highly recommended to use cell lines for which siRNA transfection
conditions have been specified and validated.
[0434] Plate the cells. Approximately 24 hours prior to
transfection, plate the cells at the appropriate density so that
they will be approximately 70-90% confluent, or approximately
1.times.10.sup.5 cells/ml at the time of transfection. Cell
densities that are too low may lead to toxicity due to excess
exposure and uptake of transfection reagent-siRNA complexes. Cell
densities that are too high may lead to low transfection
efficiencies and little or no silencing. Incubate the cells
overnight. Standard incubation conditions for mammalian cells are
37.degree. C. in 5% CO.sub.2. Other cell types, such as insect
cells, require different temperatures and CO.sub.2 concentrations
that are readily ascertainable by persons skilled in the art. Use
conditions appropriate for the cell type of interest.
[0435] siRNA re-suspension. Add 20 .mu.l siRNA universal buffer to
each siRNA to generate a final concentration of 50 .mu.M.
[0436] siRNA-lipid complex formation. Use RNase-free solutions and
tubes. Using the following table, Table XI:
TABLE-US-00009 TABLE XI 96-WELL 24-WELL MIXTURE 1
(TRANSIT-TKO-PLASMID DILUTION MIXTURE) Opti-MEM 9.3 .mu.l 46.5
.mu.l TransIT-TKO (1 .mu.g/.mu.l) 0.5 .mu.l 2.5 .mu.l MIXTURE 1
10.0 .mu.l 50.0 .mu.l FINAL VOLUME MIXTURE 2 (SIRNA DILUTION
MIXTURE) Opti-MEM 9.0 .mu.l 45.0 .mu.l siRNA (1 .mu.M) 1.0 .mu.l
5.0 .mu.l MIXTURE 2 10.0 .mu.l 50.0 .mu.l FINAL VOLUME MIXTURE 3
(SIRNA-TRANSFECTION REAGENT MIXTURE) Mixture 1 10 .mu.l 50 .mu.l
Mixture 2 10 .mu.l 50 .mu.l MIXTURE 3 20 .mu.l 100 .mu.l FINAL
VOLUME Incubate 20 minutes at room temperature MIXTURE 4
(MEDIA-SIRNA/TRANSFECTION REAGENT MIXTURE) Mixture 3 20 .mu.l 100
.mu.l Complete media 80 .mu.l 400 .mu.l MIXTURE 4 100 .mu.l 500
.mu.l FINAL VOLUME Incubate 48 hours at 37.degree. C.
[0437] Transfection. Create a Mixture 1 by combining the specified
amounts of OPTI-MEM serum free media and transfection reagent in a
sterile polystyrene tube. Create a Mixture 2 by combining specified
amounts of each siRNA with OPTI-MEM media in sterile 1 ml tubes.
Create a Mixture 3 by combining specified amounts of Mixture I and
Mixture 2. Mix gently (do not vortex) and incubate at room
temperature for 20 minutes. Create a Mixture 4 by combining
specified amounts of Mixture 3 to complete media. Add appropriate
volume to each cell culture well. Incubate cells with transfection
reagent mixture for 24-72 hours at 37.degree. C. This incubation
time is flexible. The ratio of silencing will remain consistent at
any point in the time period. Assay for gene silencing using an
appropriate detection method such as RT-PCR, Western blot analysis,
immunohistochemistry, phenotypic analysis, mass spectrometry,
fluorescence, radioactive decay, or any other method that is now
known or that comes to be known to persons skilled in the art and
that from reading this disclosure would useful with the present
invention. The optimal window for observing a knockdown phenotype
is related to the mRNA turnover of the gene of interest, although
24-72 hours is standard. Final Volume reflects amount needed in
each well for the desired cell culture format. When adjusting
volumes for a Stock Mix, an additional 10% should be used to
accommodate variability in pipetting, etc. Duplicate or triplicate
assays should be carried out when possible.
Example XVII
siRNAs that Target PCSK9
[0438] siRNAs that target PCSK9 [NCBI accession number
NM.sub.--174936] having sequences generated in silico by the
algorithms herein, are provided. In various embodiments, the siRNAs
are rationally designed. In various embodiments, the siRNAs are
functional or hyperfunctional. These siRNA that have been generated
by the algorithms of the present invention include:
TABLE-US-00010 AAUGGAGGCUUAGCUUUCU; (SEQ. ID NO. 438)
ACACCAGCAUACAGAGUGA; (SEQ. ID NO. 439) AGAUUGGGCUGGCUCUGAA; (SEQ.
ID NO. 440) AGCAGGAACUGAGCCAGAA; (SEQ. ID NO. 441)
CAACUGCAGCGUCCACACA; (SEQ. ID NO. 442) CAACUUUUCUAGACCUGUU; (SEQ.
ID NO. 443) CAAGCAAGCAGACAUUUAU; (SEQ. ID NO. 444)
CAAGCAGACAUUUAUCUUU; (SEQ. ID NO. 445) CAAGGAGGCAGGAUUCUUC; (SEQ.
ID NO. 446) CAAGGGAAGGGCACGGUUA; (SEQ. ID NO. 447)
CACCAAGGAGGCAGGAUUC; (SEQ. ID NO. 448) CACCUUUACUCUGCUCUAU; (SEQ.
ID NO. 449) CAGAAUGACUUUUAUUGAG; (SEQ. ID NO. 450)
CAGAGUGACCACCGGGAAA; (SEQ. ID NO. 451) CAGCCAACCCGCUCCACUA; (SEQ.
ID NO. 452) CAGGAAGCUCGGUGAGUGA; (SEQ. ID NO. 453)
CCAAGCAAGCAGACAUUUA; (SEQ. ID NO. 454) CCAAGCCUCUUCUUACUUC; (SEQ.
ID NO. 455) CCAAGGAGGCAGGAUUCUU; (SEQ. ID NO. 456)
CCAAGGAUCCGUGGAGGUU; (SEQ. ID NO. 457) CCACCAAGGAGGCAGGAUU; (SEQ.
ID NO. 458) CCACGUGGCUGGCAUUGCA; (SEQ. ID NO. 459)
CCACUUCUCUGCCAAAGAU; (SEQ. ID NO. 460) CCAGCUAACUGUGGAGAAG; (SEQ.
ID NO. 461) CCCAGAGCAUCCCGUGGAA; (SEQ. ID NO. 462)
CCCAGGAGCUCCAGUGACA; (SEQ. ID NO. 463) CCUCAUAGGCCUGGAGUUU; (SEQ.
ID NO. 464) CCUGAUUAAUGGAGGCUUA; (SEQ. ID NO. 465)
CCUUUACACUGCUCUAUGC; (SEQ. ID NO. 466) CGGCUGGGCUCCUCAUUUU; (SEQ.
ID NO. 467) CGUCGAGGCGCUCAUGGUU; (SEQ. ID NO. 468)
CUAGACACCAGCAUACAGA; (SEQ. ID NO. 469) CUCAUAGGCCUGGAGUUUA; (SEQ.
ID NO. 470) GAACACAGACCAGGAAGCU; (SEQ. ID NO. 471)
GAACCACAGCCACCUUCCA; (SEQ. ID NO. 472) GACAGGCCAGCAAGUGUGA; (SEQ.
ID NO. 473) GACGAUGCCUGCCUGUACU; (SEQ. ID NO. 474)
GAGCCAGAAACGCAGAUUG; (SEQ. ID NO. 475) GAGGGUGUCUACGCCAUUG; (SEQ.
ID NO. 476) GAGUUGAGGCAGAGACUGA; (SEQ. ID NO. 477)
GAUCCACUUCUCUGCCAAA; (SEQ. ID NO. 478) GAUCCUGCAUGUCUUCCAU; (SEQ.
ID NO. 479) GCAAGCAGACAUUUAUCUU; (SEQ. ID NO. 480)
GCAGAAUGACUUUUAUUGA; (SEQ. ID NO. 481) GCAGAGACUGAUCCACUUC; (SEQ.
ID NO. 482) GCAGCCUCCUUGCCUGGAA; (SEQ. ID NO. 483)
GCAGCCUGGUGGAGGUGUA; (SEQ. ID NO. 484) GCAGGAACUGAGCCAGAAA; (SEQ.
ID NO. 485) GCAGGAUUCUUCCCAUGGA; (SEQ. ID NO. 486)
GCAUUUCACCAUUCAAACA; (SEQ. ID NO. 487) GCCAAGCCUCUUCUUACUU; (SEQ.
ID NO. 488) GCCAGCUGCUCCCAAUGUG; (SEQ. ID NO. 489)
GCCCUCAUCUCCAGCUAAC; (SEQ. ID NO. 490) GCGCCCUGCUCCUGAACUU; (SEQ.
