siRNA targeting pituitary tumor-transforming 1 (PTTG1)

Abstract

Efficient sequence specific gene silencing is possible through the use of siRNA technology. By selecting particular siRNAs by rational design, one can maximize the generation of an effective gene silencing reagent, as well as methods for silencing genes. Methods, compositions, and kits generated through rational design of siRNAs are disclosed including those directed to PTTG1.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No. 10/714,333, filed Nov. 14, 2003, which claims the benefit of U.S. Provisional Application No. 60/426,137, filed Nov. 14, 2002, and also claims the benefit of U.S. Provisional Application No. 60/502,050, filed Sep. 10, 2003; this application is also a continuation-in-part of U.S. Ser. No. 10/940,892, filed Sep. 14, 2004, which is a continuation of PCT Application No. PCT/US 04/14885, international filing date May 12, 2004. The disclosures of the priority applications, including the sequence listings and tables submitted in electronic form in lieu of paper, are incorporated by reference into the instant specification.

SEQUENCE LISTING

[0002] The sequence listing for this application has been submitted in accordance with 37 CFR .sctn. 1.52(e) and 37 CFR .sctn. 1.821 on CD-ROM in lieu of paper on a disk containing the sequence listing file entitled "DHARMA.sub.--2100-US45_CRF.txt" created May 30, 2007, 88 kb. Applicants hereby incorporate by reference the sequence listing provided on CD-ROM in lieu of paper into the instant specification.

FIELD OF INVENTION

[0003] The present invention relates to RNA interference ("RNAi").

BACKGROUND OF THE INVENTION

[0004] Relatively recently, researchers observed that double stranded RNA ("dsRNA") could be used to inhibit protein expression. This ability to silence a gene has broad potential for treating human diseases, and many researchers and commercial entities are currently investing considerable resources in developing therapies based on this technology.

[0005] Double stranded RNA induced gene silencing can occur on at least three different levels: (i) transcription inactivation, which refers to RNA guided DNA or histone methylation; (ii) siRNA induced mRNA degradation; and (iii) mRNA induced transcriptional attenuation.

[0006] It is generally considered that the major mechanism of RNA induced silencing (RNA interference, or RNAi) in mammalian cells is mRNA degradation. Initial attempts to use RNAi in mammalian cells focused on the use of long strands of dsRNA. However, these attempts to induce RNAi met with limited success, due in part to the induction of the interferon response, which results in a general, as opposed to a target-specific, inhibition of protein synthesis. Thus, long dsRNA is not a viable option for RNAi in mammalian systems.

[0007] More recently it has been shown that when short (18-30 bp) RNA duplexes are introduced into mammalian cells in culture, sequence-specific inhibition of target mRNA can be realized without inducing an interferon response. Certain of these short dsRNAs, referred to as small inhibitory RNAs ("siRNAs"), can act catalytically at sub-molar concentrations to cleave greater than 95% of the target mRNA in the cell. A description of the mechanisms for siRNA activity, as well as some of its applications are described in Provost et al. (2002) Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, EMBO J. 21(21): 5864-5874; Tabara et al. (2002) The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 109(7):861-71; Ketting et al. (2002) Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. elegans; Martinez et al., Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 110(5):563; Hutvagner & Zamore (2002) A microRNA in a multiple-turnover RNAi enzyme complex, Science 297:2056.

[0008] From a mechanistic perspective, introduction of long double stranded RNA into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer. Sharp, RNA interference--2001, Genes Dev. 2001, 15:485. Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs. Bernstein, Caudy, Hammond, & Hannon (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409:363. The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition. Nykanen, Haley, & Zamore (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway, Cell 107:309. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing. Elbashir, Lendeckel, & Tuschl (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev. 15:188, FIG. 1.

[0009] The interference effect can be long lasting and may be detectable after many cell divisions. Moreover, RNAi exhibits sequence specificity. Kisielow, M. et al. (2002) Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA, J. Biochem. 363:1-5. Thus, the RNAi machinery can specifically knock down one type of transcript, while not affecting closely related mRNA. These properties make siRNA a potentially valuable tool for inhibiting gene expression and studying gene function and drug target validation. Moreover, siRNAs are potentially useful as therapeutic agents against: (1) diseases that are caused by over-expression or misexpression of genes; and (2) diseases brought about by expression of genes that contain mutations.

