U.S. patent application number 11/022055 was filed with the patent office on 2006-06-22 for methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended sirna.
This patent application is currently assigned to UNIVERSITY OF MASSACHUSETTS. Invention is credited to Dianne Schwarz, Phillip D. Zamore.
Application Number | 20060134787 11/022055 |
Document ID | / |
Family ID | 36596445 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134787 |
Kind Code |
A1 |
Zamore; Phillip D. ; et
al. |
June 22, 2006 |
Methods and compositions for enhancing the efficacy and specificity
of single and double blunt-ended siRNA
Abstract
The present invention provides methods of enhancing the efficacy
and specificity of RNAi using single or double blunt-ended siRNA.
The invention also provides single and double-blunt ended siRNA
compositions, vectors, and transgenes containing the same for
mediating silencing of a target gene. Therapeutic methods are also
featured.
Inventors: |
Zamore; Phillip D.;
(Northboro, MA) ; Schwarz; Dianne; (Westborough,
MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
UNIVERSITY OF MASSACHUSETTS
Boston
MA
|
Family ID: |
36596445 |
Appl. No.: |
11/022055 |
Filed: |
December 22, 2004 |
Current U.S.
Class: |
435/455 ;
514/44A |
Current CPC
Class: |
C12N 2310/14 20130101;
C12N 15/111 20130101; C12N 15/113 20130101; C12Y 115/01001
20130101; C12N 2320/50 20130101 |
Class at
Publication: |
435/455 ;
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/87 20060101 C12N015/87 |
Claims
1. A method of enhancing the ability of a first strand of a RNAi
agent to act as a guide strand in mediating RNAi, the RNAi agent
derived from an RNA duplex having at least one blunt end,
comprising lessening the base pair strength between the 5' end of
the first strand and the 3' end of a second strand of the duplex as
compared to the base pair strength between the 3' end of the first
strand and the 5' end of the second strand.
2. A method of enhancing the efficacy of a siRNA duplex having at
least one blunt end, the siRNA duplex comprising a sense and an
antisense strand, comprising lessening the base pair strength
between the antisense strand 5' end (AS 5') and the sense strand 3'
end (S 3') as compared to the base pair strength between the
antisense strand 3' end (AS 3') and the sense strand 5' end (S '5),
such that efficacy is enhanced.
3. A method of enhancing the ability of a first strand of a RNAi
agent to act as a guide strand in mediating RNAi, the RNAi agent
derived from an RNA duplex having 5' and 3' blunt ends, comprising
lessening the base pair strength between the 5' end of the first
strand and the 3' end of a second strand of the duplex as compared
to the base pair strength between the 3' end of the first strand
and the 5' end of the second strand.
4. A method of enhancing the efficacy of a siRNA duplex having 5'
and 3' blunt ends, the siRNA duplex comprising a sense and an
antisense strand, comprising lessening the base pair strength
between the antisense strand 5' end (AS 5') and the sense strand 3'
end (S 3') as compared to the base pair strength between the
antisense strand 3' end (AS 3') and the sense strand 5' end (S '5),
such that efficacy is enhanced.
5. A method of promoting entry of a desired strand of an siRNA
duplex having at least one blunt end into a RISC complex,
comprising enhancing the asymmetry of the siRNA duplex, such that
entry of the desired strand is promoted.
6. The method of claim 5, wherein the siRNA duplex has 5' and 3'
blunt ends.
7. The method of claim 5, wherein asymmetry is enhanced by
lessening the base pair strength between the 5' end of the desired
strand and the 3' end of a complementary strand of the duplex as
compared to the base pair strength between the 3' end of the
desired strand and the 5' end of the complementary strand.
8. The method of any one of claims 1, 2, 3, or 4, wherein the
base-pair strength is less due to fewer G:C base pairs between the
5' end of the first or antisense strand and the 3' end of the
second or sense strand than between the 3' end of the first or
antisense strand and the 5' end of the second or sense strand.
9. The method of any one of claims 1, 2, 3, or 4, wherein the base
pair strength is less due to at least one mismatched base pair
between the 5' end of the first or antisense strand and the 3' end
of the second or sense strand.
10. The method of claim 9, wherein the mismatched base pair is
selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C,
U:U, I:A, I:U, and I:C.
11. The method of any one of claims 1, 2, 3, or 4, wherein the base
pair strength is less due to at least one wobble base pair between
the 5' end of the first or antisense strand and the 3' end of the
second or sense strand.
12. The method of claim 11, wherein the wobble base pair is
G:U.
13. The method of claim 11, wherein the wobble base pair is
G:T.
14. The method of any one of claims 1, 2, 3, or 4, wherein the base
pair strength is less due to: (a) at least one mismatched base pair
between the 5' end of the first or antisense strand and the 3' end
of the second or sense strand; and (b) at least one wobble base
pair between the 5' end of the first or antisense strand and the 3'
end of the second or sense strand.
15. The method of claim 14, wherein the mismatched base pair is
selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C
and U:U.
16. The method of claim 14, wherein the mismatched base pair is
selected from the group consisting of G:A, C:A, C:T, G:G, A:A, C:C
and U:T.
17. The method of claim 14, wherein the wobble base pair is
G:U.
18. The method of claim 14, wherein the wobble base pair is
G:T.
19. The method of any one of claims 1, 2, 3, or 4, wherein the base
pair strength is less due to at least one base pair comprising a
rare nucleotide.
20. The method of claim 19, wherein the modified nucleotide is
selected from the group consisting of 2-amino-G, 2-amino-A,
2,6-diamino-G, and 2,6-diamino-A.
21. The method of claim 1, wherein the RNAi agent is a siRNA
duplex.
22. The method of any one of claims 1, 2, 3, or 4, wherein the RNAi
agent or siRNA duplex is chemically synthesized.
23. The method of any one of claims 1, 2, 3, or 4, wherein the RNAi
agent or siRNA duplex is enzymatically synthesized.
24. The method of any one of claims 1, 2, 3, or 4, wherein the RNAi
agent or siRNA duplex is derived from an engineered precursor.
25. A method of enhancing silencing of a target mRNA, comprising
contacting a cell having an RNAi pathway with the RNAi agent or
siRNA duplex of claims 1, 2, 3, or 4 under conditions such that
silencing is enhanced.
26. A method of enhancing silencing of a target mRNA in a subject,
comprising administering to the subject a pharmaceutical
composition comprising the RNAi agent or siRNA duplex of claims 1,
2, 3, or 4 such that silencing is enhanced.
27. A method of decreasing silencing of an inadvertent target mRNA
by a dsRNAi agent, the dsRNAi agent comprising a sense strand, an
antisense strand, and having at least one blunt end comprising: (a)
detecting a significant degree of complementarity between the sense
strand and the inadvertent target; and (b) enhancing the base pair
strength between the 5' end of the sense strand and the 3' end of
the antisense strand relative to the base pair strength between the
3' end of the sense strand and the 5' end of the antisense strand;
such that silencing of the inadvertent target mRNA is
decreased.
28. The method of claim 27, wherein silencing of the inadvertent
target mRNA is decreased relative to silencing of a desired target
mRNA.
29. An RNAi agent or siRNA duplex having 5' and 3' blunt ends
comprising a sense strand and an antisense strand, wherein the base
pair strength between the antisense strand 3' end (AS 3') and the
sense strand 5' end (S 5') is less than the base pair strength
between the antisense strand 5' end (AS 5') and the sense strand 3'
end (S '3), such that the antisense strand preferentially guides
cleavage of a target mRNA.
30. The siRNA duplex of claim 29, wherein the base-pair strength is
less due to fewer G:C base pairs between the AS 5' and the S 3'
than between the AS 3' and the S 5'.
31. The siRNA duplex of claim 29, wherein the base pair strength is
less due to at least one mismatched base pair between the AS 5' and
the S 3'.
32. The siRNA duplex of claim 31, wherein the mismatched base pair
is selected from the group consisting of G:A, C:A, C:T, U:T, C:U,
G:G, A:A, C:C, U:U, I:A, I:U, and I:C.
33. The siRNA duplex of claim 29, wherein the base pair strength is
less due to at least one wobble base pair between the AS 5' and the
S 3'.
34. The siRNA duplex of claim 29, wherein the wobble base pair is
G:U.
35. The siRNA duplex of claim 29, wherein the wobble base pair is
G:T.
36. The siRNA duplex of claim 29, wherein the base pair strength is
less due to at least one base pair comprising a modified
nucleotide.
37. The siRNA duplex of claim 36, wherein the modified nucleotide
is selected from the group consisting of 2-amino-G, 2-amino-A,
2,6-diamino-G, and 2,6-diamino-A.
38. A composition comprising the RNAi agent or siRNA duplex of
claim 29, formulated to facilitate entry of the RNAi agent or siRNA
duplex into a cell.
39. A pharmaceutical composition comprising the RNAi agent or siRNA
duplex of claim 38.
40. An engineered pre-miRNA comprising the RNAi agent or siRNA
duplex of claim 38.
41. A vector encoding the pre-miRNA of claim 40.
42. A pre-miRNA comprising the pre-miRNA of claim 41.
43. A vector encoding the pre-miRNA of claim 42.
44. A small hairpin RNA (shRNA) comprising nucleotide sequence
identical to the sense and antisense strand of the siRNA duplex of
claim 29.
45. The shRNA of claim 44, wherein the nucleotide sequence
identical to the sense strand is upstream of the nucleotide
sequence identical to the antisense strand.
46. The shRNA of claim 44, wherein the nucleotide sequence
identical to the antisense strand is upstream of the nucleotide
sequence identical to the sense strand.
47. A vector encoding the shRNA of any one of claims 44-46.
48. A cell comprising the vector of any one of claims 41, 43, or
47.