ID NO. 491) GCUAGCAACACCCAAAGGU; (SEQ. ID NO. 492)
GGACCCGCUUCCACAGACA; (SEQ. ID NO. 493) GGACGAUGCCUGCCUCUAC; (SEQ.
ID NO. 494) GGAGAGGGCCAACAACUGU; (SEQ. ID NO. 495)
GGAGCUGGCCUUGAAGUUG; (SEQ. ID NO. 496) GGAGUGAGCCAGGCAGUGA; (SEQ.
ID NO. 497) GGAUUCUUCCCAUGGAUAG; (SEQ. ID NO. 498)
GGCAGAGACUGAUCCACUU; (SEQ. ID NO. 499) GGGAGAGGGCCAACAACUG; (SEQ.
ID NO. 500) GGGCAUUUCACCAUUCAAA; (SEQ. ID NO. 501)
GGGCUGAGCUUUAAAAUGG; (SEQ. ID NO. 502) GGGCUGGGGCUGAGCUUUA; (SEQ.
ID NO. 503) GGGGAUACCUCACCAAGAU; (SEQ. ID NO. 504)
GGUCACCGACUUCGAGAAU; (SEQ. ID NO. 505) GGUCAUGGUCACCGACUUC; (SEQ.
ID NO. 506) GGUCUGGAAUGCAAAGUCA; (SEQ. ID NO. 507)
GGUUAGCGGCACCCUCAUA; (SEQ. ID NO. 508) GUACAGCCGCGUCCUCAAC; (SEQ.
ID NO. 509) UAAUGGAGGCUUAGCUUUC; (SEQ. ID NO. 510)
UCACCAAGAUCCUGCAUGU; (SEQ. ID NO. 511) UCACCGACUUCGAGAAUGU; (SEQ.
ID NO. 512) UCACUGGCCUGGCGGAGAU; (SEQ. ID NO. 513)
UCAUAGGCCUGGAGUUUAU; (SEQ. ID NO. 514) UCUCCUAGACACCAGCAUA; (SEQ.
ID NO. 515) UGACAGCCGUUGCCAUCUG; (SEQ. ID NO. 516)
UGCUGGAGCUGGCCUUGAA; (SEQ. ID NO. 517) UGGCGGAGAUGCUUCUAAG; (SEQ.
ID NO. 518) UGUCCUCUCUGUUGCCUUU; (SEQ. ID NO. 519)
UGUCUUCCAUGGCCUUCUU; (SEQ. ID NO. 520) UUAAAAUGGUUCCGACUUG; (SEQ.
ID NO. 521) UUAAUGGAGGCUUAGCUUU; (SEQ. ID NO. 522)
AAGUCAAGGAGCAUGGAAU; (SEQ. ID NO. 523) AAUGCAAAGUCAAGGAGCA; (SEQ.
ID NO. 524) ACACCAGCAUACAGAGUGA; (SEQ. ID NO. 525)
AGAAUGUGCCCGAGGAGGA; (SEQ. ID NO. 526) CAACUGCAGCGUCCACACA; (SEQ.
ID NO. 527) CAAGGGAAGGGCACGGUUA; (SEQ. ID NO. 528)
CAGAGUGACCACCGGGAAA; (SEQ. ID NO. 529) CCAAGGAUCCGUGGAGGUU; (SEQ.
ID NO. 530) CCACGUGGCUGGCAUUGCA; (SEQ. ID NO. 531)
CCACUUCUCUGCCAAAGAU; (SEQ. ID NO. 532) CCCAGAGCAUCCCGUGGAA; (SEQ.
ID NO. 533) CCUACGUGGUGGUGCUGAA; (SEQ. ID NO. 534)
CCUCAUAGGCCUGGAGUUU; (SEQ. ID NO. 535) CCUGGAGUUUAUUCGGAAA; (SEQ.
ID NO. 536) CGUGGAACCUGGAGCGGAU; (SEQ. ID NO. 537)
CUAGACACCAGCAUACAGA; (SEQ. ID NO. 538) CUCAUAGGCCUGGAGUUUA; (SEQ.
ID NO. 539) GAACCACAGCCACCUUCCA; (SEQ. ID NO. 540)
GACAGGCCAGCAAGUGUGA; (SEQ. ID NO. 541) GACGAUGCCUGCCUCUACU; (SEQ.
ID NO. 542) GAGGGUGUCUACGCCAUUG; (SEQ. ID NO. 543)
GAGUUGAGGCAGAGACUGA; (SEQ. ID NO. 544) GAUCCACUUCUCUGCCAAA; (SEQ.
ID NO. 545) GAUCCUGCAUGUCUUCCAU; (SEQ. ID NO. 546)
GCAGAGACUGAUCCACUUC; (SEQ. ID NO. 547) GCAGCCUGGUGGAGGUGUA; (SEQ.
ID NO. 548) GGACCCGCUUCCACAGACA; (SEQ. ID NO. 549)
GGACGAUGCCUGCCUCUAC; (SEQ. ID NO. 550) GGAGCUGGCCUUGAAGUUG; (SEQ.
ID NO. 551) GGCAGAGACUGAUCCACUU; (SEQ. ID NO. 552)
GGGGAUACCUCACCAAGAU; (SEQ. ID NO. 553) GGUCACCGACUUCGAGAAU; (SEQ.
ID NO. 554) GGUCAUGGUCACCGACUUC; (SEQ. ID NO. 555)
GGUCUGGAAUGCAAAGUCA; (SEQ. ID NO. 556) GGUUAGCGGCACCCUCAUA; (SEQ.
ID NO. 557) GUACAGCCGCGUCCUCAAC; (SEQ. ID NO. 558)
UCACCAAGAUCCUGCAUGU; (SEQ. ID NO. 559) UCACCGACUUCGAGAAUGU; (SEQ.
ID NO. 560) UCAUAGGCCUGGAGUUUAU; (SEQ. ID NO. 561)
UCUCCAUGACACCAGCAUA; (SEQ. ID NO. 562)
UGACAGCCGUUGCCAUCUG; (SEQ. ID NO. 563) UGCUGGAGCUGGCCUUGAA; (SEQ.
ID NO. 564) and UGUCUUCCAUGGCCUUCUU. (SEQ. ID NO. 565)
[0439] Thus, consistent with Example XVII, the present invention
provides an siRNA that targets PCSK9 provided, wherein the siRNA is
selected from the group consisting of SEQ. ID NOs. 438-565.
[0440] In another embodiment, an siRNA is provided, said siRNA
comprising a sense region and an antisense region, wherein said
sense region and said antisense region are at least 90%
complementary, said sense region and said antisense region together
form a duplex region comprising 18-30 base pairs, and said sense
region comprises a sequence that is at least 90% similar to a
sequence selected from the group consisting of: SEQ. ID NOs
438-565.
[0441] In another embodiment, an siRNA is provided wherein the
siRNA comprises a sense region and an antisense region, wherein
said sense region and said antisense region are at least 90%
complementary, said sense region and said antisense region together
form a duplex region comprising 18-30 base pairs, and said sense
region comprises a sequence that is identical to a contiguous
stretch of at least 18 bases of a sequence selected from the group
consisting of: SEQ. ID NOs 438-565.
[0442] In another embodiment, an siRNA is provided wherein the
siRNA comprises a sense region and an antisense region, wherein
said sense region and said antisense region are at least 90%
complementary, said sense region and said antisense region together
form a duplex region comprising 19-30 base pairs, and said sense
region comprises a sequence that is identical to a contiguous
stretch of at least 18 bases of a sequence selected from the group
consisting of: SEQ. ID NOs 438-565.
[0443] In another embodiment, a pool of at least two siRNAs is
provided, wherein said pool comprises a first siRNA and a second
siRNA, said first siRNA comprises a duplex region of length 18-30
base pairs that has a first sense region that is at least 90%
similar to 18 bases of a first sequence selected from the group
consisting of: SEQ. ID NOs 438-565 and said second siRNA comprises
a duplex region of length 18-30 base pairs that has a second sense
region that is at least 90% similar to 18 bases of a second
sequence selected from the group consisting of: SEQ. ID NOs 438-565
and wherein said first sense region and said second sense region
are not identical.
[0444] In another embodiment, a pool of at least two siRNAs is
provided, wherein said pool comprises a first siRNA and a second
siRNA, said first siRNA comprises a duplex region of length 18-30
base pairs that has a first sense region that is identical to at
least 18 bases of a sequence selected from the group consisting of:
SEQ. ID NOs 438-565 and wherein the second siRNA comprises a second
sense region that comprises a sequence that is identical to at
least 18 bases of a sequence selected from the group consisting of:
SEQ. ID NOs 438-565.