[0010] Successful siRNA-dependent gene silencing depends on a number of factors. One of the most contentious issues in RNAi is the question of the necessity of siRNA design, i.e., considering the sequence of the siRNA used. Early work in C. elegans and plants circumvented the issue of design by introducing long dsRNA (see, for instance, Fire, A. et al. (1998) Nature 391:806-811). In this primitive organism, long dsRNA molecules are cleaved into siRNA by Dicer, thus generating a diverse population of duplexes that can potentially cover the entire transcript. While some fraction of these molecules are non-functional (i.e., induce little or no silencing) one or more have the potential to be highly functional, thereby silencing the gene of interest and alleviating the need for siRNA design. Unfortunately, due to the interferon response, this same approach is unavailable for mammalian systems. While this effect can be circumvented by bypassing the Dicer cleavage step and directly introducing siRNA, this tactic carries with it the risk that the chosen siRNA sequence may be non-functional or semi-functional.

[0011] A number of researches have expressed the view that siRNA design is not a crucial element of RNAi. On the other hand, others in the field have begun to explore the possibility that RNAi can be made more efficient by paying attention to the design of the siRNA. Unfortunately, none of the reported methods have provided a satisfactory scheme for reliably selecting siRNA with acceptable levels of functionality. Accordingly, there is a need to develop rational criteria by which to select siRNA with an acceptable level of functionality, and to identify siRNA that have this improved level of functionality, as well as to identify siRNAs that are hyperfunctional.

SUMMARY OF THE INVENTION

[0012] The present invention is directed to increasing the efficiency of RNAi, particularly in mammalian systems. Accordingly, the present invention provides kits, siRNAs and methods for increasing siRNA efficacy.

[0013] According to a first embodiment, the present invention provides a kit for gene silencing, wherein said kit is comprised of a pool of at least two siRNA duplexes, each of which is comprised of a sequence that is complementary to a portion of the sequence of one or more target messenger RNA, and each of which is selected using non-target specific criteria.

[0014] According to a second embodiment, the present invention provides a method for selecting an siRNA, said method comprising applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; and determining the relative functionality of the at least two siRNAs.