49. The cell of claim 48, which is a mammalian cell.
50. The cell of claim 48, which is a human cell.
51. A transgene encoding the shRNA of any one of claims 44-46.
Description
RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application Ser. No. 60/532,116, entitled
"Methods and Compositions for Enhancing the Efficacy and
Specificity of Single and Double Blunt-Ended siRNA", filed Dec. 22,
2003. The entire contents of the above-referenced provisional
patent application are incorporated herein by this reference.
RELATED INFORMATION
[0002] The contents of any patents, patent applications, and
references cited throughout this specification are hereby
incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Small interfering RNAs (siRNAs) are produced by the cleavage
of double-stranded RNA (dsRNA) precursors by Dicer, a member of the
RNase III family of dsRNA-specific endonucleases. Typically, siRNAs
result when transposons, viruses, or endogenous genes express long
dsRNA or when dsRNA is introduced experimentally into plant or
animal cells to trigger gene silencing, a process known as RNA
interference (RNAi).
[0004] siRNAs were first identified as the specificity determinants
of the RNA interference (RNAi) pathway, where they act as guides to
direct endonucleolytic cleavage of their target RNAs. Prototypical
siRNA duplexes are 21 nucleotide, double-stranded RNAs that contain
19 base pairs, with two-nucleotide, 3' overhanging ends. Active
siRNAs contain 5' phosphates and 3' hydroxyls.
[0005] siRNAs are typically found in the RNA-induced silencing
complex (RISC) that mediates both cleavage and translational
control. siRNA duplexes can assemble into RISC in the absence of
target mRNA, both in vivo and in vitro. Each RISC contains only one
of the two strands of the siRNA duplex. Since siRNA duplexes have
no foreknowledge of which siRNA strand will guide target cleavage,
both strands must assemble with the appropriate proteins to form a
RISC.
[0006] It has been observed that both siRNA strands are competent
to direct RNAi (Tuschl et al., Genes Dev 13, 3191-3197 (1999);
Hammond et al., Nature 404, 293-296 (2000); Zamore et al., Cell
101, 25-33 (2000); Elbashir et al., Genes Dev 15, 188-200 (2001);
Elbashir et al., EMBO J 20, 6877-6888 (2001); Nykanen et al., Cell
107, 309-321 (2001). That is, the antisense strand of an siRNA can
direct cleavage of a corresponding sense RNA target, whereas the
sense siRNA strand directs cleavage of an antisense target. In this
way, siRNA duplexes appear to be functionally symmetric.
[0007] The ability to control which strand of an siRNA duplex
enters into the RISC complex to direct cleavage of a corresponding
RNA target would provide a significant advance for both research
and therapeutic applications of RNAi technology.
SUMMARY OF THE INVENTION
[0008] The invention solves the foregoing problems of siRNA gene
targeting by determining the structural and functional
characteristics of single and blunt-ended siRNAs and in particular,
their strand specificity for a gene target. Accordingly, an
entirely new constellation of single and double blunt-ended siRNA
agents, e.g., siRNA duplexes, can be designed to efficiently and
specifically modulate a sense and/or antisense gene target.
[0009] In addition, the invention provides a method for introducing
alterations in either the 5', 3', or both the 5' and 3' of a single
or double blunt-ended siRNA such that either the sense, the
antisense, or both the sense and antisense strand will enter the
RNAi pathway (e.g., RISC) and target a cognate gene target(s) for
cleavage and destruction. Typically, the alteration takes the form
of a mismatched base pair that allows for a portion of the siRNA
duplex, e.g., the 5' end of the antisense strand, to separate or
fray.
[0010] Accordingly, the invention has several advantages which
include, but are not limited to, the following: [0011] providing
methods for designing single and double blunt-ended siRNA agents,
e.g., siRNA duplexes, have a characteristic strand specificity;
[0012] providing single and double blunt-ended siRNA agents, e.g.,
siRNA duplexes or small hairpin RNAs (shRNAs) with at least one
blunt end, suitable for gene modulation in plant or animal cells;
and [0013] methods for modulating gene expression in a subject in
need thereof using the single or double blunt-ended siRNA
compositions of the invention, e.g., in the form of a
pharmaceutical composition suitable for administering to a
patient.
[0014] Accordingly, in one aspect, the invention provides methods
for improving the efficiency (or specificity) of an RNAi reaction
comprising modifying (e.g., increasing) the asymmetry of an RNAi
agent (i.e., an RNA duplex having at least one blunt end) such that
the ability of the sense or second strand to mediate RNAi (e.g.,
mediate cleavage of a target RNA) is lessened.
[0015] In one embodiment, the asymmetry is increased in favor of
the 5' end of the first strand, e.g., by lessening the bond
strength (e.g., the strength of the interaction) between the 5' end
of the first strand and 3' end of the second strand relative to the
bond strength (e.g., the strength of the interaction) between the
5' end of the second strand and the 3' end of the first strand.
[0016] In another embodiment, the asymmetry is increased in favor
of the 5' end of the first strand by increasing bond strength
(e.g., the strength of the interaction) between the 5' end of the
second or sense strand and the 3' end of the first or antisense
strand, relative to the bond strength (e.g., the strength of the
interaction) between the 5' end of the first and the 3' end of the
second strand.
[0017] In another embodiment, the bond strength is increased, e.g.,
the hydrogen bonding is increased between nucleotides or analogs at
the 5' end, e.g., within 5 nucleotides of the second or sense
strand (numbered from the 5' end of the second strand) and
complementary nucleotides of the first or antisense strand. It is
understood that the asymmetry can be zero (i.e., no asymmetry), for
example, when the bonds or base pairs between the 5' and 3'
terminal bases are of the same nature, strength or structure. More
routinely, however, there exists some asymmetry due to the
different nature, strength or structure of at least one nucleotide
(often one or more nucleotides) between terminal nucleotides or
nucleotide analogs.
[0018] Accordingly, in one aspect, the instant invention provides a
method of enhancing the ability of a first strand of a single or
double blunt-ended RNAi agent to act as a guide strand in mediating
RNAi, involving lessening the base pair strength between the 5' end
of the first strand and the 3' end of a second strand of the duplex
as compared to the base pair strength between the 3' end of the
first strand and the 5' end of the second strand.
[0019] In a related aspect, the invention provides a method of
enhancing the efficacy of a single or double blunt-ended siRNA
duplex, the siRNA duplex comprising a sense and an antisense
strand, involving lessening the base pair strength between the
antisense strand 5' end (AS 5') and the sense strand 3' end (S 3')
as compared to the base pair strength between the antisense strand
3' end (AS 3') and the sense strand 5' end (S '5), such that
efficacy is enhanced.
[0020] In another aspect of the invention, a method is provided for
promoting entry of a desired strand of an single or double
blunt-ended siRNA duplex into a RISC complex, comprising enhancing
the asymmetry of the single or double blunt-ended siRNA duplex,
such that entry of the desired strand is promoted. In one
embodiment of this aspect of the invention, the asymmetry is
enhanced by lessening the base pair strength between the 5' end of
the desired strand and the 3' end of a complementary strand of the
duplex as compared to the base pair strength between the 3' end of
the desired strand and the 5' end of the complementary strand.
[0021] In another aspect of the invention, a single or double
blunt-ended siRNA duplex is provided comprising a sense strand and
an antisense strand, wherein the base pair strength between the
antisense strand 5' end (AS 5') and the sense strand 3' end (S 3')
is less than the base pair strength between the antisense strand 3'
end (AS 3') and the sense strand 5' end (S '5), such that the
antisense strand preferentially guides cleavage of a target
mRNA.
[0022] In one embodiment of these aspects of the invention, the
base-pair strength is less due to fewer G:C base pairs between the
5' end of the first or antisense strand and the 3' end of the
second or sense strand than between the 3' end of the first or
antisense strand and the 5' end of the second or sense strand.
[0023] In another embodiment, the base pair strength is less due to
at least one mismatched base pair between the 5' end of the first
or antisense strand and the 3' end of the second or sense strand.
Preferably, the mismatched or wobble base pair is selected from the
group consisting of G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U,
and I:C.
[0024] In yet another embodiment, the base pair strength is less
due to at least one base pair comprising a modified nucleotide. In
preferred embodiments, the modified nucleotide is selected from the
group consisting of 2-amino-G (e.g.,
2,2-diamino-1,2-dihydro-purin-6-one), 2-amino-A, 2,6-diamino-G, and
2,6-diamino-A.
[0025] In other embodiments of the above aspects, the single or
double blunt-ended RNAi agent or siRNA duplex is derived from an
engineered precursor, and can be chemically synthesized or
enzymatically synthesized.
[0026] In another aspect of the instant invention, compositions are
provided comprising a single or double blunt-ended siRNA duplex of
the invention formulated to facilitate entry of the siRNA duplex
into a cell. Also provided are pharmaceutical composition
comprising a siRNA duplex of the invention.
[0027] Further provided are an engineered pre-miRNA comprising the
siRNA duplex of any one of the preceding claims, as well as a
vector encoding the pre-miRNA. In related aspects, the invention
provides a pre-miRNA comprising the pre-miRNA, as well as a vector
encoding the pre-miRNA.
[0028] Also featured in the instant invention are small hairpin RNA
(shRNA) capable of forming at least a single blunt end comprising
nucleotide sequence identical to the sense and antisense strand of
the siRNA duplex as described above.
[0029] In one embodiment, the nucleotide sequence identical to the
sense strand is upstream of the nucleotide sequence identical to
the antisense strand. In another embodiment, the nucleotide
sequence identical to the antisense strand is upstream of the
nucleotide sequence identical to the sense strand. Further provided
are vectors and transgenes encoding the shRNAs of the
invention.
[0030] In yet another aspect, the invention provides cells
comprising the vectors featured in the instant invention.
Preferably, the cell is a mammalian cell, e.g., a human cell.
[0031] In other aspects of the invention, methods of enhancing
silencing of a target mRNA, comprising contacting a cell having an
RNAi pathway with any of the foregoing single or double blunt-ended
RNAi agents such that silencing is enhanced.