[0445] In another embodiment, a pool of at least two siRNAs is
provided, wherein said pool comprises a first siRNA and a second
siRNA, said first siRNA comprises a duplex region of length 19-30
base pairs and has a first sense region comprising a sequence that
is at least 90% similar to a sequence selected from the group
consisting of: SEQ. ID NOs 438-565, and said duplex of said second
siRNA is 19-30 base pairs and comprises a second sense region that
comprises a sequence that is at least 90% similar to a sequence
selected from the group consisting of: SEQ. ID NOs 438-565.
[0446] In another embodiment, a pool of at least two siRNAs is
provided, wherein said pool comprises a first siRNA and a second
siRNA, said first siRNA comprises a duplex region of length 19-30
base pairs and has a first sense region comprising a sequence that
is identical to at least 18 bases of a sequence selected the group
consisting of: SEQ. ID NOs 438-565 and said duplex of said second
siRNA is 19-30 base pairs and comprises a second sense region
comprising a sequence that is identical to a sequence selected from
the group consisting of: SEQ. ID NOs 438-565.
[0447] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departure from the present disclosure as come within
known or customary practice within the art to which the invention
pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
Sequence CWU 1
1
565119RNAArtificial SequenceSynthetic 1nnanannnnu cnaannnna
19219RNAArtificial SequenceSynthetic 2nnanannnnu gnaannnna
19319RNAArtificial SequenceSynthetic 3nnanannnnu unaannnna
19419RNAArtificial SequenceSynthetic 4nnanannnnu cncannnna
19519RNAArtificial SequenceSynthetic 5nnanannnnu gncannnna
19619RNAArtificial SequenceSynthetic 6nnanannnnu uncannnna
19719RNAArtificial SequenceSynthetic 7nnanannnnu cnuannnna
19819RNAArtificial SequenceSynthetic 8nnanannnnu gnuannnna
19919RNAArtificial SequenceSynthetic 9nnanannnnu unuannnna
191019RNAArtificial SequenceSynthetic 10nnancnnnnu cnaannnna
191119RNAArtificial SequenceSynthetic 11nnancnnnnu gnaannnna
191219RNAArtificial SequenceSynthetic 12nnancnnnnu unaannnna
191319RNAArtificial SequenceSynthetic 13nnancnnnnu cncannnna
191419RNAArtificial SequenceSynthetic 14nnancnnnnu gncannnna
191519RNAArtificial SequenceSynthetic 15nnancnnnnu uncannnna
191619RNAArtificial SequenceSynthetic 16nnancnnnnu cnuannnna
191719RNAArtificial SequenceSynthetic 17nnancnnnnu gnuannnna
191819RNAArtificial SequenceSynthetic 18nnancnnnnu unuannnna
191919RNAArtificial SequenceSynthetic 19nnangnnnnu cnaannnna
192019RNAArtificial SequenceSynthetic 20nnangnnnnu gnaannnna
192119RNAArtificial SequenceSynthetic 21nnangnnnnu unaannnna
192219RNAArtificial SequenceSynthetic 22nnangnnnnu cncannnna
192319RNAArtificial SequenceSynthetic 23nnangnnnnu gncannnna
192419RNAArtificial SequenceSynthetic 24nnangnnnnu uncannnna
192519RNAArtificial SequenceSynthetic 25nnangnnnnu cnuannnna
192619RNAArtificial SequenceSynthetic 26nnangnnnnu gnuannnna
192719RNAArtificial SequenceSynthetic 27nnangnnnnu unuannnna
192822RNAArtificial SequenceSynthetic 28gucnnanann nnucnaannn na
2229208DNAHomo Sapiensmisc_feature(1)...(108)Human cyclophilin
fragment 29gttccaaaaa cagtggataa ttttgtggcc ttagctacag gagagaaagg
atttggctac 60aaaaacagca aattccatcg tgtaatcaag gacttcatga tccagggcgg
agacttcacc 120aggggagatg gcacaggagg aaagagcatc tacggtgagc
gcttccccga tgagaacttc 180aaactgaagc actacgggcc tggctggg
20830200DNAPhotinus pyralismisc_feature(1)...(200)Firefly
luciferase fragment 30tgaacttccc gccgccgttg ttgttttgga gcacggaaag
acgatgacgg aaaaagagat 60cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag
ttgcgcggag gagttgtgtt 120tgtggacgaa gtaccgaaag gtcttaccgg
aaaactcgac gcaagaaaaa tcagagagat 180cctcataaag gccaagaagg
20031108DNAHomo sapiensmisc_feature(1)...(108)Human DBL fragment
31acgggcaagg ccaagtggga tgcctggaat gagctgaaag ggacttccaa ggaagatgcc
60atgaaagctt acatcaacaa agtagaagag ctaaagaaaa aatacggg
1083219RNAArtificial SequenceSynthetic 32guuccaaaaa caguggaua
193319RNAArtificial SequenceSynthetic 33uccaaaaaca guggauaau
193419RNAArtificial SequenceSynthetic 34caaaaacagu ggauaauuu
193519RNAArtificial SequenceSynthetic 35aaaacagugg auaauuuug
193619RNAArtificial SequenceSynthetic 36aacaguggau aauuuugug
193719RNAArtificial SequenceSynthetic 37caguggauaa uuuuguggc
193819RNAArtificial SequenceSynthetic 38guggauaauu uuguggccu
193919RNAArtificial SequenceSynthetic 39ggauaauuuu guggccuua
194019RNAArtificial SequenceSynthetic 40auaauuuugu ggccuuagc
194119RNAArtificial SequenceSynthetic 41aauuuugugg ccuuagcua
194219RNAArtificial SequenceSynthetic 42uuuuguggcc uuagcuaca
194319RNAArtificial SequenceSynthetic 43uuguggccuu agcuacagg
194419RNAArtificial SequenceSynthetic 44guggccuuag cuacaggag
194519RNAArtificial SequenceSynthetic 45ggccuuagcu acaggagag
194619RNAArtificial SequenceSynthetic 46ccuuagcuac aggagagaa
194719RNAArtificial SequenceSynthetic 47uuagcuacag gagagaaag
194819RNAArtificial SequenceSynthetic 48agcuacagga gagaaagga
194919RNAArtificial SequenceSynthetic 49cuacaggaga gaaaggauu
195019RNAArtificial SequenceSynthetic 50acaggagaga aaggauuug
195119RNAArtificial SequenceSynthetic 51aggagagaaa ggauuuggc
195219RNAArtificial SequenceSynthetic 52gagagaaagg auuuggcua
195319RNAArtificial SequenceSynthetic 53gagaaaggau uuggcuaca
195419RNAArtificial SequenceSynthetic 54gaaaggauuu ggcuacaaa
195519RNAArtificial SequenceSynthetic 55aaggauuugg cuacaaaaa
195619RNAArtificial SequenceSynthetic 56ggauuuggcu acaaaaaca
195719RNAArtificial SequenceSynthetic 57auuuggcuac aaaaacagc
195819RNAArtificial SequenceSynthetic 58uuggcuacaa aaacagcaa
195919RNAArtificial SequenceSynthetic 59ggcuacaaaa acagcaaau
196019RNAArtificial SequenceSynthetic 60cuacaaaaac agcaaauuc
196119RNAArtificial SequenceSynthetic 61acaaaaacag caaauucca
196219RNAArtificial SequenceSynthetic 62aaaaacagca aauuccauc
196319RNAArtificial SequenceSynthetic 63aaacagcaaa uuccaucgu
196419RNAArtificial SequenceSynthetic 64acagcaaauu ccaucgugu
196519RNAArtificial SequenceSynthetic 65agcaaauucc aucguguaa
196619RNAArtificial SequenceSynthetic 66caaauuccau cguguaauc
196719RNAArtificial SequenceSynthetic 67aauuccaucg uguaaucaa
196819RNAArtificial SequenceSynthetic 68uuccaucgug uaaucaagg
196919RNAArtificial SequenceSynthetic 69ccaucgugua aucaaggac