[0015] According to a third embodiment, the present invention also provides a method for selecting an siRNA wherein said selection criteria are embodied in a formula comprising: (-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-10- *C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-9*U.sub.9-9*C.sub.18-8*G.sub.10-- 7*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.19+7*U.sub.17+8*A.sub.2+8*A.sub.4+- 8*A.sub.5+8*C.sub.4+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.- 9+15*G.sub.1+18*A.sub.3+19*U.sub.10-Tm-3*(GC.sub.total)-6*(GC.sub.15-19)-3- 0*X; or Formula VIII: (-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5- )*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18- +(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8- +(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-10)*U16+(- 3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-20)*C6+(-18- )*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-- 5)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-4)*G5- +(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-11)*G10+(1)*G11+(9)*G12+(-24)*G13+(18)*G14+- (11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number of A+U in position 15-19)-3*(number of G+C in whole siRNA), Formula X wherein position numbering begins at the 5'-most position of a sense strand, and [0016] A.sub.1=1 if A is the base at position 1 of the sense strand, otherwise its value is 0; [0017] A.sub.2=1 if A is the base at position 2 of the sense strand, otherwise its value is 0; [0018] A.sub.3=1 if A is the base at position 3 of the sense strand, otherwise its value is 0; [0019] A.sub.4=1 if A is the base at position 4 of the sense strand, otherwise its value is 0; [0020] A.sub.5=1 if A is the base at position 5 of the sense strand, otherwise its value is 0; [0021] A.sub.6=1 if A is the base at position 6 of the sense strand, otherwise its value is 0; [0022] A.sub.7=1 if A is the base at position 7 of the sense strand, otherwise its value is 0; [0023] A.sub.10=1 if A is the base at position 10 of the sense strand, otherwise its value is 0; [0024] A.sub.11=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; [0025] A.sub.13=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; [0026] A.sub.19=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; [0027] C.sub.3=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; [0028] C.sub.4=1 if C is the base at position 4 of the sense strand, otherwise its value is 0; [0029] C.sub.5=1 if C is the base at position 5 of the sense strand, otherwise its value is 0; [0030] C.sub.6=1 if C is the base at position 6 of the sense strand, otherwise its value is 0; [0031] C.sub.7=1 if C is the base at position 7 of the sense strand, otherwise its value is 0; [0032] C.sub.9=1 if C is the base at position 9 of the sense strand, otherwise its value is 0; [0033] C.sub.17=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; [0034] C.sub.18=1 if C is the base at position 18 of the sense strand, otherwise its value is 0; [0035] C.sub.19=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; [0036] G.sub.1=1 if G is the base at position 1 on the sense strand, otherwise its value is 0; [0037] G.sub.2=1 if G is the base at position 2 of the sense strand, otherwise its value is 0; [0038] G.sub.8=1 if G is the base at position 8 on the sense strand, otherwise its value is 0; [0039] G.sub.10=1 if G is the base at position 10 on the sense strand, otherwise its value is 0; [0040] G.sub.13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0; [0041] G.sub.19=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0; [0042] U.sub.1=1 if U is the base at position 1 on the sense strand, otherwise its value is 0; [0043] U.sub.2=1 if U is the base at position 2 on the sense strand, otherwise its value is 0; [0044] U.sub.3=1 if U is the base at position 3 on the sense strand, otherwise its value is 0; [0045] U.sub.4=1 if U is the base at position 4 on the sense strand, otherwise its value is 0; [0046] U.sub.7=1 if U is the base at position 7 on the sense strand, otherwise its value is 0; [0047] U.sub.9=1 if U is the base at position 9 on the sense strand, otherwise its value is 0; [0048] U.sub.10=1 if U is the base at position 10 on the sense strand, otherwise its value is 0; [0049] U.sub.15=1 if U is the base at position 15 on the sense strand, otherwise its value is 0; [0050] U.sub.16=1 if U is the base at position 16 on the sense strand, otherwise its value is 0; [0051] U.sub.17=1 if U is the base at position 17 on the sense strand, otherwise its value is 0; [0052] U.sub.18=1 if U is the base at position 18 on the sense strand, otherwise its value is 0. [0053] GC.sub.15-19=the number of G and C bases within positions 15-19 of the sense strand, or within positions 15-18 if the sense strand is only 18 base pairs in length; [0054] GC.sub.total=the number of G and C bases in the sense strand; [0055] Tm=100 if the siRNA oligo has the internal repeat longer then 4 base pairs, otherwise its value is 0; and [0056] X=the number of times that the same nucleotide repeats four or more times in a row.

[0057] According to a fourth embodiment, the invention provides a method for developing an algorithm for selecting siRNA, said method comprising: (a) selecting a set of siRNA; (b) measuring gene silencing ability of each siRNA from said set; (c) determining relative functionality of each siRNA; (d) determining improved functionality by the presence or absence of at least one variable selected from the group consisting of the presence or absence of a particular nucleotide at a particular position, the total number of As and Us in positions 15-19, the number of times that the same nucleotide repeats within a given sequence, and the total number of Gs and Cs; and (e) developing an algorithm using the information of step (d).

[0058] According to a fifth embodiment, the present invention provides a kit, wherein said kit is comprised of at least two siRNAs, wherein said at least two siRNAs comprise a first optimized siRNA and a second optimized siRNA, wherein said first optimized siRNA and said second optimized siRNA are optimized according a formula comprising Formula X.

[0059] The present invention also provides a method for identifying a hyperfunctional siRNA, comprising applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; determining the relative functionality of the at least two siRNAs and assigning each of the at least two siRNAs a functionality score; and selecting siRNAs from the at least two siRNAs that have a functionality score that reflects greater than 80 percent silencing at a concentration in the picomolar range, wherein said greater than 80 percent silencing endures for greater than 120 hours.

[0060] According to a sixth embodiment, the present invention provides a hyperfunctional siRNA that is capable of silencing Bcl2.

[0061] According to a seventh embodiment, the present invention provides a method for developing an siRNA algorithm for selecting functional and hyperfunctional siRNAs for a given sequence. The method comprises:

[0062] (a) selecting a set of siRNAs;

[0063] (b) measuring the gene silencing ability of each siRNA from said set;

[0064] (c) determining the relative functionality of each siRNA;

[0065] (d) determining the amount of improved functionality by the presence or absence of at least one variable selected from the group consisting of the total GC content, melting temperature of the siRNA, GC content at positions 15-19, the presence or absence of a particular nucleotide at a particular position, relative thermodynamic stability at particular positions in a duplex, and the number of times that the same nucleotide repeats within a given sequence; and

[0066] (e) developing an algorithm using the information of step (d).