[0032] Also provided are methods of enhancing silencing of a target
mRNA in a subject, comprising administering to the subject a
pharmaceutical composition comprising any of the foregoing single
or double blunt-ended RNAi agents such that silencing is
enhanced.
[0033] Further provided is a method of decreasing silencing of an
inadvertent target mRNA by a single or double blunt-ended RNAi
agents the RNAi agent comprising a sense strand and an antisense
strand involving the steps of: (a) detecting a significant degree
of complementarity between the sense strand and the inadvertent
target; and (b) enhancing the base pair strength between the 5' end
of the sense strand and the 3' end of the antisense strand relative
to the base pair strength between the 3' end of the sense strand
and the 5' end of the antisense strand; such that silencing of the
inadvertent target mRNA is decreased. In a preferred embodiment,
the silencing of the inadvertent target mRNA is decreased relative
to silencing of a desired target mRNA.
[0034] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows a schematic of the structural and functional
characteristics of classical siRNA (i.e., having 3' dinucleotide
overhangs) with either a 5' or 3' frayed end as compared to the
siRNAs of the invention having at least one blunt end. Selected
single blunt-ended siRNAs with either a 5' or 3' frayed end are
shown as well as their corresponding ability to target cleavage of
a test sense and/or antisense target. Numbers on the left
correspond to the siRNA shown in further detail structurally in
FIG. 4 and as tested for target specificity in FIG. 5.
[0036] FIG. 2 shows a schematic of the structural and functional
characteristics of siRNAs of the invention having both 5' and 3'
blunt ends. Selected double blunt-ended siRNAs with either a 5' or
3' frayed end are shown as well as their corresponding ability to
target cleavage of a sense and/or antisense gene target. Numbers on
the left correspond to the siRNA shown in further detail
structurally in FIG. 4 and tested for target specificity in FIG.
5.
[0037] FIG. 3 shows the structure of all siRNA duplexes tested, in
particular, the single and double blunt-ended siRNA duplexes of the
invention and their correspondence with sense or antisense gene
targets to determine their efficacy and specificity. Each siRNA
duplex tested is identified by a number which corresponds to
functional target specificity results obtained in vitro using
Drosophila extracts (and shown in FIG. 5). Single blunt-ended siRNA
duplexes and double blunted-ended siRNA duplexes and their
alignment with sense targets are numbered, respectively, 1-18 and
19-22. The foregoing and their alignment with antisense targets are
numbered, respectively, 23-44.
[0038] FIG. 4 shows the efficacy and specificity of the single
blunt-ended siRNA duplexes and their ability to cleave sense and
antisense gene targets using Drosophila extracts that provide a
functional RISC-mediated RNAi pathway. Black (x) data points show %
antisense gene target cleaved (SOD1 sense target; i.e., gene
knockdown) whereas red (o) data points show % sense gene target
cleaved.
[0039] FIG. 5 shows the efficacy and specificity of the double
blunt-ended siRNA duplexes and their ability to cleave sense and
antisense gene targets using Drosophila extracts that provide a
functional RISC-mediated RNAi pathway. Black (x) data points show %
antisense gene target cleaved (SOD1 sense target; i.e., gene
knockdown) whereas red (o) data points show % sense gene target
cleaved.
DETAILED DESCRIPTION OF THE INVENTION
[0040] In order to provide a clear understanding of the
specification and claims, the following definitions are
conveniently provided below.
Definitions
[0041] As used herein the term "blunt end", for example, "single
blunt-end" or "double blunt-ended siRNA" refers to, e.g., an siRNA
duplex where at least one end of the duplex lacks any overhang,
e.g., a 3' dinucleotide overhang, such that both the 5' and 3'
strand end together, i.e., are flush or as referred to herein, are
blunt. The molecules of the invention have at least one blunt end
and, preferably, two blunt ends, i.e., are double blunt-ended (See
FIGS. 1-3 which show schematically classical siRNA duplexes having
3' dinucleotide overhangs as compared with the single and double
blunt-ended siRNAs of the invention).
[0042] The term "small interfering RNA" ("siRNA") (also referred to
in the art as "short interfering RNAs") refers to an RNA (or RNA
analog) comprising between about 10-50 nucleotides (or nucleotide
analogs) which is capable of directing or mediating RNA
interference. Preferably, an siRNA comprises between about 15-30
nucleotides or nucleotide analogs, more preferably between about
16-25 nucleotides (or nucleotide analogs), even more preferably
between about 18-23 nucleotides (or nucleotide analogs), and even
more preferably between about 19-22 nucleotides (or nucleotide
analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide
analogs). As mentioned above, at least one end if not both ends of
the siRNA of the invention, is blunt. Preferred single blunt-ended
siRNA molecules comprise a 21 nucleotide (nt) strand paired with a
strand that is 19 nt, 18 nt, or 17 nt. In another embodiment,
single blunt-ended siRNA molecules comprise a 19 nt strand paired
with a 18 nt strand or, preferably, a 17 nt strand, wherein the 19
nt strand is favored to enter the RISC pathway. It is also
understood that a blunt ended siRNA, if base paired or matched, is
more prone to separating or fraying, then an end that is matched
but also has a one or more nucleotide overhang, e.g., a
dinucleotide overhang, because of the unpaired helical nature of
the overhang and the stacking forces which contribute to
maintaining the base pairs immediately downstream.
[0043] The term "RNA interference" ("RNAi") (also referred to in
the art as "gene silencing" and/or "target silencing", e.g.,
"target mRNA silencing") refers to a selective intracellular
degradation of RNA. RNAi occurs in cells naturally to remove
foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via
fragments cleaved from free dsRNA which direct the degradative
mechanism to other similar RNA sequences. Alternatively, RNAi can
be initiated by the hand of man, for example, to silence the
expression of target genes.
[0044] The term "antisense strand" of an siRNA or RNAi agent refers
to a strand that is substantially complementary to a section of
about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22
nucleotides of the mRNA of the gene targeted for silencing. The
antisense strand or first strand has sequence sufficiently
complementary to the desired target mRNA sequence to direct
target-specific RNA interference (RNAi), e.g., complementarity
sufficient to trigger the destruction of the desired target mRNA by
the RNAi machinery or process.
[0045] The term "sense strand" or "second strand" of an siRNA or
RNAi agent refers to a strand that is complementary to the
antisense strand or first strand. Antisense and sense strands can
also be referred to as first or second strands, the first or second
strand having complementarity to the target sequence and the
respective second or first strand having complementarity to said
first or second strand.
[0046] The term "guide strand" refers to a strand of an RNAi agent,
e.g., an antisense strand of an siRNA duplex, that enters into the
RISC complex and directs cleavage of the target mRNA.
[0047] The term "target gene" is a gene whose expression is to be
selectively inhibited or "silenced". This silencing is achieved by
cleaving the mRNA of the target gene by an siRNA or miRNA, e.g., an
siRNA or miRNA that is created from an engineered RNA precursor by
a cell's RNAi system. One portion or segment of a duplex stem of
the RNA precursor is an antisense strand that is complementary,
e.g., sufficiently complementary to trigger the destruction of the
desired target mRNA by the RNAi machinery or process, to a section
of about 18 to about 40 or more nucleotides of the mRNA of the
target gene.
[0048] The term "asymmetry", as in the asymmetry of a single or
double blunt-ended siRNA duplex, refers to an inequality of bond
strength or base pairing strength between the siRNA termini (e.g.,
between terminal nucleotides on a first strand and terminal
nucleotides on an opposing second strand, e.g., a base pair
mismatch that allows for a separation or fraying of the end(s)),
such that the 5' end of one strand of the duplex is more frequently
in a transient unpaired, e.g., single-stranded, state than the 5'
end of the complementary strand. This structural difference
determines that one strand of the duplex is preferentially
incorporated into a RISC complex. The strand whose 5' end is less
tightly paired to the complementary strand will preferentially be
incorporated into RISC and mediate RNAi.
[0049] The term "bond strength" or "base pair strength" refers to
the strength of the interaction between pairs of nucleotides (or
nucleotide analogs) on opposing strands of an oligonucleotide
duplex (e.g., an siRNA duplex), due primarily to hydrogen-bonding,
Van der Waals interactions, and the like between such nucleotides
(or nucleotide analogs).
[0050] The term "fray" or "fraying" refers to the ability of a
portion of the siRNA duplex of the invention to separate, typically
at the end, preferably at the 5' end of the first or antisense
strand, due to a base pair mismatch. For determining the
thermodynamic stability or local thermodynamic stability of such
ends, energy rules can be based on nearest neighbor analysis and/or
amount of stacking.
DETAILED DESCRIPTION
Overview
[0051] The present invention features "small interfering RNA
molecules" ("siRNA molecules" or "siRNA") having at least one blunt
end, methods of making such siRNA molecules and methods for using
the single or double blunt-ended siRNA molecules (e.g., research
and/or therapeutic methods). A blunt-ended siRNA molecule of the
invention is a duplex consisting of a sense strand and
complementary antisense strand, the antisense strand having
sufficient complementarity to a target mRNA to mediate RNAi and
having at least one end (5', 3', or both 5' and 3') without an
overhang. Accordingly, the molecules of the invention are
distinguished from typical siRNA molecules which have a 3'
dinucleotide overhang at each end of the molecule.
[0052] Preferably, the strands are aligned such that, at one end,
preferably at both ends, there are no bases at the end of the
strands which do not align (i.e., for which no complementary bases
occur in the opposing strand) such that no overhang occurs at one
or both ends of the duplex when the strands are annealed.
Preferably, the single or double blunt-ended siRNA molecule has a
length from about 10-50 or more nucleotides, i.e., each strand
comprises 10-50 nucleotides (or nucleotide analogs). More
preferably, the siRNA molecule has a length from about 15-45 or
15-30 nucleotides. Even more preferably, the siRNA molecule has a
length from about 16-25 nucleotides, 18-23 nucleotides, or 19
nucleotides. The single or double blunt-ended siRNA molecules of
the invention further have a sequence that is "sufficiently
complementary" to a target mRNA sequence to direct target-specific
RNA interference (RNAi), as defined herein, i.e., the single or
double blunt-ended siRNA has a sequence sufficient to trigger the
destruction of the target mRNA by the RNAi machinery or
process.