197019RNAArtificial SequenceSynthetic 70aucguguaau caaggacuu
197119RNAArtificial SequenceSynthetic 71cguguaauca aggacuuca
197219RNAArtificial SequenceSynthetic 72uguaaucaag gacuucaug
197319RNAArtificial SequenceSynthetic 73uaaucaagga cuucaugau
197419RNAArtificial SequenceSynthetic 74aucaaggacu ucaugaucc
197519RNAArtificial SequenceSynthetic 75caaggacuuc augauccag
197619RNAArtificial SequenceSynthetic 76aggacuucau gauccaggg
197719RNAArtificial SequenceSynthetic 77gacuucauga uccagggcg
197819RNAArtificial SequenceSynthetic 78cuucaugauc cagggcgga
197919RNAArtificial SequenceSynthetic 79ucaugaucca gggcggaga
198019RNAArtificial SequenceSynthetic 80augauccagg gcggagacu
198119RNAArtificial SequenceSynthetic 81gauccagggc ggagacuuc
198219RNAArtificial SequenceSynthetic 82uccagggcgg agacuucac
198319RNAArtificial SequenceSynthetic 83cagggcggag acuucacca
198419RNAArtificial SequenceSynthetic 84gggcggagac uucaccagg
198519RNAArtificial SequenceSynthetic 85gcggagacuu caccagggg
198619RNAArtificial SequenceSynthetic 86ggagacuuca ccaggggag
198719RNAArtificial SequenceSynthetic 87agacuucacc aggggagau
198819RNAArtificial SequenceSynthetic 88acuucaccag gggagaugg
198919RNAArtificial SequenceSynthetic 89uucaccaggg gagauggca
199019RNAArtificial SequenceSynthetic 90caccagggga gauggcaca
199119RNAArtificial SequenceSynthetic 91ccaggggaga uggcacagg
199219RNAArtificial SequenceSynthetic 92aggggagaug gcacaggag
199319RNAArtificial SequenceSynthetic 93gggagauggc acaggagga
199419RNAArtificial SequenceSynthetic 94gagauggcac aggaggaaa
199519RNAArtificial SequenceSynthetic 95gauggcacag gaggaaaga
199619RNAArtificial SequenceSynthetic 96uggcacagga ggaaagagc
199719RNAArtificial SequenceSynthetic 97gcacaggagg aaagagcau
199819RNAArtificial SequenceSynthetic 98acaggaggaa agagcaucu
199919RNAArtificial SequenceSynthetic 99aggaggaaag agcaucuac
1910019RNAArtificial SequenceSynthetic 100gaggaaagag caucuacgg
1910119RNAArtificial SequenceSynthetic 101ggaaagagca ucuacggug
1910219RNAArtificial SequenceSynthetic 102aaagagcauc uacggugag
1910319RNAArtificial SequenceSynthetic 103agagcaucua cggugagcg
1910419RNAArtificial SequenceSynthetic 104agcaucuacg gugagcgcu
1910519RNAArtificial SequenceSynthetic 105caucuacggu gagcgcuuc
1910619RNAArtificial SequenceSynthetic 106ucuacgguga gcgcuuccc
1910719RNAArtificial SequenceSynthetic 107uacggugagc gcuuccccg
1910819RNAArtificial SequenceSynthetic 108cggugagcgc uuccccgau
1910919RNAArtificial SequenceSynthetic 109gugagcgcuu ccccgauga
1911019RNAArtificial SequenceSynthetic 110gagcgcuucc ccgaugaga
1911119RNAArtificial SequenceSynthetic 111gcgcuucccc gaugagaac
1911219RNAArtificial SequenceSynthetic 112gcuuccccga ugagaacuu
1911319RNAArtificial SequenceSynthetic 113uuccccgaug agaacuuca
1911419RNAArtificial SequenceSynthetic 114ccccgaugag aacuucaaa
1911519RNAArtificial SequenceSynthetic 115ccgaugagaa cuucaaacu
1911619RNAArtificial SequenceSynthetic 116gaugagaacu ucaaacuga
1911719RNAArtificial SequenceSynthetic 117ugagaacuuc aaacugaag
1911819RNAArtificial SequenceSynthetic 118agaacuucaa acugaagca
1911919RNAArtificial SequenceSynthetic 119aacuucaaac ugaagcacu
1912019RNAArtificial SequenceSynthetic 120cuucaaacug aagcacuac
1912119RNAArtificial SequenceSynthetic 121ucaaacugaa gcacuacgg
1912219RNAArtificial SequenceSynthetic 122acgggcaagg ccaaguggg
1912319RNAArtificial SequenceSynthetic 123cgggcaaggc caaguggga
1912419RNAArtificial SequenceSynthetic 124gggcaaggcc aagugggau
1912519RNAArtificial SequenceSynthetic 125ggcaaggcca agugggaug
1912619RNAArtificial SequenceSynthetic 126gcaaggccaa gugggaugc
1912719RNAArtificial SequenceSynthetic 127caaggccaag ugggaugcc
1912819RNAArtificial SequenceSynthetic 128aaggccaagu gggaugccu
1912919RNAArtificial SequenceSynthetic 129aggccaagug ggaugccug
1913019RNAArtificial SequenceSynthetic 130ggccaagugg gaugccugg
1913119RNAArtificial SequenceSynthetic 131gccaaguggg augccugga
1913219RNAArtificial SequenceSynthetic 132ccaaguggga ugccuggaa
1913319RNAArtificial SequenceSynthetic 133caagugggau gccuggaau
1913419RNAArtificial SequenceSynthetic 134aagugggaug ccuggaaug
1913519RNAArtificial SequenceSynthetic 135agugggaugc cuggaauga
1913619RNAArtificial SequenceSynthetic 136gugggaugcc uggaaugag
1913719RNAArtificial SequenceSynthetic 137ugggaugccu ggaaugagc
1913819RNAArtificial SequenceSynthetic 138gggaugccug gaaugagcu
1913919RNAArtificial SequenceSynthetic 139ggaugccugg aaugagcug
1914019RNAArtificial SequenceSynthetic 140gaugccugga augagcuga
1914119RNAArtificial SequenceSynthetic 141augccuggaa ugagcugaa
1914219RNAArtificial SequenceSynthetic 142ugccuggaau gagcugaaa
1914319RNAArtificial SequenceSynthetic 143gccuggaaug agcugaaag
1914419RNAArtificial SequenceSynthetic 144ccuggaauga gcugaaagg
1914519RNAArtificial SequenceSynthetic 145cuggaaugag cugaaaggg
1914619RNAArtificial SequenceSynthetic 146uggaaugagc ugaaaggga
1914719RNAArtificial SequenceSynthetic 147ggaaugagcu gaaagggac
1914819RNAArtificial SequenceSynthetic 148gaaugagcug aaagggacu
1914919RNAArtificial SequenceSynthetic 149aaugagcuga aagggacuu
1915019RNAArtificial SequenceSynthetic 150augagcugaa agggacuuc
1915119RNAArtificial SequenceSynthetic 151ugagcugaaa gggacuucc
1915219RNAArtificial SequenceSynthetic 152gagcugaaag ggacuucca
1915319RNAArtificial SequenceSynthetic 153agcugaaagg gacuuccaa
1915419RNAArtificial SequenceSynthetic 154gcugaaaggg acuuccaag
1915519RNAArtificial SequenceSynthetic 155cugaaaggga cuuccaagg
1915619RNAArtificial SequenceSynthetic 156ugaaagggac uuccaagga
1915719RNAArtificial SequenceSynthetic 157gaaagggacu uccaaggaa
1915819RNAArtificial SequenceSynthetic 158aaagggacuu ccaaggaag
1915919RNAArtificial SequenceSynthetic 159aagggacuuc caaggaaga
1916019RNAArtificial SequenceSynthetic 160agggacuucc aaggaagau
1916119RNAArtificial SequenceSynthetic 161gggacuucca aggaagaug
1916219RNAArtificial SequenceSynthetic 162ggacuuccaa ggaagaugc
1916319RNAArtificial SequenceSynthetic 163gacuuccaag gaagaugcc
1916419RNAArtificial SequenceSynthetic 164acuuccaagg aagaugcca
1916519RNAArtificial SequenceSynthetic 165cuuccaagga agaugccau
1916619RNAArtificial SequenceSynthetic 166uuccaaggaa gaugccaug
1916719RNAArtificial SequenceSynthetic 167uccaaggaag augccauga
1916819RNAArtificial SequenceSynthetic 168ccaaggaaga ugccaugaa
1916919RNAArtificial SequenceSynthetic 169caaggaagau gccaugaaa