[0067] According to this embodiment, preferably the set of siRNAs comprises at least 90 siRNAs from at least one gene, more preferably at least 180 siRNAs from at least two different genes, and most preferably at least 270 and 360 siRNAs from at least three and four different genes, respectively. Additionally, in step (d) the determination is made with preferably at least two, more preferably at least three, even more preferably at least four, and most preferably all of the variables. The resulting algorithm is not target sequence specific.

[0068] In another embodiment, the present invention provides rationally designed siRNAs identified using the formulas above.

[0069] In yet another embodiment, the present invention is directed to hyperfunctional siRNA.

[0070] The ability to use the above algorithms, which are not sequence or species specific, allows for the cost-effective selection of optimized siRNAs for specific target sequences. Accordingly, there will be both greater efficiency and reliability in the use of siRNA technologies.

[0071] In various embodiments, siRNAs that target pituitary tumor-transforming 1 (PTTG1) are provided. In various embodiments, the siRNAs are rationally designed. In various embodiments, the siRNAs are functional or hyperfunctional.

[0072] In various embodiments, an siRNA that targets PTTG1 is provided, wherein the siRNA is selected from the group consisting of various siRNA sequences targeting PTTG1 that are disclosed herein. In various embodiments, the siRNA sequence is selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498.

[0073] In various embodiments, siRNA comprising a sense region and an antisense region are provided, wherein said sense region and said antisense region are at least 90% complementary, said sense region and said antisense region together form a duplex region comprising 18-30 base pairs, and said sense region comprises a sequence that is at least 90% similar to a sequence selected from the group consisting of siRNA sequences targeting PTTG1 that are disclosed herein. In various embodiments, the siRNA sequence is selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498.

[0074] In various embodiments, an siRNA comprising a sense region and an antisense region is provided, wherein said sense region and said antisense region are at least 90% complementary, said sense region and said antisense region together form a duplex region comprising 18-30 base pairs, and said sense region comprises a sequence that is identical to a contiguous stretch of at least 18 bases of a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498. In various embodiments, the duplex region is 19-30 base pairs, and the sense region comprises a sequence that is identical to a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498.

[0075] In various embodiments, a pool of at least two siRNAs is provided, wherein said pool comprises a first siRNA and a second siRNA, said first siRNA comprising a duplex region of length 18-30 base pairs that has a first sense region that is at least 90% similar to 18 bases of a first sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498, and said second siRNA comprises a duplex region of length 18-30 base pairs that has a second sense region that is at least 90% similar to 18 bases of a second sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498, wherein said first sense region and said second sense region are not identical.

[0076] In various embodiments, the first sense region comprises a sequence that is identical to at least 18 bases of a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498, and said second sense region comprises a sequence that is identical to at least 18 bases of a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498. In various embodiments, the duplex of said first siRNA is 19-30 base pairs, and said first sense region comprises a sequence that is at least 90% similar to a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498, and said duplex of said second siRNA is 19-30 base pairs and comprises a sequence that is at least 90% similar to a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498.

[0077] In various embodiments, the duplex of said first siRNA is 19-30 base pairs and said first sense region comprises a sequence that is identical to at least 18 bases of a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498, and said duplex of said second siRNA is 19-30 base pairs and said second region comprises a sequence that is identical to a sequence selected from the group consisting of SEQ ID NO. 438 to SEQ ID NO. 498.

[0078] For a better understanding of the present invention together with other and further advantages and embodiments, reference is made to the following description taken in conjunction with the examples, the scope of which is set forth in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0079] FIG. 1 shows a model for siRNA-RISC interactions. RISC has the ability to interact with either end of the siRNA or miRNA molecule. Following binding, the duplex is unwound, and the relevant target is identified, cleaved, and released.

[0080] FIG. 2 is a representation of the functionality of two hundred and seventy siRNA duplexes that were generated to target human cyclophilin, human diazepam-binding inhibitor (DB), and firefly luciferase.