1. Preferred RNA Molecules
[0053] The single or double blunt-ended siRNAs featured in the
invention provide enhanced specificity and efficacy for mediating
RISC-mediated cleavage of a desired target gene. In a preferred
aspect, the base pair strength between the antisense strand 5' end
(AS 5') and the sense strand 3' end (S 3') of the siRNAs is less
than the bond strength or base pair strength between the antisense
strand 3' end (AS 3') and the sense strand 5' end (S '5), such that
the antisense strand preferentially guides cleavage of a target
mRNA. In one embodiment, the bond strength or base-pair strength is
less due to fewer G:C base pairs between the 5' end of the first or
antisense strand and the 3' end of the second or sense strand than
between the 3' end of the first or antisense strand and the 5' end
of the second or sense strand.
[0054] In another embodiment, the bond strength or base pair
strength is less due to at least one mismatched base pair between
the 5' end of the first or antisense strand and the 3' end of the
second or sense strand. Preferably, the mismatched base pair is
selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C
and U:U. In a related embodiment, the bond strength or base pair
strength is less due to at least one wobble base pair, e.g., G:U,
between the 5' end of the first or antisense strand and the 3' end
of the second or sense strand.
[0055] In yet another embodiment, the bond strength or base pair
strength is less due to at least one base pair comprising a rare
nucleotide, e.g., inosine (I). Preferably, the base pair is
selected from the group consisting of an I:A, I:U, and I:C.
[0056] In yet another embodiment, the bond strength or base pair
strength is less due to at least one base pair comprising a
modified nucleotide. In preferred embodiments, the modified
nucleotide is selected from the group consisting of 2-amino-G,
2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.
[0057] In general, single or double blunt-ended siRNAs containing
nucleotide sequences sufficiently identical to a portion of the
target gene to effect RISC-mediated cleavage of the target gene are
preferred. 2. Gene Target Sequence Identity
[0058] Typically, 100% sequence identity between the single or
double blunt-ended siRNA and the target gene is not required to
practice the present invention. The invention has the advantage of
being able to tolerate preferred sequence variations of the methods
and compositions of the invention in order to enhance efficiency
and specificity of RNAi. For example, single or double blunt-ended
siRNA sequences with insertions, deletions, and single point
mutations relative to the target sequence can also be effective for
inhibition. Alternatively, single or double blunt-ended siRNA
sequences with nucleotide analog substitutions or insertions can be
effective for inhibition.
[0059] Sequence identity may be determined by sequence comparison
and alignment algorithms known in the art. To determine the percent
identity of two nucleic acid sequences (or of two amino acid
sequences), the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the first sequence or
second sequence for optimal alignment). The nucleotides (or amino
acid residues) at corresponding nucleotide (or amino acid)
positions are then compared. When a position in the first sequence
is occupied by the same residue as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), optionally penalizing the score for the
number of gaps introduced and/or length of gaps introduced.
[0060] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In one embodiment, the alignment generated
over a certain portion of the sequence aligned having sufficient
identity but not over portions having low degree of identity (i.e.,
a local alignment). A preferred, non-limiting example of a local
alignment algorithm utilized for the comparison of sequences is the
algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into
the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10.
[0061] In another embodiment, the alignment is optimized by
introducing appropriate gaps and percent identity is determined
over the length of the aligned sequences (i.e., a gapped
alignment). To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment,
the alignment is optimized by introducing appropriate gaps and
percent identity is determined over the entire length of the
sequences aligned (i.e., a global alignment). A preferred,
non-limiting example of a mathematical algorithm utilized for the
global comparison of sequences is the algorithm of Myers and
Miller, CABIOS (1989). Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
[0062] Greater than 80% sequence identity, e.g., 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or even 100% sequence identity, between the
siRNA antisense strand and the portion of the target gene is
preferred. Alternatively, the siRNA may be defined functionally as
a nucleotide sequence (or oligonucleotide sequence) that is capable
of hybridizing with a portion of the target gene transcript (e.g.,
400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or
70.degree. C. hybridization for 12-16 hours; followed by washing).
Additional preferred hybridization conditions include hybridization
at 70.degree. C. in 1.times.SSC or 50.degree. C. in 1.times.SSC,
50% formamide followed by washing at 70.degree. C. in 0.3.times.SSC
or hybridization at 70.degree. C. in 4.times.SSC or 50.degree. C.
in 4.times.SSC, 50% formamide followed by washing at 67.degree. C.
in 1.times.SSC. The hybridization temperature for hybrids
anticipated to be less than 50 base pairs in length should be
5-10.degree. C. less than the melting temperature (Tm) of the
hybrid, where Tm is determined according to the following
equations. For hybrids less than 18 base pairs in length,
Tm(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids
between 18 and 49 base pairs in length, Tm(.degree.
C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the
number of bases in the hybrid, and [Na+] is the concentration of
sodium ions in the hybridization buffer ([Na+] for
1.times.SSC=0.165 M). Additional examples of stringency conditions
for polynucleotide hybridization are provided in Sambrook, J., E.
F. Fritsch, and T. Maniatis, 1989, Molecular Cloning. A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., chapters 9 and 11, and Current Protocols in Molecular
Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons,
Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
The length of the identical nucleotide sequences may be at least
about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45,
47 or 50 bases.
3. Other Modifications for RNA Stability
[0063] The RNA molecules of the present invention can be modified
to improve stability in serum or in growth medium for cell
cultures. In order to enhance the stability, the 3'-residues may be
stabilized against degradation, e.g., they may be selected such
that they consist of purine nucleotides, particularly adenosine or
guanosine nucleotides. Alternatively, substitution of pyrimidine
nucleotides by modified analogues, e.g., substitution of uridine by
2'-deoxythymidine is tolerated and does not affect the efficiency
of RNA interference.
[0064] In a preferred aspect, the invention features small
interfering RNAs (siRNAs) that include a sense strand and an
antisense strand, wherein the antisense strand has a sequence
sufficiently complementary to a target mRNA sequence to direct
target-specific RNA interference (RNAi) and wherein the sense
strand and/or antisense strand is modified by the substitution of
internal nucleotides with modified nucleotides, such that in vivo
stability is enhanced as compared to a corresponding unmodified
siRNA. As defined herein, an "internal" nucleotide is one occurring
at any position other than the 5' end or 3' end of nucleic acid
molecule, polynucleotide or oligonucleotide. An internal nucleotide
can be within a single-stranded molecule or within a strand of a
duplex or double-stranded molecule. In one embodiment, the sense
strand and/or antisense strand is modified by the substitution of
at least one internal nucleotide. In another embodiment, the sense
strand and/or antisense strand is modified by the substitution of
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In
another embodiment, the sense strand and/or antisense strand is
modified by the substitution of at least 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or more of the internal nucleotides. In yet another embodiment,
the sense strand and/or antisense strand is modified by the
substitution of all of the internal nucleotides.
[0065] In a preferred embodiment of the present invention the RNA
molecule may contain at least one modified nucleotide analogue. The
nucleotide analogues may be located at positions where the
target-specific activity, e.g., the RNAi mediating activity is not
substantially effected, e.g., in a region at the 5'-end and/or the
3'-end of the RNA molecule. Particularly, the ends may be
stabilized by incorporating modified nucleotide analogues.
[0066] Preferred nucleotide analogues include sugar- and/or
backbone-modified ribonucleotides (i.e., include modifications to
the phosphate-sugar backbone). For example, the phosphodiester
linkages of natural RNA may be modified to include at least one of
a nitrogen or sulfur heteroatom. In preferred backbone-modified
ribonucleotides the phosphoester group connecting to adjacent
ribonucleotides is replaced by a modified group, e.g., of
phosphothioate group. In preferred sugar-modified ribonucleotides,
the 2' OH-group is replaced by a group selected from H, OR, R,
halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or ON, wherein R is
C.sub.1-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or
I.
[0067] Also preferred are nucleobase-modified ribonucleotides,
i.e., ribonucleotides, containing at least one non-naturally
occurring nucleobase instead of a naturally occurring nucleobase.
Bases may be modified to block the activity of adenosine deaminase.
Exemplary modified nucleobases include, but are not limited to,
uridine and/or cytidine modified at the 5-position, e.g.,
5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or
guanosines modified at the 8 position, e.g., 8-bromo guanosine;
deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated
nucleotides, e.g., N6-methyl adenosine are suitable. It should be
noted that the above modifications may be combined.
4. RNA Synthesis
[0068] RNA may be produced enzymatically or by partial/total
organic synthesis, any modified ribonucleotide can be introduced by
in vitro enzymatic or organic synthesis. In one embodiment, an RNAi
agent is prepared chemically. Methods of synthesizing RNA molecules
are known in the art, in particular, the chemical synthesis methods
as de scribed in Verma and Eckstein (1998) Annul Rev. Biochem.
67:99-134.
[0069] In another embodiment, a ss-siRNA is prepared enzymatically.
For example, a ds-siRNA can be prepared by enzymatic processing of
a long ds RNA having sufficient complementarity to the desired
target mRNA. Processing of long ds RNA can be accomplished in
vitro, for example, using appropriate cellular lysates and
ds-siRNAs can be subsequently purified by gel electrophoresis or
gel filtration. ds-siRNA can then be denatured according to
art-recognized methodologies.
[0070] In an exemplary embodiment, RNA can be purified from a
mixture by extraction with a solvent or resin, precipitation,
electrophoresis, chromatography, or a combination thereof.
Alternatively, the RNA may be used with no or a minimum of
purification to avoid losses due to sample processing.