1917019RNAArtificial SequenceSynthetic 170aaggaagaug ccaugaaag
1917119RNAArtificial SequenceSynthetic 171aggaagaugc caugaaagc
1917219RNAArtificial SequenceSynthetic 172ggaagaugcc augaaagcu
1917319RNAArtificial SequenceSynthetic 173gaagaugcca ugaaagcuu
1917419RNAArtificial SequenceSynthetic 174aagaugccau gaaagcuua
1917519RNAArtificial SequenceSynthetic 175agaugccaug aaagcuuac
1917619RNAArtificial SequenceSynthetic 176gaugccauga aagcuuaca
1917719RNAArtificial SequenceSynthetic 177augccaugaa agcuuacau
1917819RNAArtificial SequenceSynthetic 178ugccaugaaa gcuuacauc
1917919RNAArtificial SequenceSynthetic 179gccaugaaag cuuacauca
1918019RNAArtificial SequenceSynthetic 180ccaugaaagc uuacaucaa
1918119RNAArtificial SequenceSynthetic 181caugaaagcu uacaucaac
1918219RNAArtificial SequenceSynthetic 182augaaagcuu acaucaaca
1918319RNAArtificial SequenceSynthetic 183ugaaagcuua caucaacaa
1918419RNAArtificial SequenceSynthetic 184gaaagcuuac aucaacaaa
1918519RNAArtificial SequenceSynthetic 185aaagcuuaca ucaacaaag
1918619RNAArtificial SequenceSynthetic 186aagcuuacau caacaaagu
1918719RNAArtificial SequenceSynthetic 187agcuuacauc aacaaagua
1918819RNAArtificial SequenceSynthetic 188gcuuacauca acaaaguag
1918919RNAArtificial SequenceSynthetic 189cuuacaucaa caaaguaga
1919019RNAArtificial SequenceSynthetic 190uuacaucaac aaaguagaa
1919119RNAArtificial SequenceSynthetic 191uacaucaaca aaguagaag
1919219RNAArtificial SequenceSynthetic 192acaucaacaa aguagaaga
1919319RNAArtificial SequenceSynthetic 193caucaacaaa guagaagag
1919419RNAArtificial SequenceSynthetic 194aucaacaaag uagaagagc
1919519RNAArtificial SequenceSynthetic 195ucaacaaagu agaagagcu
1919619RNAArtificial SequenceSynthetic 196caacaaagua gaagagcua
1919719RNAArtificial SequenceSynthetic 197aacaaaguag aagagcuaa
1919819RNAArtificial SequenceSynthetic 198acaaaguaga agagcuaaa
1919919RNAArtificial SequenceSynthetic 199caaaguagaa gagcuaaag
1920019RNAArtificial SequenceSynthetic 200aaaguagaag agcuaaaga
1920119RNAArtificial SequenceSynthetic 201aaguagaaga gcuaaagaa
1920219RNAArtificial SequenceSynthetic 202aguagaagag cuaaagaaa
1920319RNAArtificial SequenceSynthetic 203guagaagagc uaaagaaaa
1920419RNAArtificial SequenceSynthetic 204uagaagagcu aaagaaaaa
1920519RNAArtificial SequenceSynthetic 205agaagagcua aagaaaaaa
1920619RNAArtificial SequenceSynthetic 206gaagagcuaa agaaaaaau
1920719RNAArtificial SequenceSynthetic 207aagagcuaaa gaaaaaaua
1920819RNAArtificial SequenceSynthetic 208agagcuaaag aaaaaauac
1920919RNAArtificial SequenceSynthetic 209gagcuaaaga aaaaauacg
1921019RNAArtificial SequenceSynthetic 210agcuaaagaa aaaauacgg
1921119RNAArtificial SequenceSynthetic 211gcuaaagaaa aaauacggg
1921219RNAArtificial SequenceSynthetic 212auccucauaa aggccaaga
1921319RNAArtificial SequenceSynthetic 213agauccucau aaaggccaa
1921419RNAArtificial SequenceSynthetic 214agagauccuc auaaaggcc
1921519RNAArtificial SequenceSynthetic 215agagagaucc ucauaaagg
1921619RNAArtificial SequenceSynthetic 216ucagagagau ccucauaaa
1921719RNAArtificial SequenceSynthetic 217aaucagagag auccucaua
1921819RNAArtificial SequenceSynthetic 218aaaaucagag agauccuca
1921919RNAArtificial SequenceSynthetic 219gaaaaaucag agagauccu
1922019RNAArtificial SequenceSynthetic 220aagaaaaauc agagagauc
1922119RNAArtificial SequenceSynthetic 221gcaagaaaaa ucagagaga
1922219RNAArtificial SequenceSynthetic 222acgcaagaaa aaucagaga
1922319RNAArtificial SequenceSynthetic 223cgacgcaaga aaaaucaga
1922419RNAArtificial SequenceSynthetic 224cucgacgcaa gaaaaauca
1922519RNAArtificial SequenceSynthetic 225aacucgacgc aagaaaaau
1922619RNAArtificial SequenceSynthetic 226aaaacucgac gcaagaaaa
1922719RNAArtificial SequenceSynthetic 227ggaaaacucg acgcaagaa
1922819RNAArtificial SequenceSynthetic 228ccggaaaacu cgacgcaag
1922919RNAArtificial SequenceSynthetic 229uaccggaaaa cucgacgca
1923019RNAArtificial SequenceSynthetic 230cuuaccggaa aacucgacg
1923119RNAArtificial SequenceSynthetic 231gucuuaccgg aaaacucga
1923219RNAArtificial SequenceSynthetic 232aggucuuacc ggaaaacuc
1923319RNAArtificial SequenceSynthetic 233aaaggucuua ccggaaaac
1923419RNAArtificial SequenceSynthetic 234cgaaaggucu uaccggaaa
1923519RNAArtificial SequenceSynthetic 235accgaaaggu cuuaccgga
1923619RNAArtificial SequenceSynthetic 236guaccgaaag gucuuaccg
1923719RNAArtificial SequenceSynthetic 237aaguaccgaa aggucuuac
1923819RNAArtificial SequenceSynthetic 238cgaaguaccg aaaggucuu
1923919RNAArtificial SequenceSynthetic 239gacgaaguac cgaaagguc
1924019RNAArtificial SequenceSynthetic 240uggacgaagu accgaaagg
1924119RNAArtificial SequenceSynthetic 241uguggacgaa guaccgaaa
1924219RNAArtificial SequenceSynthetic 242uuuguggacg aaguaccga
1924319RNAArtificial SequenceSynthetic 243uguuugugga cgaaguacc
1924419RNAArtificial SequenceSynthetic 244uguguuugug gacgaagua
1924519RNAArtificial SequenceSynthetic 245guuguguuug uggacgaag
1924619RNAArtificial SequenceSynthetic 246gaguuguguu uguggacga
1924719RNAArtificial SequenceSynthetic 247aggaguugug uuuguggac
1924819RNAArtificial SequenceSynthetic 248ggaggaguug uguuugugg
1924919RNAArtificial SequenceSynthetic 249gcggaggagu uguguuugu
1925019RNAArtificial SequenceSynthetic 250gcgcggagga guuguguuu
1925119RNAArtificial SequenceSynthetic 251uugcgcggag gaguugugu
1925219RNAArtificial SequenceSynthetic 252aguugcgcgg aggaguugu
1925319RNAArtificial SequenceSynthetic 253aaaguugcgc ggaggaguu
1925419RNAArtificial SequenceSynthetic 254aaaaaguugc gcggaggag
1925519RNAArtificial SequenceSynthetic 255cgaaaaaguu gcgcggagg
1925619RNAArtificial SequenceSynthetic 256cgcgaaaaag uugcgcgga
1925719RNAArtificial SequenceSynthetic 257accgcgaaaa aguugcgcg
1925819RNAArtificial SequenceSynthetic 258caaccgcgaa aaaguugcg
1925919RNAArtificial SequenceSynthetic 259aacaaccgcg aaaaaguug
1926019RNAArtificial SequenceSynthetic 260guaacaaccg cgaaaaagu
1926119RNAArtificial SequenceSynthetic 261aaguaacaac cgcgaaaaa
1926219RNAArtificial SequenceSynthetic 262ucaaguaaca accgcgaaa
1926319RNAArtificial SequenceSynthetic 263agucaaguaa caaccgcga
1926419RNAArtificial SequenceSynthetic 264ccagucaagu aacaaccgc
1926519RNAArtificial SequenceSynthetic 265cgccagucaa guaacaacc
1926619RNAArtificial SequenceSynthetic 266gucgccaguc aaguaacaa
1926719RNAArtificial SequenceSynthetic 267acgucgccag ucaaguaac
1926819RNAArtificial SequenceSynthetic 268uuacgucgcc agucaagua