[0081] FIG. 3a is a representation of the silencing effect of 30 siRNAs in three different cells lines, HEK293, DU145, and Hela. FIG. 3b shows the frequency of different functional groups (>95% silencing (black), >80% silencing (gray), >50% silencing (dark gray), and <50% silencing (white)) based on GC content. In cases where a given bar is absent from a particular GC percentage, no siRNA were identified for that particular group. FIG. 3c shows the frequency of different functional groups based on melting temperature (Tm).

[0082] FIGS. 4A-4E are representations of a statistical analysis that revealed correlations between silencing and five sequence-related properties of siRNA: (A) an A at position 19 of the sense strand, (B) an A at position 3 of the sense strand, (C) a U at position 10 of the sense strand, (D) a base other than G at position 13 of the sense strand, and (E) a base other than C at position 19 of the sense strand. All variables were correlated with siRNA silencing of firefly luciferase and human cyclophilin. siRNAs satisfying the criterion are grouped on the left (Selected) while those that do not, are grouped on the right (Eliminated). Y-axis is "% Silencing of Control." Each position on the X-axis represents a unique siRNA.

[0083] FIGS. 5A and 5B are representations of firefly luciferase and cyclophilin siRNA panels sorted according to functionality and predicted values using Formula VIII. The siRNA found within the circle represent those that have Formula VIII values (SMARTSCORES.TM., or siRNA rank) above zero. siRNA outside the indicated area have calculated Formula VIII values that are below zero. Y-axis is "Expression (% Control)." Each position on the X-axis represents a unique siRNA.

[0084] FIG. 6A is a representation of the average internal stability profile (AISP) derived from 270 siRNAs taken from three separate genes (cyclophilin B, DBI and firefly luciferase). Graphs represent AISP values of highly functional, functional, and non-functional siRNA. FIG. 6B is a comparison between the AISP of naturally derived GFP siRNA (filled squares) and the AISP of siRNA from cyclophilin B, DBI, and luciferase having >90% silencing properties (no fill) for the antisense strand. "DG" is the symbol for .DELTA.G, free energy.

[0085] FIG. 7 is a histogram showing the differences in duplex functionality upon introduction of base pair mismatches. The X-axis shows the mismatch introduced in the siRNA and the position it is introduced (e.g., 8C>A reveals that position 8 (which normally has a C) has been changed to an A). The Y-axis is "% Silencing (Normalized to Control)." The samples on the X-axis represent siRNAs at 100 nM and are, reading from left to right: 1A to C, 1A to G, 1A to U; 2A to C, 2A to G, 2A to U; 3A to C, 3A to G, 3A to U; 4G to A, 4G to C; 4G to U; 5U to A, 5U to C, 5U to G; 6U to A, 6U to C, 6U to G; 7G to A, 7G to C, 7G to U; 8C to A, 8C to G, 8C to U; 9G to A, 9G to C, 9G to U; 10C to A, 10C to G, 10C to U; 11G to A, 11G to C, 11G to U; 12G to A, 12G to C, 12G to U; 13A to C, 13A to G, 13A to U; 14G to A, 14G to C, 14G to U; 15G to A, 15G to C, 15G to U; 16A to C, 16A to G, 16A to U; 17G to A, 17G to C, 17G to U; 18U to A, 18U to C, 18U to G; 19U to A, 19U to C, 19U to G; 20 wt; Control.

[0086] FIG. 8A is histogram that shows the effects of 5'sense and antisense strand modification with 2'-O-methylation on functionality. FIG. 8B is an expression profile showing a comparison of sense strand off-target effects for IGF1R-3 and 2'-O-methyl IGF1R-3. Sense strand off-targets (lower box) are not induced when the 5' end of the sense strand is modified with 2'-O-methyl groups (top box).

[0087] FIG. 9 shows a graph of SMARTSCORES.TM., or siRNA rank, versus RNAi silencing values for more than 360 siRNA directed against 30 different genes. SiRNA to the right of the vertical bar represent those siRNA that have desirable SMARTSCORES.TM., or siRNA rank.