Alternatively, the siRNA can also be prepared by enzymatic
transcription from synthetic DNA templates or from DNA plasmids
isolated from recombinant bacteria. Typically, phage RNA
polymerases are used such as T7, T3 or SP6 RNA polymerase (Milligan
and Uhlenbeck (1989) Methods Enzymol. 180:51-62). The RNA may be
dried for storage or dissolved in an aqueous solution. The solution
may contain buffers or salts to inhibit annealing, and/or promote
stabilization of the single strands.
[0071] In one embodiment, the single or double blunt-ended siRNAs
are synthesized either in vivo, in situ, or in vitro. Endogenous
RNA polymerase of the cell may mediate transcription in vivo or in
situ, or cloned RNA polymerase can be used for transcription in
vivo or in vitro. For transcription from a transgene in vivo or an
expression construct, a regulatory region (e.g., promoter,
enhancer, silencer, splice donor and acceptor, polyadenylation) may
be used to transcribe the ss-siRNA. Inhibition may be targeted by
specific transcription in an organ, tissue, or cell type;
stimulation of an environmental condition (e.g., infection, stress,
temperature, chemical inducers); and/or engineering transcription
at a developmental stage or age. A transgenic organism that
expresses ss-siRNA from a recombinant construct may be produced by
introducing the construct into a zygote, an embryonic stem cell, or
another multipotent cell derived from the appropriate organism.
5. Selecting a Gene Target
[0072] In one embodiment, the target mRNA of the invention encodes
the amino acid sequence of a cellular protein, e.g., a protein
involved in cell growth or suppression, e.g., a nuclear,
cytoplasmic, transmembrane, membrane-associated protein, or
cellular ligand. In another embodiment, the target mRNA of the
invention specifies the amino acid sequence of an extracellular
protein (e.g., an extracellular matrix protein or secreted
protein). Typical classes of proteins are listed for illustrative
purposes.
[0073] Developmental proteins suitable for targeting according to
the invention include e.g., adhesion molecules, cyclin kinase
inhibitors, Wnt family members, Pax family members, Winged helix
family members, Hox family members, cytokines/lymphokines and their
receptors, growth/differentiation factors and their receptors,
neurotransmitters and their receptors).
[0074] Oncogene-encoded proteins suitable for targeting according
to the invention include, e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL,
CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR,
HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS,
PIM I, PML, RET, SRC, TALI, TCL3, and YES).
[0075] Tumor suppressor proteins suitable for targeting according
to the invention include e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I,
NF2, RB I, TP53, and WTI).
[0076] Enzymatic proteins suitable for targeting according to the
invention include, e.g., ACC synthases and oxidases, ACP
desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases,
alcohol dehydrogenases, amylases, amyloglucosidases, catalases,
cellulases, chalcone synthases, chitinases, cyclooxygenases,
decarboxylases, dextriinases, DNA and RNA polymerases,
galactosidases, glucanases, glucose oxidases, granule-bound starch
synthases, GTPases, helicases, hernicellulases, integrases,
inulinases, invertases, isomerases, kinases, lactases, lipases,
lipoxygenases, lysozymes, nopaline synthases, octopine synthases,
pectinesterases, peroxidases, phosphatases, phospholipases,
phosphorylases, phytases, plant growth regulator synthases,
polygalacturonases, proteinases and peptidases, pullanases,
recombinases, reverse transcriptases, RUBISCOs, topoisomerases,
xylanases, and telomerases.
[0077] In a preferred aspect of the invention, the target mRNA
molecule of the invention specifies the amino acid sequence of a
protein associated with a pathological condition. For example, the
protein may be a pathogen-associated protein (e.g., a viral protein
involved in immunosuppression of the host, replication of the
pathogen, transmission of the pathogen, or maintenance of the
infection), or a host protein which facilitates entry of the
pathogen into the host, drug metabolism by the pathogen or host,
replication or integration of the pathogen's genome, establishment
or spread of infection in the host, or assembly of the next
generation of pathogen. Alternatively, the protein may be a
tumor-associated protein or an autoimmune disease-associated
protein.
[0078] By modulating the expression of the foregoing proteins,
valuable information regarding the function of such proteins and
therapeutic benefits which may be obtained from such modulation can
be obtained.
6. Assay for Testing Engineered RNA Precursors
[0079] Drosophila embryo lysates can be used to determine if the
engineered siRNAs of the invention, e.g., single or double
blunt-ended siRNA duplexes (but also, e.g., expressed shRNAs) have
their intended function (see also Examples 1-3). This lysate assay
is described in Tuschl et al., 1999, supra, Zamore et al., 2000,
supra, and Hutvdgner et al., Science 293, 834-838 (2001). These
lysates recapitulate RNAi in vitro, thus permitting investigation
into, e.g., which strand enters the complex, is assembled into
RISC, and is used as a guide strand for target destruction.
Briefly, the test siRNA is incubated with Drosophila embryo lysate
for various times, then assayed for the production of the mature
siRNA by primer extension or Northern hybridization. As in the in
vivo setting, mature RNA accumulates in the cell-free reaction.
Thus, an RNA corresponding to the proposed precursor can be shown
to be converted into a siRNA duplex in the Drosophila embryo
lysate.
[0080] Furthermore, an engineered RNA precursor can be functionally
tested in the Drosophila embryo lysates. In this case, the
engineered RNA precursor is incubated in the lysate in the presence
of a 5' radiolabeled target mRNA in a standard in vitro RNAi
reaction for various lengths of time. The target mRNA can be 5'
radiolabeled using guanylyl transferase (as described in Tuschl et
al., 1999, supra and references therein) or other suitable methods.
The products of the in vitro reaction are then isolated and
analyzed on a denaturing acrylamide or agarose gel to determine if
the target mRNA has been cleaved in response to the presence of the
engineered RNA precursor in the reaction. The extent and position
of such cleavage of the mRNA target will indicate if the
engineering of the precursor created a pre-siRNA capable of
mediating sequence-specific RNAi.
7. Methods of Introducing RNAs, Vectors, and Host Cells
[0081] Physical methods of introducing nucleic acids include
injection of a solution containing the RNA, bombardment by
particles covered by the RNA, soaking the cell or organism in a
solution of the RNA, or electroporation of cell membranes in the
presence of the RNA. A viral construct packaged into a viral
particle would accomplish both efficient introduction of an
expression construct into the cell and transcription of RNA encoded
by the expression construct. Other methods known in the art for
introducing nucleic acids to cells may be used, such as
lipid-mediated carrier transport, chemical-mediated transport, such
as calcium phosphate, and the like. Thus the RNA may be introduced
along with components that perform one or more of the following
activities: enhance RNA uptake by the cell, inhibit annealing of
single strands, stabilize the single strands, or other-wise
increase inhibition of the target gene.
[0082] RNA may be directly introduced into the cell (i.e.,
intracellularly); or introduced extracellularly into a cavity,
interstitial space, into the circulation of an organism, introduced
orally, or may be introduced by bathing a cell or organism in a
solution containing the RNA. Vascular or extravascular circulation,
the blood or lymph system, and the cerebrospinal fluid are sites
where the RNA may be introduced.
[0083] The cell with the target gene may be derived from or
contained in any organism. The organism may a plant, animal,
protozoan, bacterium, virus, or fungus. The plant may be a monocot,
dicot or gymnosperm; the animal may be a vertebrate or
invertebrate. Preferred microbes are those used in agriculture or
by industry, and those that are pathogenic for plants or
animals
[0084] Alternatively, vectors, e.g., transgenes encoding a siRNA of
the invention, i.e., having at least one blunt end, can be
engineered into a host cell or transgenic animal using art
recognized techniques.
8. Methods of Treatment:
[0085] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted target gene expression or activity. It is
understood that "treatment" or "treating" as used herein, is
defined as the application or administration of a therapeutic agent
(e.g., a RNAi agent or vector or transgene encoding same) to a
patient, or application or administration of a therapeutic agent to
an isolated tissue or cell line from a patient, who has a disease
or disorder, a symptom of disease or disorder or a predisposition
toward a disease or disorder, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disease or disorder, the symptoms of the disease or disorder,
or the predisposition toward disease.
9. Prophylactic Methods
[0086] In another aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted target gene expression or activity, by
administering to the subject a therapeutic agent (e.g., a RNAi
agent or vector or transgene encoding same). Subjects at risk for a
disease which is caused or contributed to by aberrant or unwanted
target gene expression or activity can be identified by, for
example, any or a combination of diagnostic or prognostic assays as
described herein. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the target
gene aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
target gene aberrancy, for example, a target gene, target gene
agonist or target gene antagonist agent can be used for treating
the subject. The appropriate agent can be determined based on
screening assays described herein.
10. Therapeutic Methods
[0087] In yet another aspect, the invention pertains to methods of
modulating target gene expression, protein expression or activity
for therapeutic purposes. Accordingly, in an exemplary embodiment,
the modulatory method of the invention involves contacting a cell
capable of expressing target gene with a therapeutic agent (e.g., a
RNAi agent or vector or transgene encoding same) that is specific
for the target gene or protein (e.g., is specific for the mRNA
encoded by said gene or specifying the amino acid sequence of said
protein) such that expression or one or more of the activities of
target protein is modulated. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant or unwanted expression or activity of a
target gene polypeptide or nucleic acid molecule. Inhibition of
target gene activity is desirable in situations in which target
gene is abnormally unregulated and/or in which decreased target
gene activity is likely to have a beneficial effect.
11. Pharmacogenomics
[0088] The therapeutic agents (e.g., a RNAi agent or vector or
transgene encoding same) of the invention can be administered to
individuals to treat (prophylactically or therapeutically)
disorders associated with aberrant or unwanted target gene
activity. In conjunction with such treatment, pharmacogenomics
(i.e., the study of the relationship between an individual's
genotype and that individual's response to a foreign compound or
drug) may be considered. Differences in metabolism of therapeutics
can lead to severe toxicity or therapeutic failure by altering the
relation between dose and blood concentration of the
pharmacologically active drug. Thus, a physician or clinician may
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer a therapeutic agent as
well as tailoring the dosage and/or therapeutic regimen of
treatment with a therapeutic agent.