1926919RNAArtificial SequenceSynthetic 269gauuacgucg ccagucaag
1927019RNAArtificial SequenceSynthetic 270uggauuacgu cgccaguca
1927119RNAArtificial SequenceSynthetic 271cguggauuac gucgccagu
1927219RNAArtificial SequenceSynthetic 272aucguggauu acgucgcca
1927319RNAArtificial SequenceSynthetic 273agaucgugga uuacgucgc
1927419RNAArtificial SequenceSynthetic 274agagaucgug gauuacguc
1927519RNAArtificial SequenceSynthetic 275aaagagaucg uggauuacg
1927619RNAArtificial SequenceSynthetic 276aaaaagagau cguggauua
1927719RNAArtificial SequenceSynthetic 277ggaaaaagag aucguggau
1927819RNAArtificial SequenceSynthetic 278acggaaaaag agaucgugg
1927919RNAArtificial SequenceSynthetic 279ugacggaaaa agagaucgu
1928019RNAArtificial SequenceSynthetic 280gaugacggaa aaagagauc
1928119RNAArtificial SequenceSynthetic 281acgaugacgg aaaaagaga
1928219RNAArtificial SequenceSynthetic 282agacgaugac ggaaaaaga
1928319RNAArtificial SequenceSynthetic 283aaagacgaug acggaaaaa
1928419RNAArtificial SequenceSynthetic 284ggaaagacga ugacggaaa
1928519RNAArtificial SequenceSynthetic 285acggaaagac gaugacgga
1928619RNAArtificial SequenceSynthetic 286gcacggaaag acgaugacg
1928719RNAArtificial SequenceSynthetic 287gagcacggaa agacgauga
1928819RNAArtificial SequenceSynthetic 288uggagcacgg aaagacgau
1928919RNAArtificial SequenceSynthetic 289uuuggagcac ggaaagacg
1929019RNAArtificial SequenceSynthetic 290guuuuggagc acggaaaga
1929119RNAArtificial SequenceSynthetic 291uuguuuugga gcacggaaa
1929219RNAArtificial SequenceSynthetic 292uguuguuuug gagcacgga
1929319RNAArtificial SequenceSynthetic 293guuguuguuu uggagcacg
1929419RNAArtificial SequenceSynthetic 294ccguuguugu uuuggagca
1929519RNAArtificial SequenceSynthetic 295cgccguuguu guuuuggag
1929619RNAArtificial SequenceSynthetic 296gccgccguug uuguuuugg
1929719RNAArtificial SequenceSynthetic 297ccgccgccgu uguuguuuu
1929819RNAArtificial SequenceSynthetic 298ucccgccgcc guuguuguu
1929919RNAArtificial SequenceSynthetic 299cuucccgccg ccguuguug
1930019RNAArtificial SequenceSynthetic 300aacuucccgc cgccguugu
1930119RNAArtificial SequenceSynthetic 301ugaacuuccc gccgccguu
1930219RNAArtificial SequenceSynthetic 302gggagauagu gaugaagua
1930319RNAArtificial SequenceSynthetic 303gaaguacauc cauuauaag
1930419RNAArtificial SequenceSynthetic 304guacgacaac cgggagaua
1930519RNAArtificial SequenceSynthetic 305agauagugau gaaguacau
1930619RNAArtificial SequenceSynthetic 306ugaagacucu gcucaguuu
1930719RNAArtificial SequenceSynthetic 307gcaugcggcc ucuguuuga
1930819RNAArtificial SequenceSynthetic 308ugcggccucu guuugauuu
1930919RNAArtificial SequenceSynthetic 309gagauaguga ugaaguaca
1931019RNAArtificial SequenceSynthetic 310ggagauagug augaaguac
1931119RNAArtificial SequenceSynthetic 311gaagacucug cucaguuug
1931219DNAArtificial SequenceSynthetic 312gaaagaatct gtagagaaa
1931319DNAArtificial SequenceSynthetic 313gcaatgagct gtttgaaga
1931419DNAArtificial SequenceSynthetic 314tgacaaaggt ggataaatt
1931519DNAArtificial SequenceSynthetic 315ggaaatggat ctctttgaa
1931619DNAArtificial SequenceSynthetic 316ggaaagtaat ggtccaaca
1931719DNAArtificial SequenceSynthetic 317agacagttat gcagctatt
1931819DNAArtificial SequenceSynthetic 318ccaattctcg gaagcaaga
1931919DNAArtificial SequenceSynthetic 319gaaagtaatg gtccaacag
1932019DNAArtificial SequenceSynthetic 320gcgccagagt gaacaagta
1932119DNAArtificial SequenceSynthetic 321gaaggtggcc cagctatgt
1932219DNAArtificial SequenceSynthetic 322ggaaccagcg ccagagtga
1932319DNAArtificial SequenceSynthetic 323gagcgagatt gcaggcata
1932419DNAArtificial SequenceSynthetic 324gttagtatct gatgacttg
1932519DNAArtificial SequenceSynthetic 325gaaatggaac cactaagaa
1932619DNAArtificial SequenceSynthetic 326ggaaatggaa ccactaaga
1932719DNAArtificial SequenceSynthetic 327caactacact ttccaatgc
1932819DNAArtificial SequenceSynthetic 328ccaccaagat ttcatgata
1932919DNAArtificial SequenceSynthetic 329gatcggaact ccaacaaga
1933019DNAArtificial SequenceSynthetic 330aaacggagct acagattat
1933119DNAArtificial SequenceSynthetic 331ccacacagca ttcttgtaa
1933219DNAArtificial SequenceSynthetic 332gaagttacct tgagcaatc
1933319DNAArtificial SequenceSynthetic 333ggacttggcc gatccagaa
1933419DNAArtificial SequenceSynthetic 334gcacttggat cgagatgag
1933519DNAArtificial SequenceSynthetic 335caaagaccaa ttcgcgtta
1933619DNAArtificial SequenceSynthetic 336ccgaatcaat cgcatcttc
1933719DNAArtificial SequenceSynthetic 337gacatgatcc tgcagttca
1933819DNAArtificial SequenceSynthetic 338gagcgaatcg tcaccactt
1933919DNAArtificial SequenceSynthetic 339cctccgagct ggcgtctac
1934019DNAArtificial SequenceSynthetic 340tcacatggtt aacctctaa
1934119DNAArtificial SequenceSynthetic 341gatgagggac gccataatc
1934219DNAArtificial SequenceSynthetic 342cctctaacta caaatctta
1934319DNAArtificial SequenceSynthetic 343ggaaggtgct atccaaaat
1934419DNAArtificial SequenceSynthetic 344gcaagcaagt cctaacatt
1934519DNAArtificial SequenceSynthetic 345ggaagaggag tagacctta
1934619DNAArtificial SequenceSynthetic 346aggaatcagt gttgtagta
1934719DNAArtificial SequenceSynthetic 347gaagaggagt agaccttac
1934819DNAArtificial SequenceSynthetic 348gaaagtcaag cctggtatt
1934919DNAArtificial SequenceSynthetic 349aaagtcaagc ctggtatta
1935019DNAArtificial SequenceSynthetic 350gctatgaacg tgaatgatc
1935119DNAArtificial SequenceSynthetic 351caagcctggt attacgttt
1935219DNAArtificial SequenceSynthetic 352ggaacaagat ctgtcaatt
1935319DNAArtificial SequenceSynthetic 353gcaatgaacg tgaacgaaa
1935419DNAArtificial SequenceSynthetic 354caatgaacgt gaacgaaat
1935519DNAArtificial SequenceSynthetic 355ggacaggagc ggtatcaca
1935619DNAArtificial SequenceSynthetic 356agacagagct tgagaataa
1935719DNAArtificial SequenceSynthetic 357gagaagatct ttatgcaaa
1935819DNAArtificial SequenceSynthetic 358gaagagaaat cagcagata
1935919DNAArtificial SequenceSynthetic 359gcaagtaact caactaaca
1936019DNAArtificial SequenceSynthetic 360gagctaatct gccacattg
1936119DNAArtificial SequenceSynthetic 361gcagatgagt tactagaaa
1936219DNAArtificial SequenceSynthetic 362caacttaatt gtccagaaa
1936319DNAArtificial SequenceSynthetic 363caacacagga ttctgataa
1936419DNAArtificial SequenceSynthetic 364agattgtgcc taagtctct
1936519DNAArtificial SequenceSynthetic 365atgaagatct ggaggtgaa
1936619DNAArtificial SequenceSynthetic 366tttgagactt cttgcctaa
1936719DNAArtificial SequenceSynthetic 367agatcaccct ccttaaata