[0088] FIGS. 10A-E compare the RNAi of five different genes (SEAP, DBI, PLK, Firefly Luciferase, and Renilla Luciferase) by varying numbers of randomly selected siRNA and four rationally designed (SMART-selected) siRNA chosen using the algorithm described in Formula VIII. In addition, RNAi induced by a pool of the four SMART-selected siRNA is reported at two different concentrations (100 and 400 nM). 10F is a comparison between a pool of randomly selected EGFR siRNA (Pool 1) and a pool of SMART-selected EGFR siRNA (Pool 2). Pool 1, S1-S4 and Pool 2 S1-S4 represent the individual members that made up each respective pool. Note that numbers for random siRNAs represent the position of the 5' end of the sense strand of the duplex. The Y-axis represents the % expression of the control(s). The X-axis is the percent expression of the control.

[0089] FIG. 11 shows the Western blot results from cells treated with siRNA directed against twelve different genes involved in the clathrin-dependent endocytosis pathway (CHC, DynII, CALM, CLCa, CLCb, Eps15, Eps15R, Rab5a, Rab5b, Rab5c, .beta.2 subunit of AP-2 and EEA.1). siRNA were selected using Formula VIII. "Pool" represents a mixture of duplexes 1-4. Total concentration of each siRNA in the pool is 25 nM. Total concentration=4.times.25=100 nM.

[0090] FIG. 12 is a representation of the gene silencing capabilities of rationally-selected siRNA directed against ten different genes (human and mouse cyclophilin, C-myc, human lamin A/C, QB (ubiquinol-cytochrome c reductase core protein I), MEK1 and MEK2, ATE1 (arginyl-tRNA protein transferase), GAPDH, and Eg5). The Y-axis is the percent expression of the control. Numbers 1, 2, 3 and 4 represent individual rationally selected siRNA. "Pool" represents a mixture of the four individual siRNA.

[0091] FIG. 13 is the sequence of the top ten Bcl2 siRNAs as determined by Formula VIII. Sequences are listed 5' to 3'.

[0092] FIG. 14 is the knockdown by the top ten Bcl2 siRNAs at 100 nM concentrations. The Y-axis represents the amount of expression relative to the non-specific (ns) and transfection mixture control.

[0093] FIG. 15 represents a functional walk where siRNA beginning on every other base pair of a region of the luciferase gene are tested for the ability to silence the luciferase gene. The Y-axis represents the percent expression relative to a control. The X-axis represents the position of each individual siRNA. Reading from left to right across the X-axis, the position designations are 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.

[0094] FIGS. 16A and 16B are histograms demonstrating the inhibition of target gene expression by pools of 2 (16A) and 3 (16B) siRNA duplexes taken from the walk described in FIG. 15. The Y-axis in each represents the percent expression relative to control. The X-axis in each represents the position of the first siRNA in paired pools, or trios of siRNAs. For instance, the first paired pool contains siRNAs 1 and 3. The second paired pool contains siRNAs 3 and 5. Pool 3 (of paired pools) contains siRNAs 5 and 7, and so on. For each of 16A and 16B, the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.

[0095] FIGS. 17A and 17B are histograms demonstrating the inhibition of target gene expression by pools of 4 (17A) and 5 (17B) siRNA duplexes. The Y-axis in each represents the percent expression relative to control. The X-axis in each represents the position of the first siRNA in each pool. For each of 17A and 17B, the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.

[0096] FIGS. 18A and 18B are histograms demonstrating the inhibition of target gene expression by siRNAs that are ten (18A) and twenty (18B) base pairs base pairs apart. The Y-axis represents the percent expression relative to a control. The X-axis represents the position of the first siRNA in each pool. For each of 18A and 18B, the X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.

[0097] FIG. 19 shows that pools of siRNAs (dark gray bar) work as well (or better) than the best siRNA in the pool (light gray bar). The Y-axis represents the percent expression relative to a control. The X-axis represents the position of the first siRNA in each pool. The X-axis from left to right reads 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and Plasmid.

[0098] FIG. 20 shows that the combination of several semifunctional siRNAs (dark gray) result in a significant improvement of gene expression inhibition over individual (semi-functional; light gray) siRNA. The Y-axis represents the percent expression relative to a control.