[0089] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266
12. Pharmaceutical Compositions
[0090] The invention pertains to uses of the above-described agents
for therapeutic treatments as described infra. Accordingly, the
modulators of the present invention can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the nucleic acid molecule, protein,
antibody, or modulatory compound and a pharmaceutically acceptable
carrier. As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration.
[0091] The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
13. Knockout and/or Knockdown Cells or Organisms
[0092] A further preferred use for the RNAi agents of the present
invention (or vectors or transgenes encoding same) is a functional
analysis to be carried out in eukaryotic cells, or eukaryotic
non-human organisms, preferably mammalian cells or organisms and
most preferably human cells, e.g. cell lines such as HeLa or 293 or
rodents, e.g. rats and mice. By administering a suitable RNAi agent
which is sufficiently complementary to a target mRNA sequence to
direct target-specific RNA interference, a specific knockout or
knockdown phenotype can be obtained in a target cell, e.g. in cell
culture or in a target organism.
[0093] Thus, a further subject matter of the invention is a
eukaryotic cell or a eukaryotic non-human organism exhibiting a
target gene-specific knockout or knockdown phenotype comprising a
fully or at least partially deficient expression of at least one
endogeneous target gene wherein said cell or organism is
transfected with at least one vector comprising DNA encoding an
RNAi agent capable of inhibiting the expression of the target gene.
It should be noted that the present invention allows a
target-specific knockout or knockdown of several different
endogeneous genes due to the specificity of the RNAi agent.
[0094] Gene-specific knockout or knockdown phenotypes of cells or
non-human organisms, particularly of human cells or non-human
mammals may be used in analytic to procedures, e.g. in the
functional and/or phenotypical analysis of complex physiological
processes such as analysis of gene expression profiles and/or
proteomes. Preferably the analysis is carried out by high
throughput methods using oligonucleotide based chips.
14. Transgenic Organisms
[0095] Engineered RNA precursors of the invention can be expressed
in transgenic animals. These animals represent a model system for
the study of disorders that are caused by, or exacerbated by,
overexpression or underexpression (as compared to wildtype or
normal) of nucleic acids (and their encoded polypeptides) targeted
for destruction by the RNAi agents, e.g., siRNAs and shRNAs, and
for the development of therapeutic agents that modulate the
expression or activity of nucleic acids or polypeptides targeted
for destruction.
[0096] Transgenic animals can be farm animals (pigs, goats, sheep,
cows, horses, rabbits, and the like), rodents (such as rats, guinea
pigs, and mice), non-human primates (for example, baboons, monkeys,
and chimpanzees), and domestic animals (for example, dogs and
cats). Invertebrates such as Caenorhabditis elegans or Drosophila
can be used as well as non-mammalian vertebrates such as fish
(e.g., zebrafish) or birds (e.g., chickens).
[0097] Engineered RNA precursors with stems of 18 to 30 nucleotides
in length are preferred for use in mammals, such as mice. A
transgenic founder animal can be identified based upon the presence
of a transgene that encodes the new RNA precursors in its genome,
and/or expression of the transgene in tissues or cells of the
animals, for example, using PCR or Northern analysis. Expression is
confirmed by a decrease in the expression (RNA or protein) of the
target sequence.
[0098] Methods for generating transgenic animals include
introducing the transgene into the germ line of the animal. One
method is by microinjection of a gene construct into the pronucleus
of an early stage embryo (e.g., before the four-cell stage; Wagner
et al., 1981, Proc. Natl. Acad. Sci. USA 78:5016; Brinster et al.,
1985, Proc. Natl. Acad. Sci. USA 82:4438). Alternatively, the
transgene can be introduced into the pronucleus by retroviral
infection. A detailed procedure for producing such transgenic mice
has been described (see e.g., Hogan et al., Manipulating the Mouse
Embryo. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1986); U.S. Pat. No. 5,175,383 (1992)). This procedure has also
been adapted for other animal species (e.g., Hammer et al., 1985,
Nature 315:680; Murray et al., 1989, Reprod. Fert. Devl. 1:147;
Pursel et al., 1987, Vet. Immunol. Histopath. 17:303; Rexroad et
al., 1990, J. Reprod. Fert. 41 (suppl): 1 19; Rexroad et al., 1989,
Molec. Reprod. Devl. 1:164; Simons et al., 1988, BioTechnology
6:179; Vize et al., 1988, J. Cell. Sci. 90:295; and Wagner, 1989,
J. Cell. Biochem. 13B (suppl): 164). Clones of the non-human
transgenic animals described herein can be produced according to
the methods described in Wilmut et al. ((1997) Nature, 385:810-813)
and PCT publication Nos. WO 97/07668 and WO 97/07669.
15. Screening Assays
[0099] The methods of the invention are also suitable for use in
methods to identify and/or characterize potential pharmacological
agents, e.g. identifying new pharmacological agents from a
collection of test substances and/or characterizing mechanisms of
action and/or side effects of known pharmacological agents.
[0100] Thus, the present invention also relates to a system for
identifying and/or characterizing pharmacological agents acting on
at least one target protein comprising: (a) a eukaryotic cell or a
eukaryotic non-human organism capable of expressing at least one
endogeneous target gene coding for said so target protein, (b) at
least one RNAi agent molecule capable of inhibiting the expression
of said at least one endogeneous target gene, and (c) a test
substance or a collection of test substances wherein
pharmacological properties of said test substance or said
collection are to be identified and/or characterized. Further, the
system as described above preferably comprises: (d) at least one
exogenous target nucleic acid coding for the target protein or a
variant or mutated form of the target protein wherein said
exogenous target nucleic acid differs from the endogeneous target
gene on the nucleic acid level such that the expression of the
exogenous target nucleic acid is substantially less inhibited by
the RNAi agent than the expression of the endogeneous target
gene.
[0101] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[0102] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0103] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.)).
[0104] In a preferred embodiment, the library is a natural product
library, e.g., a library produced by a bacterial, fungal, or yeast
culture. In another preferred embodiment, the library is a
synthetic compound library.
[0105] This invention is further illustrated by the following
examples which should not be construed as limiting.
EXEMPLIFICATION
[0106] Throughout the examples, the following materials and methods
were used unless otherwise stated.
Materials and Methods
[0107] In general, the practice of the present invention employs,
unless otherwise indicated, conventional techniques of nucleic acid
chemistry, recombinant DNA technology, molecular biology,
biochemistry, and cell and cell extract preparation. See, e.g., DNA
Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985); Oligonucleotide
Synthesis (M. J. Gait, Ed. 1984); Oxford Handbook of Nucleic Acid
Structure, Neidle, Ed., Oxford Univ Press (1999); RNA Interference:
The Nuts & Bolts of siRNA Technology, by D. Engelke, DNA Press,
(2003); Gene Silencing by RNA Interference: Technology and
Application, by M. Sohail, CRC Press (2004); Sambrook, Fritsch and
Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press
(1989); and Current Protocols in Molecular Biology, eds. Ausubel et
al., John Wiley & Sons (1992). See also PCT/US03/24768
(Attorney Docket No. UMY-033PC); U.S. Ser. No. 60/475,331 (Attorney
Docket No. UMY-066-1); U.S. Ser. No. 60/507,928, (Attorney Docket
No. UMY-066-2); and U.S. Ser. No. 60/475,386 (Attorney Docket No.
UMY-050-1), of which all are incorporated in their entireties by
reference herein.
siRNA Preparation
[0108] Synthetic RNAs (Dharmacon) were deprotected according to the
manufacturer's protocol. siRNA strands were annealed (Elbashir et
al., Genes Dev 15, 188-200 (2001) and used at 50 nM final
concentration unless otherwise noted. siRNA single strands were
phosphorylated with polynucleotide kinase (New England Biolabs) and
1 mM ATP according to the manufacturer's directions and used at 500
nM final concentration.
Sense and Anti-Sense Target Preparation
[0109] Target RNAs were transcribed with recombinant,
histidine-tagged, T7 RNA Polymerase from PCR products as described
(Nykanen et al., 2001, supra; Hutvagner and Zamore, Science 297,
2056-2060 (2002), except for sense sod1 mRNA, which was transcribed
from a plasmid template (Crow et al., J Neurochem 69, 1936-1944
(1997)) linearized with Bam HI. PCR templates for htt sense and
antisense and sod1 antisense target RNAs were generated by
amplifying 0.1 ng/ml (final concentration) plasmid template
encoding htt or sod1 cDNA using the following primer pairs: htt
sense target, 5'-GCG TAA TAC GAC TCA CTA TAG GAA CAG TAT GTC TCA
GAC ATC-3' and 5'-UUCG AAG UAU UCC GCG UAC GU-3'; htt antisense
target, 5'-GCG TAA TAC GAC TCA CTA TAG GAC AAG CCT AAT TAG
TGATGC-3' and 5'-GAA CAG TAT GTC TCA GAC ATC-3'; sod1 antisense
target, 5'-GCG TAA TAC GAC TCA CTA TAG GGC TTT GTT AGC AGC CGG
AT-3' and 5'-GGG AGA CCA CAA CGG TTT CCC-3'.
RISC Extract Preparation
[0110] Drosophila embryo lysate preparation, in vitro RNAi
reactions, and cap-labeling of target RNAs using guanylyl
transferase were carried out as previously described (Tuschl et
al., 1999, supra; Zamore et al., 2000, supra). Target RNAs were
used at .about.5 nM concentration to ensure that reactions occurred
under single-turnover conditions. Target cleavage under these
conditions was proportionate to siRNA concentrations. Cleavage
products of RNAi reactions were analyzed by electrophoresis on 5%
or 8% denaturing acrylamide gels. 5' end labeling and determination
of siRNA unwinding status were according to Nykanen et al. (Nykanen
et al., 2001, supra) except that unlabeled competitor RNA was used
at 100-fold molar excess. Gels were dried, then exposed to image
plates (Fuji), which were scanned with a Fuji FLA-5000
phosphorimager. Images were analyzed using Image Reader FLA-5000
version 1.0 (Fuji) and Image Gauge version 3.45 or 4.1 (Fuji). Data
analysis was performed using Excel (Microsoft) and IgorPro 5.0
(Wavemetrics).