1936819DNAArtificial SequenceSynthetic 368caacggattt ggtcgtatt
1936919DNAArtificial SequenceSynthetic 369gaaatcccat caccatctt
1937019DNAArtificial SequenceSynthetic 370gacctcaact acatggttt
1937119DNAArtificial SequenceSynthetic 371tggtttacat gttccaata
1937219DNAArtificial SequenceSynthetic 372gaagaaatcg atgttgttt
1937319DNAArtificial SequenceSynthetic 373acacaaactt gaacagcta
1937419DNAArtificial SequenceSynthetic 374ggaagaaatc gatgttgtt
1937519DNAArtificial SequenceSynthetic 375gaaacgacga gaacagttg
1937619DNAArtificial SequenceSynthetic 376gcacatggat ggaggttct
1937719DNAArtificial SequenceSynthetic 377gcagagagag cagatttga
1937819DNAArtificial SequenceSynthetic 378gaggttctct ggatcaagt
1937919DNAArtificial SequenceSynthetic 379gagcagattt gaagcaact
1938019DNAArtificial SequenceSynthetic 380caaagacgat gacttcgaa
1938119DNAArtificial SequenceSynthetic 381gatcagcatt tgcatggaa
1938219DNAArtificial SequenceSynthetic 382tccaggagtt tgtcaataa
1938319DNAArtificial SequenceSynthetic 383ggaagctgat ccaccttga
1938419DNAArtificial SequenceSynthetic 384gcagaaatct aaggatata
1938519DNAArtificial SequenceSynthetic 385caacaaggat gaagtctat
1938619DNAArtificial SequenceSynthetic 386cagcagaaat ctaaggata
1938719DNAArtificial SequenceSynthetic 387ctagatggct ttctcagta
1938819DNAArtificial SequenceSynthetic 388agacaaggtc ccaaagaca
1938919DNAArtificial SequenceSynthetic 389ggaatggcaa gaccagcaa
1939019DNAArtificial SequenceSynthetic 390agaattattc cagggttta
1939119DNAArtificial SequenceSynthetic 391gcagacaagg tcccaaaga
1939219DNAArtificial SequenceSynthetic 392agaagcagct tcaggatga
1939319DNAArtificial SequenceSynthetic 393gagcttgact tccagaaga
1939419DNAArtificial SequenceSynthetic 394ccaccgaagt tcaccctaa
1939519DNAArtificial SequenceSynthetic 395gagaagagct cctccatca
1939619DNAArtificial SequenceSynthetic 396gaaagagcat ctacggtga
1939719DNAArtificial SequenceSynthetic 397gaaaggattt ggctacaaa
1939819DNAArtificial SequenceSynthetic 398acagcaaatt ccatcgtgt
1939919DNAArtificial SequenceSynthetic 399ggaaagactg ttccaaaaa
1940019DNAArtificial SequenceSynthetic 400caacacgcct catcctcta
1940119DNAArtificial SequenceSynthetic 401catgaaagct tacatcaac
1940219DNAArtificial SequenceSynthetic 402aagatgccat gaaagctta
1940319DNAArtificial SequenceSynthetic 403gcacataccg cctgagtct
1940419DNAArtificial SequenceSynthetic 404gatcaaatct gaagaagga
1940519DNAArtificial SequenceSynthetic 405gccaagaagt ttcctaata
1940619DNAArtificial SequenceSynthetic 406cagcatatct tgaaccatt
1940719DNAArtificial SequenceSynthetic 407gaacaaagga aacggatga
1940819DNAArtificial SequenceSynthetic 408cggaaacggt ccaggctat
1940919DNAArtificial SequenceSynthetic 409gcttcgagca gacatgata
1941019DNAArtificial SequenceSynthetic 410cctacacggt cctcctata
1941119DNAArtificial SequenceSynthetic 411gccaagaacc tcatcatct
1941219DNAArtificial SequenceSynthetic 412gatatgggct gaatacaaa
1941319DNAArtificial SequenceSynthetic 413gcactctgat tgacaaata
1941419DNAArtificial SequenceSynthetic 414tgaagtctct gattaagta
1941519DNAArtificial SequenceSynthetic 415tcagagagat cctcataaa
1941619DNAArtificial SequenceSynthetic 416gcaagaagat caccatttc
1941719DNAArtificial SequenceSynthetic 417gagagaaatt tgaggatga
1941819DNAArtificial SequenceSynthetic 418gaaaggattt ggctataag
1941919DNAArtificial SequenceSynthetic 419gaaagaaggc atgaacatt
1942019DNAArtificial SequenceSynthetic 420gggagatagt gatgaagta
1942119DNAArtificial SequenceSynthetic 421gaagtacatc cattataag
1942219DNAArtificial SequenceSynthetic 422gtacgacaac cgggagata
1942319DNAArtificial SequenceSynthetic 423agatagtgat gaagtacat
1942419DNAArtificial SequenceSynthetic 424tgaagactct gctcagttt
1942519DNAArtificial SequenceSynthetic 425gcatgcggcc tctgtttga
1942619RNAArtificial SequenceSynthetic 426gcacacagcu uacuacauc
1942719RNAArtificial SequenceSynthetic 427gaaaugcccu gguaucuca
1942819RNAArtificial SequenceSynthetic 428gaaggaacgu gaugugauc
1942919RNAArtificial SequenceSynthetic 429gcacuacucc uguguguga
1943019RNAArtificial SequenceSynthetic 430gaacccagcu ggagaacuu
1943119RNAArtificial SequenceSynthetic 431gauauacagu gugaucuua
1943219RNAArtificial SequenceSynthetic 432guacuacgau ccugauuau
1943319RNAArtificial SequenceSynthetic 433gugccgaccu uuacaauuu
1943419DNAArtificial SequenceSynthetic 434gaaggaaact gaattcaaa
1943519DNAArtificial SequenceSynthetic 435ggaaatatgt actacgaaa
1943619DNAArtificial SequenceSynthetic 436ccacaaagca gtgaattta
1943719DNAArtificial SequenceSynthetic 437gtaacaagct cacgcagtt
1943819RNAArtificial SequenceSynthetic 438aauggaggcu uagcuuucu
1943919RNAArtificial SequenceSynthetic 439acaccagcau acagaguga
1944019RNAArtificial SequenceSynthetic 440agauugggcu ggcucugaa
1944119RNAArtificial SequenceSynthetic 441agcaggaacu gagccagaa
1944219RNAArtificial SequenceSynthetic 442caacugcagc guccacaca
1944319RNAArtificial SequenceSynthetic 443caacuuuucu agaccuguu
1944419RNAArtificial SequenceSynthetic 444caagcaagca gacauuuau
1944519RNAArtificial SequenceSynthetic 445caagcagaca uuuaucuuu
1944619RNAArtificial SequenceSynthetic 446caaggaggca ggauucuuc
1944719RNAArtificial SequenceSynthetic 447caagggaagg gcacgguua
1944819RNAArtificial SequenceSynthetic 448caccaaggag gcaggauuc
1944919RNAArtificial SequenceSynthetic 449caccuuuacu cugcucuau
1945019RNAArtificial SequenceSynthetic 450cagaaugacu uuuauugag
1945119RNAArtificial SequenceSynthetic 451cagagugacc accgggaaa
1945219RNAArtificial SequenceSynthetic 452cagccaaccc gcuccacua
1945319RNAArtificial SequenceSynthetic 453caggaagcuc ggugaguga
1945419RNAArtificial SequenceSynthetic 454ccaagcaagc agacauuua
1945519RNAArtificial SequenceSynthetic 455ccaagccucu ucuuacuuc
1945619RNAArtificial SequenceSynthetic 456ccaaggaggc aggauucuu
1945719RNAArtificial
SequenceSynthetic 457ccaaggaucc guggagguu 1945819RNAArtificial
SequenceSynthetic 458ccaccaagga ggcaggauu 1945919RNAArtificial
SequenceSynthetic 459ccacguggcu ggcauugca 1946019RNAArtificial
SequenceSynthetic 460ccacuucucu gccaaagau 1946119RNAArtificial
SequenceSynthetic 461ccagcuaacu guggagaag 1946219RNAArtificial
SequenceSynthetic 462cccagagcau cccguggaa 1946319RNAArtificial
SequenceSynthetic 463cccaggagcu ccagugaca 1946419RNAArtificial
SequenceSynthetic 464ccucauaggc cuggaguuu 1946519RNAArtificial
SequenceSynthetic 465ccugauuaau ggaggcuua 1946619RNAArtificial
SequenceSynthetic 466ccuuuacucu gcucuaugc 1946719RNAArtificial