[0099] FIGS. 21A, 21B and 21C show both pools (Library, Lib) and individual siRNAs in inhibition of gene expression of Beta-Galactosidase, Renilla Luciferase and SEAP (alkaline phosphatase). Numbers on the X-axis indicate the position of the 5'-most nucleotide of the sense strand of the duplex. The Y-axis represents the percent expression of each gene relative to a control. Libraries contain 19 nucleotide long siRNAs (not including overhangs) that begin at the following nucleotides: SEAP: Lib 1: 206, 766, 812, 923, Lib 2: 1117, 1280, 1300, 1487, Lib 3: 206, 766, 812, 923, 1117, 1280, 1300, 1487, Lib 4: 206, 812, 1117, 1300, Lib 5: 766, 923, 1280, 1487, Lib 6: 206, 1487; Bgal: Lib 1: 979, 1339, 2029, 2590, Lib 2: 1087, 1783, 2399, 3257, Lib 3: 979, 1783, 2590, 3257, Lib 4: 979, 1087, 1339, 1783, 2029, 2399, 2590, 3257, Lib 5: 979, 1087, 1339, 1783, Lib 6: 2029, 2399, 2590, 3257; Renilla: Lib 1: 174, 300, 432, 568, Lib 2: 592, 633, 729, 867, Lib 3: 174, 300, 432, 568, 592, 633, 729, 867, Lib 4: 174, 432, 592, 729, Lib 5: 300, 568, 633, 867, Lib 6: 592, 568.

[0100] FIG. 22 shows the results of an EGFR and TfnR internalization assay when single gene knockdowns are performed. The Y-axis represents percent internalization relative to control.

[0101] FIG. 23 shows the results of an EGFR and TfnR internalization assay when multiple genes are knocked down (e.g., Rab5a, b, c). The Y-axis represents the percent internalization relative to control.

[0102] FIG. 24 shows the simultaneous knockdown of four different genes. siRNAs directed against G6PD, GAPDH, PLK, and UQC were simultaneously introduced into cells. Twenty-four hours later, cultures were harvested and assayed for mRNA target levels for each of the four genes. A comparison is made between cells transfected with individual siRNAs vs. a pool of siRNAs directed against all four genes.

[0103] FIG. 25 shows the functionality of ten siRNAs at 0.3 nM concentrations.

DETAILED DESCRIPTION

Definitions

[0104] Unless stated otherwise, the following terms and phrases have the meanings provided below:

Complementary

[0105] The term "complementary" refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine. However, when a U is denoted in the context of the present invention, the ability to substitute a T is implied, unless otherwise stated.

[0106] Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 90% complementarity.

Deoxynucleotide

[0107] The term "deoxynucleotide" refers to a nucleotide or polynucleotide lacking a hydroxyl group (OH group) at the 2' and/or 3' position of a sugar moiety. Instead, it has a hydrogen bonded to the 2' and/or 3' carbon. Within an RNA molecule that comprises one or more deoxynucleotides, "deoxynucleotide" refers to the lack of an OH group at the 2' position of the sugar moiety, having instead a hydrogen bonded directly to the 2' carbon.

Deoxyribonucleotide

[0108] The terms "deoxyribonucleotide" and "DNA" refer to a nucleotide or polynucleotide comprising at least one sugar moiety that has an H, rather than an OH, at its 2' and/or 3'position.

Duplex Region

[0109] The phrase "duplex region" refers to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between polynucleotide strands that are complementary or substantially complementary. For example, a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the "duplex region" has 19 base pairs. The remaining bases may, for example, exist as 5' and 3' overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to 79% or greater complementarity. For example, a mismatch in a duplex region consisting of 19 base pairs results in 94.7% complementarity, rendering the duplex region substantially complementary.

Filters

[0110] The term "filter" refers to one or more procedures that are performed on sequences that are identified by the algorithm. In some instances, filtering includes in silico procedures where sequences identified by the algorithm can be screened to identify duplexes carrying desirable or undesirable motifs. Sequences carrying such motifs can be selected for, or selected against, to obtain a final set with the preferred properties. In other instances, filtering includes wet lab experiments. For instance, sequences identified by one or more versions of the algorithm can be screened using any one of a number of procedures to identify duplexes that have hyperfunctional traits (e.g., they exhibit a high degree of silencing at subnanomolar concentrations and/or exhibit high degrees of silencing longevity).