Example 1
Functionally Asymmetric siRNA Duplexes Having a Single Blunt
End
[0111] The following example describes methods for constructing
single blunt-ended siRNA duplexes capable of selectively entering a
RISC-mediated RNAi pathway and selectively cleaving a test target
for destruction.
[0112] Briefly, to assess quantitatively if the two strands of an
siRNA duplex having a single 5' or 3' blunt end are equally
competent to direct RNAi, the individual rates of sense and
antisense target cleavage for a single blunt-ended siRNA duplex
directed against the SOD1 target gene were examined (FIG. 4). The
relevant portions of the sense and antisense target RNA sequences
are shown in FIG. 4 and in schematic form in FIG. 1 (see lower
panel). The single blunt-ended siRNA duplex effectively silences
SOD1 expression in Drosophila extracts when having a weakened 5'
end (i.e., "frayed end") (compare 4 with 1 in FIGS. 1 and 4).
[0113] Accordingly, these results indicate that 1) a single blunt
end siRNA is functional and 2) that weakening the 5' antisense base
pair interaction with the 3' sense strand dramatically increases
entry of the antisense strand into the complex and subsequent gene
knockdown activity.
Example 2
Functionally Asymmetric siRNA Duplexes Having a Double Blunt
Ends
[0114] The following example describes methods for constructing
double blunt-ended siRNA duplexes capable of selectively entering a
RISC-mediated RNAi pathway and selectively cleaving a test target
for destruction.
[0115] Briefly, to assess quantitatively if the two strands of an
double blunt-ended siRNA duplex are equally competent to direct
RNAi, the individual rates of sense and antisense target cleavage
for a single blunt-ended siRNA duplex directed against the SOD1
target gene were examined (FIG. 5). The relevant portions of the
sense and antisense target RNA sequences are shown in FIG. 3 and in
schematic form in FIG. 2 (see lower panel). The double blunt-ended
siRNA duplexes effectively silence SOD1 expression in Drosophila
extracts and this activity is increased in the when having a
weakened 5' end (i.e., "frayed end") (compare 4 with 1 in FIGS. 1
and 4).
[0116] Accordingly, these results indicate that 1) double blunt-end
siRNA molecules are functional and 2) that weakening the 5'
antisense base pair interaction with the 3' sense strand or the 5'
sense base pair interaction with the 3' antisense strand modulates
the entry of the antisense and sense strand into the complex and
subsequent gene knockdown activity (see FIGS. 2 and 5).
Example 3
Single and Double Blunt-Ended siRNA Strand Contribution in RISC
Assembly
[0117] The following example describes methods for determining
RISC-mediated selectivity regarding the single and double
blunt-ended siRNAs of the invention.
[0118] To identify the source of asymmetry in the function of such
an single or double blunt-ended siRNA duplex, the unwinding of the
two siRNA strands when the duplex is incubated in a standard in
vitro RNAi reaction is measured. This assay has been observed to
determine accurately the fraction of siRNA that is unwound in an
ATP-dependent step in the RNAi pathway and that no functional RISC
is assembled in the absence of ATP (Nykanen et al., 2001). Other
observations have noted that siRNA unwinding correlates with
capacity of an siRNA to function in target cleavage (Nykanen et
al., 2001, supra; Martinez et al., Cell 110, 563-574 (2002)),
demonstrating that siRNA duplex unwinding is required to assemble a
RISC competent to base pair with its target RNA.
[0119] Accordingly, the accumulation of single stranded siRNA
against a test gene such as luciferase after 1 hour incubation in
an in vitro RNAi reaction in the absence of target RNA is measured.
After one hour of incubation with Drosophila embryo lysate in a
standard RNAi reaction, the antisense strand of the luciferase
siRNA is converted to single-strand. In control experiments,
single-stranded RNA is assayed without incubation in lysate. Since
the production of single-stranded antisense siRNA must be
accompanied by an equal amount of single-stranded sense siRNA, the
missing sense-strand is calculated to have been destroyed after
unwinding.
[0120] To establish that the observed asymmetry in the accumulation
of the two single-strands is not an artifact of our unwinding
assay, an independent method for measuring the fraction of siRNA
present as single-strands in protein-RNA complexes is performed. In
this assay, double-stranded siRNA is incubated with Drosophila
embryo lysate in a standard RNAi reaction for 1 h, then a 31 nt
2'-O-methyl RNA oligonucleotide containing a 21 nt sequence
complementary to the radiolabeled siRNA strand is added.
2'-O-methyl oligonucleotides are not cleaved by the RNAi machinery,
but can bind stably to complementary siRNA within the RISC. To
allow recovery of RISC, the 2'-O-methyl oligonucleotide is tethered
to a magnetic bead via a biotin-streptavidin linkage. After washing
away unbound RNA and protein, the amount of radioactive siRNA bound
to the bead is measured. The assay is performed with separate siRNA
duplexes in which either the sense or the antisense strand is
5'-.sup.32P-radiolabeled. Capture of .sup.32P-siRNA is observed
when the 2'-O-methyl oligonucleotide contained a 21-nt region
complementary to the radiolabeled siRNA strand, but not when an
unrelated oligonucleotide is used.
[0121] Thus, the above assay captures all RISC activity directed by
the siRNA strand complementary to the tethered oligonucleotide,
demonstrating that it measures siRNA present in the lysate as
single-strand complexed with RISC proteins.
[0122] Accordingly, this assay can determine the contribution each
strand from a single or double blunt-ended siRNA of the invention
makes to RISC assembly.
EQUIVALENTS
[0123] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 138 <210>
SEQ ID NO 1 <211> LENGTH: 42 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
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ggaacagtat gtctcagaca tc 42 <210> SEQ ID NO 2 <211>
LENGTH: 21 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic Primer <400>
SEQUENCE: 2 uucgaaguau uccgcguacg u 21 <210> SEQ ID NO 3
<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic Primer
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TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic Primer <400> SEQUENCE: 4 gaacagtatg
tctcagacat c 21 <210> SEQ ID NO 5 <211> LENGTH: 41
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
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Primer <400> SEQUENCE: 6 gggagaccac aacggtttcc c 21
<210> SEQ ID NO 7 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 7 gagacuuggg
caaugugac 19 <210> SEQ ID NO 8 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <400> SEQUENCE: 8
gucacauugc ccaagucuct t 21 <210> SEQ ID NO 9 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 9 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 10 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 10 gagacuuggg caaugugac 19 <210> SEQ ID
NO 11 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 11 uucacauugc ccaagucuct t 21
<210> SEQ ID NO 12 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 12
gaugaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54
<210> SEQ ID NO 13 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 13 gagacuuggg
caaugugac 19 <210> SEQ ID NO 14 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER
INFORMATION: inosine <400> SEQUENCE: 14 nucacauugc ccaagucuct
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TYPE: RNA <213> ORGANISM: Drosophila sp. <400>
SEQUENCE: 15 gaugaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga
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TYPE: RNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <400> SEQUENCE: 16
gagacuuggg caaugugac 19 <210> SEQ ID NO 17 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
17 gucacauugc ccaagucuat t 21
<210> SEQ ID NO 18 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 18
gaugaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54
<210> SEQ ID NO 19 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 19 gagacuuggg
caaugugaa 19 <210> SEQ ID NO 20 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <400> SEQUENCE: 20
gucacauugc ccaagucuct t 21 <210> SEQ ID NO 21 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 21 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 22 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 22 gagacuuggg caaugugaa 19 <210> SEQ ID
NO 23 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 23 uucacauugc ccaagucuct t 21
<210> SEQ ID NO 24 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 24
gaugaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54
<210> SEQ ID NO 25 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 25 gagacuuggg
caaugugaa 19 <210> SEQ ID NO 26 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER
INFORMATION: inosine <400> SEQUENCE: 26 ucacauugc ccaagucuct
t 21 <210> SEQ ID NO 27 <211> LENGTH: 54 <212>
TYPE: RNA <213> ORGANISM: Drosophila sp. <400>
SEQUENCE: 27 augaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga
uggu 54 <210> SEQ ID NO 28 <211> LENGTH: 19 <212>
TYPE: RNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <400> SEQUENCE: 28
gagacuuggg caaugugaa 19 <210> SEQ ID NO 29 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
29 gucacauugc ccaagucuat t 21 <210> SEQ ID NO 30 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 30 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 31 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
31 gagacuuggg caaugugact t 21 <210> SEQ ID NO 32 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 32 gucacauugc ccaagucuc 19 <210> SEQ ID
NO 33 <211> LENGTH: 54 <212> TYPE: RNA <213>
ORGANISM: Drosophila sp. <400> SEQUENCE: 33 gaugaagaga
ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54 <210> SEQ
ID NO 34 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 34 uagacuuggg caaugugact t 21
<210> SEQ ID NO 35 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 35 gucacauugc
ccaagucuc 19 <210> SEQ ID NO 36
<211> LENGTH: 54 <212> TYPE: RNA <213> ORGANISM:
Drosophila sp. <400> SEQUENCE: 36 gaugaagaga ggcauguugg
agacuugggc aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 37
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
siRNA Molecule <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (1) <223> OTHER INFORMATION: inosine <400>
SEQUENCE: 37 nagacuuggg caaugugact t 21 <210> SEQ ID NO 38
<211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 38 gucacauugc ccaagucuc 19
<210> SEQ ID NO 39 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 39
gaugaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54
<210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic siRNA Molecule <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 40 gagauugggc aaugugaatt 20
<210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 41 gucacaauug
cccaagucuc 20 <210> SEQ ID NO 42 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 42 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 43 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
43 gagacuuggg caaugugaut t 21 <210> SEQ ID NO 44 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 44 gucacauugc ccaagucuc 19 <210> SEQ ID
NO 45 <211> LENGTH: 54 <212> TYPE: RNA <213>
ORGANISM: Drosophila sp. <400> SEQUENCE: 45 gaugaagaga
ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54 <210> SEQ
ID NO 46 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 46 gagacuuggg caaugugact t 21
<210> SEQ ID NO 47 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 47 gucacauugc