SequenceSynthetic 467cggcugggcu ccucauuuu 1946819RNAArtificial
SequenceSynthetic 468cgucgaggcg cucaugguu 1946919RNAArtificial
SequenceSynthetic 469cuagacacca gcauacaga 1947019RNAArtificial
SequenceSynthetic 470cucauaggcc uggaguuua 1947119RNAArtificial
SequenceSynthetic 471gaacacagac caggaagcu 1947219RNAArtificial
SequenceSynthetic 472gaaccacagc caccuucca 1947319RNAArtificial
SequenceSynthetic 473gacaggccag caaguguga 1947419RNAArtificial
SequenceSynthetic 474gacgaugccu gccucuacu 1947519RNAArtificial
SequenceSynthetic 475gagccagaaa cgcagauug 1947619RNAArtificial
SequenceSynthetic 476gagggugucu acgccauug 1947719RNAArtificial
SequenceSynthetic 477gaguugaggc agagacuga 1947819RNAArtificial
SequenceSynthetic 478gauccacuuc ucugccaaa 1947919RNAArtificial
SequenceSynthetic 479gauccugcau gucuuccau 1948019RNAArtificial
SequenceSynthetic 480gcaagcagac auuuaucuu 1948119RNAArtificial
SequenceSynthetic 481gcagaaugac uuuuauuga 1948219RNAArtificial
SequenceSynthetic 482gcagagacug auccacuuc 1948319RNAArtificial
SequenceSynthetic 483gcagccuccu ugccuggaa 1948419RNAArtificial
SequenceSynthetic 484gcagccuggu ggaggugua 1948519RNAArtificial
SequenceSynthetic 485gcaggaacug agccagaaa 1948619RNAArtificial
SequenceSynthetic 486gcaggauucu ucccaugga 1948719RNAArtificial
SequenceSynthetic 487gcauuucacc auucaaaca 1948819RNAArtificial
SequenceSynthetic 488gccaagccuc uucuuacuu 1948919RNAArtificial
SequenceSynthetic 489gccagcugcu cccaaugug 1949019RNAArtificial
SequenceSynthetic 490gcccucaucu ccagcuaac 1949119RNAArtificial
SequenceSynthetic 491gcgcccugcu ccugaacuu 1949219RNAArtificial
SequenceSynthetic 492gcuagcaaca cccaaaggu 1949319RNAArtificial
SequenceSynthetic 493ggacccgcuu ccacagaca 1949419RNAArtificial
SequenceSynthetic 494ggacgaugcc ugccucuac 1949519RNAArtificial
SequenceSynthetic 495ggagagggcc aacaacugu 1949619RNAArtificial
SequenceSynthetic 496ggagcuggcc uugaaguug 1949719RNAArtificial
SequenceSynthetic 497ggagugagcc aggcaguga 1949819RNAArtificial
SequenceSynthetic 498ggauucuucc cauggauag 1949919RNAArtificial
SequenceSynthetic 499ggcagagacu gauccacuu 1950019RNAArtificial
SequenceSynthetic 500gggagagggc caacaacug 1950119RNAArtificial
SequenceSynthetic 501gggcauuuca ccauucaaa 1950219RNAArtificial
SequenceSynthetic 502gggcugagcu uuaaaaugg 1950319RNAArtificial
SequenceSynthetic 503gggcuggggc ugagcuuua 1950419RNAArtificial
SequenceSynthetic 504ggggauaccu caccaagau 1950519RNAArtificial
SequenceSynthetic 505ggucaccgac uucgagaau 1950619RNAArtificial
SequenceSynthetic 506ggucaugguc accgacuuc 1950719RNAArtificial
SequenceSynthetic 507ggucuggaau gcaaaguca 1950819RNAArtificial
SequenceSynthetic 508gguuagcggc acccucaua 1950919RNAArtificial
SequenceSynthetic 509guacagccgc guccucaac 1951019RNAArtificial
SequenceSynthetic 510uaauggaggc uuagcuuuc 1951119RNAArtificial
SequenceSynthetic 511ucaccaagau ccugcaugu 1951219RNAArtificial
SequenceSynthetic 512ucaccgacuu cgagaaugu 1951319RNAArtificial
SequenceSynthetic 513ucacuggccu ggcggagau 1951419RNAArtificial
SequenceSynthetic 514ucauaggccu ggaguuuau 1951519RNAArtificial
SequenceSynthetic 515ucuccuagac accagcaua 1951619RNAArtificial
SequenceSynthetic 516ugacagccgu ugccaucug 1951719RNAArtificial
SequenceSynthetic 517ugcuggagcu ggccuugaa 1951819RNAArtificial
SequenceSynthetic 518uggcggagau gcuucuaag 1951919RNAArtificial
SequenceSynthetic 519uguccucucu guugccuuu 1952019RNAArtificial
SequenceSynthetic 520ugucuuccau ggccuucuu 1952119RNAArtificial
SequenceSynthetic 521uuaaaauggu uccgacuug 1952219RNAArtificial
SequenceSynthetic 522uuaauggagg cuuagcuuu 1952319RNAArtificial
SequenceSynthetic 523aagucaagga gcauggaau 1952419RNAArtificial
SequenceSynthetic 524aaugcaaagu caaggagca 1952519RNAArtificial
SequenceSynthetic 525acaccagcau acagaguga 1952619RNAArtificial
SequenceSynthetic 526agaaugugcc cgaggagga 1952719RNAArtificial
SequenceSynthetic 527caacugcagc guccacaca 1952819RNAArtificial
SequenceSynthetic 528caagggaagg gcacgguua 1952919RNAArtificial
SequenceSynthetic 529cagagugacc accgggaaa 1953019RNAArtificial
SequenceSynthetic 530ccaaggaucc guggagguu 1953119RNAArtificial
SequenceSynthetic 531ccacguggcu ggcauugca 1953219RNAArtificial
SequenceSynthetic 532ccacuucucu gccaaagau 1953319RNAArtificial
SequenceSynthetic 533cccagagcau cccguggaa 1953419RNAArtificial
SequenceSynthetic 534ccuacguggu ggugcugaa 1953519RNAArtificial
SequenceSynthetic 535ccucauaggc cuggaguuu 1953619RNAArtificial
SequenceSynthetic 536ccuggaguuu auucggaaa 1953719RNAArtificial
SequenceSynthetic 537cguggaaccu ggagcggau 1953819RNAArtificial
SequenceSynthetic 538cuagacacca gcauacaga 1953919RNAArtificial
SequenceSynthetic 539cucauaggcc uggaguuua 1954019RNAArtificial
SequenceSynthetic 540gaaccacagc caccuucca 1954119RNAArtificial
SequenceSynthetic 541gacaggccag caaguguga 1954219RNAArtificial
SequenceSynthetic 542gacgaugccu gccucuacu 1954319RNAArtificial
SequenceSynthetic 543gagggugucu acgccauug 1954419RNAArtificial
SequenceSynthetic 544gaguugaggc agagacuga 1954519RNAArtificial
SequenceSynthetic 545gauccacuuc ucugccaaa 1954619RNAArtificial
SequenceSynthetic 546gauccugcau gucuuccau 1954719RNAArtificial
SequenceSynthetic 547gcagagacug auccacuuc 1954819RNAArtificial
SequenceSynthetic 548gcagccuggu ggaggugua 1954919RNAArtificial
SequenceSynthetic 549ggacccgcuu ccacagaca 1955019RNAArtificial
SequenceSynthetic 550ggacgaugcc ugccucuac 1955119RNAArtificial
SequenceSynthetic 551ggagcuggcc uugaaguug 1955219RNAArtificial
SequenceSynthetic 552ggcagagacu gauccacuu 1955319RNAArtificial
SequenceSynthetic 553ggggauaccu caccaagau 1955419RNAArtificial
SequenceSynthetic 554ggucaccgac uucgagaau 1955519RNAArtificial
SequenceSynthetic 555ggucaugguc accgacuuc 1955619RNAArtificial
SequenceSynthetic 556ggucuggaau gcaaaguca 1955719RNAArtificial
SequenceSynthetic 557gguuagcggc acccucaua 1955819RNAArtificial
SequenceSynthetic 558guacagccgc guccucaac 1955919RNAArtificial
SequenceSynthetic 559ucaccaagau ccugcaugu 1956019RNAArtificial
SequenceSynthetic 560ucaccgacuu cgagaaugu 1956119RNAArtificial
SequenceSynthetic 561ucauaggccu ggaguuuau 1956219RNAArtificial
SequenceSynthetic 562ucuccuagac accagcaua 1956319RNAArtificial
SequenceSynthetic 563ugacagccgu ugccaucug 1956419RNAArtificial
SequenceSynthetic 564ugcuggagcu ggccuugaa 1956519RNAArtificial
SequenceSynthetic 565ugucuuccau ggccuucuu 19
* * * * *