ccaagucua 19 <210> SEQ ID NO 48 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 48 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 49 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
49 uagacuuggg caaugugact t 21 <210> SEQ ID NO 50 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 50 gucacauugc ccaagucua 19 <210> SEQ ID
NO 51 <211> LENGTH: 54 <212> TYPE: RNA <213>
ORGANISM: Drosophila sp. <400> SEQUENCE: 51 gaugaagaga
ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54 <210> SEQ
ID NO 52 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1) <223> OTHER INFORMATION: inosine
<400> SEQUENCE: 52 nagacuuggg caaugugact t 21 <210> SEQ
ID NO 53 <211> LENGTH: 19 <212> TYPE: RNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 53 gucacauugc ccaagucua 19
<210> SEQ ID NO 54 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 54
gaugaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54
<210> SEQ ID NO 55 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic siRNA Molecule <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 55 gagacuuggg caaugugaat t 21
<210> SEQ ID NO 56 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 56 gucacauugc
ccaagucua 19 <210> SEQ ID NO 57 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 57 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 58 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
58 gagacuuggg caaugugaut t 21 <210> SEQ ID NO 59 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 59 gucacauugc ccaagucua 19 <210> SEQ ID
NO 60 <211> LENGTH: 54 <212> TYPE: RNA <213>
ORGANISM: Drosophila sp. <400> SEQUENCE: 60 gaugaagaga
ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54 <210> SEQ
ID NO 61 <211> LENGTH: 19 <212> TYPE: RNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 61 gagacuuggg caaugugac 19
<210> SEQ ID NO 62 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 62 gucacauugc
ccaagucuc 19 <210> SEQ ID NO 63 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 63 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 64 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 64 gagacuuggg caaugugac 19 <210> SEQ ID
NO 65 <211> LENGTH: 19 <212> TYPE: RNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 65 gucacauugc ccaagucua 19
<210> SEQ ID NO 66 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 66
gaugaagaga ggcauguugg agacuugggc aaugugacug cugacaaaga uggu 54
<210> SEQ ID NO 67 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 67 gagacuuggg
caaugugaa 19 <210> SEQ ID NO 68 <211> LENGTH: 19
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
68 gucacauugc ccaagucuc 19 <210> SEQ ID NO 69 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 69 gaugaagaga ggcauguugg agacuugggc
aaugugacug cugacaaaga uggu 54 <210> SEQ ID NO 70 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 70 gagacuuggg caaugugaa 19 <210> SEQ ID
NO 71 <211> LENGTH: 19 <212> TYPE: RNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 71 gucacauugc ccaagucua 19
<210> SEQ ID NO 72 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 72
gaugaagaga ggcauguugg agacuugggc aaugugagug cugacaaaga uggu 54
<210> SEQ ID NO 73 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 73
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 74 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 74 gagacuuggg
caaugugac 19 <210> SEQ ID NO 75 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Mol <400> SEQUENCE: 75 gucacauugc
ccaagucuct t 21 <210> SEQ ID NO 76 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 76 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 77 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 77 gagacuuggg caaugugac 19 <210> SEQ ID
NO 78 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 78 uucacauugc ccaagucuct t 21
<210> SEQ ID NO 79 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 79
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 80 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 80 gagacuuggg
caaugugac 19 <210> SEQ ID NO 81 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER
INFORMATION: inosine <400> SEQUENCE: 81 nucacauugc ccaagucuct
t 21 <210> SEQ ID NO 82 <211> LENGTH: 54 <212>
TYPE: RNA <213> ORGANISM: Drosophila sp. <400>
SEQUENCE: 82 accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu
cauc 54 <210> SEQ ID NO 83 <211> LENGTH: 19 <212>
TYPE: RNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <400> SEQUENCE: 83
gagacuuggg caaugugac 19 <210> SEQ ID NO 84 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
84 gucacauugc ccaagucuat t 21 <210> SEQ ID NO 85 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 85 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 86 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 86 gagacuuggg caaugugaa 19 <210> SEQ ID
NO 87 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 87 gucacauugc ccaagucuct t 21
<210> SEQ ID NO 88 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 88
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 89 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 89 gagacuuggg
caaugugaa 19 <210> SEQ ID NO 90 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <400> SEQUENCE: 90
uucacauugc ccaagucuct t 21 <210> SEQ ID NO 91 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 91 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 92 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 92 gagacuuggg
caaugugaa 19 <210> SEQ ID NO 93 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic siRNA Molecule <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER
INFORMATION: inosine <400> SEQUENCE: 93 nucacauugc ccaagucuct
t 21 <210> SEQ ID NO 94 <211> LENGTH: 54 <212>
TYPE: RNA <213> ORGANISM: Drosophila sp. <400>
SEQUENCE: 94 accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu
cauc 54 <210> SEQ ID NO 95 <211> LENGTH: 19 <212>
TYPE: RNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic siRNA Molecule <400> SEQUENCE: 95
gagacuuggg caaugugaa 19 <210> SEQ ID NO 96 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
96 gucacauugc ccaagucuat t 21 <210> SEQ ID NO 97 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 97 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 98 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
98 gagacuuggg caaugugact t 21 <210> SEQ ID NO 99 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 99 gucacauugc ccaagucuc 19 <210> SEQ ID
NO 100 <211> LENGTH: 54 <212> TYPE: RNA <213>
ORGANISM: Drosophila sp. <400> SEQUENCE: 100 accaucuuug
ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54 <210> SEQ
ID NO 101 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Combined DNA/RNA Molecule:
Synthetic siRNA Molecule <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 101 uagacuuggg caaugugact t 21
<210> SEQ ID NO 102 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 102 gucacauugc
ccaagucuc 19 <210> SEQ ID NO 103 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 103 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 104 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223>
OTHER INFORMATION: inosine <400> SEQUENCE: 104 nagacuuggg
caaugugact t 21 <210> SEQ ID NO 105 <211> LENGTH: 19
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
105 gucacauugc ccaagucuc 19 <210> SEQ ID NO 106 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 106 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 107 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
107 gagacuuggg caaugugaat t 21 <210> SEQ ID NO 108
<211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 108 gucacauugc ccaagucuc 19
<210> SEQ ID NO 109 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE:
109
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 110 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic siRNA Molecule <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 110 gagacuuggg caaugugaut t 21
<210> SEQ ID NO 111 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 111 gucacauugc
ccaagucuc 19 <210> SEQ ID NO 112 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 112 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 113 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
113 gagacuuggg caaugugact t 21 <210> SEQ ID NO 114
<211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 114 gucacauugc ccaagucua 19
<210> SEQ ID NO 115 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 115
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 116 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic siRNA Molecule <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 116 uagacuuggg caaugugact t 21
<210> SEQ ID NO 117 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 117 gucacauugc
ccaagucua 19 <210> SEQ ID NO 118 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 118 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 119 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223>
OTHER INFORMATION: inosine <400> SEQUENCE: 119 nagacuuggg
caaugugact t 21 <210> SEQ ID NO 120 <211> LENGTH: 19
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
120 gucacauugc ccaagucua 19 <210> SEQ ID NO 121 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 121 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 122 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic siRNA Molecule
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
122 gagacuuggg caaugugaat t 21 <210> SEQ ID NO 123
<211> LENGTH: 19 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic siRNA
Molecule <400> SEQUENCE: 123 gucacauugc ccaagucua 19
<210> SEQ ID NO 124 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 124
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 125 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic siRNA Molecule <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 125 gagacuuggg caaugugaut t 21
<210> SEQ ID NO 126 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 126 gucacauugc
ccaagucua 19 <210> SEQ ID NO 127 <211> LENGTH: 54
<212> TYPE: RNA <213> ORGANISM: Drosophila sp.
<400> SEQUENCE: 127
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 128 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 128 gagacuuggg
caaugugac 19 <210> SEQ ID NO 129 <211> LENGTH: 19
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
129 gucacauugc ccaagucuc 19 <210> SEQ ID NO 130 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 130 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 131 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 131 gagacuuggg caaugugac 19 <210> SEQ
ID NO 132 <211> LENGTH: 19 <212> TYPE: RNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 132 gucacauugc ccaagucua 19
<210> SEQ ID NO 133 <211> LENGTH: 54 <212> TYPE:
RNA <213> ORGANISM: Drosophila sp. <400> SEQUENCE: 133
accaucuuug ucagcaguca cauugcccaa gucuccaaca ugccucucuu cauc 54
<210> SEQ ID NO 134 <211> LENGTH: 19 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic siRNA Molecule <400> SEQUENCE: 134 gagacuuggg
caaugugaa 19 <210> SEQ ID NO 135 <211> LENGTH: 19
<212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic siRNA Molecule <400> SEQUENCE:
135 gucacauugc ccaagucuc 19 <210> SEQ ID NO 136 <211>
LENGTH: 54 <212> TYPE: RNA <213> ORGANISM: Drosophila
sp. <400> SEQUENCE: 136 accaucuuug ucagcaguca cauugcccaa
gucuccaaca ugccucucuu cauc 54 <210> SEQ ID NO 137 <211>
LENGTH: 19 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic siRNA Molecule
<400> SEQUENCE: 137 gagacuuggg caaugugaa 19 <210> SEQ
ID NO 138 <211> LENGTH: 19 <212> TYPE: RNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
siRNA Molecule <400> SEQUENCE: 138 gucacauugc ccaagucua
19
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