Gene Silencing

[0111] The phrase "gene silencing" refers to a process by which the expression of a specific gene product is lessened or attenuated. Gene silencing can take place by a variety of pathways. Unless specified otherwise, as used herein, gene silencing refers to decreases in gene product expression that results from RNA interference (RNAi), a defined, though partially characterized pathway whereby small inhibitory RNA (siRNA) act in concert with host proteins (e.g., the RNA induced silencing complex, RISC) to degrade messenger RNA (mRNA) in a sequence-dependent fashion. The level of gene silencing can be measured by a variety of means, including, but not limited to, measurement of transcript levels by Northern Blot Analysis, B-DNA techniques, transcription-sensitive reporter constructs, expression profiling (e.g., DNA chips), and related technologies. Alternatively, the level of silencing can be measured by assessing the level of the protein encoded by a specific gene. This can be accomplished by performing a number of studies including Western Analysis, measuring the levels of expression of a reporter protein that has e.g., fluorescent properties (e.g., GFP) or enzymatic activity (e.g., alkaline phosphatases), or several other procedures.

miRNA

[0112] The term "miRNA" refers to microRNA.

Nucleotide

[0113] The term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs.

[0114] Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2, or CN, wherein R is an alkyl moiety. Nucleotide analogs are also meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2'-methyl ribose, non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates and peptides.

[0115] Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.

[0116] The term nucleotide is also meant to include what are known in the art as universal bases. By way of example, universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine. The term "nucleotide" is also meant to include the N3' to P5' phosphoramidate, resulting from the substitution of a ribosyl 3' oxygen with an amine group.

[0117] Further, the term nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.

Off-Target Silencing and Off-Target Interference

[0118] The phrases "off-target silencing" and "off-target interference" are defined as degradation of mRNA other than the intended target mRNA due to overlapping and/or partial homology with secondary mRNA messages.

Polynucleotide

[0119] The term "polynucleotide" refers to polymers of nucleotides, and includes but is not limited to DNA, RNA, DNA/RNA hybrids including polynucleotide chains of regularly and/or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an --OH, then and --H, then an --OH, then an --H, and so on at the 2' position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included.

Polyribonucleotide

[0120] The term "polyribonucleotide" refers to a polynucleotide comprising two or more modified or unmodified ribonucleotides and/or their analogs. The term "polyribonucleotide" is used interchangeably with the term "oligoribonucleotide."

Ribonucleotide and Ribonucleic Acid

[0121] The term "ribonucleotide" and the phrase "ribonucleic acid" (RNA), refer to a modified or unmodified nucleotide or polynucleotide comprising at least one ribonucleotide unit. A ribonucleotide unit comprises an hydroxyl group attached to the 2' position of a ribosyl moiety that has a nitrogenous base attached in N-glycosidic linkage at the 1' position of a ribosyl moiety, and a moiety that either allows for linkage to another nucleotide or precludes linkage.

siRNA

[0122] The term "siRNA" refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.

[0123] siRNA may be divided into five (5) groups (non-functional, semi-functional, functional, highly functional, and hyper-functional) based on the level or degree of silencing that they induce in cultured cell lines. As used herein, these definitions are based on a set of conditions where the siRNA is transfected into said cell line at a concentration of 100 nM and the level of silencing is tested at a time of roughly 24 hours after transfection, and not exceeding 72 hours after transfection. In this context, "non-functional siRNA" are defined as those siRNA that induce less than 50% (<50%) target silencing. "Semi-functional siRNA" induce 50-79% target silencing. "Functional siRNA" are molecules that induce 80-95% gene silencing. "Highly-functional siRNA" are molecules that induce greater than 95% gene silencing. "Hyperfunctional siRNA" are a special class of molecules. For purposes of this document, hyperfunctional siRNA are defined as those molecules that: (1) induce greater than 95% silencing of a specific target when they are transfected at subnanomolar concentrations (i.e., less than one nanomolar); and/or (2) induce functional (or better) levels of silencing for greater than 96 hours. These relative functionalities (though not intended to be absolutes) may be used to compare siRNAs to a particular target for applications such as functional genomics, target identification and therapeutics.

SMARTSCORE.TM., or siRNA Rank

[0124] The term "SMARTSCORE.TM.", or "siRNA rank" refers to a number determined by applying any of the formulas to a given siRNA sequence. The term "SMART-selected" or "rationally selected" or "rational selection" refers to siRNA that have been selected on the basis of their SMARTSCORES.TM., or siRNA ranking.

Substantially Similar

[0125] The phrase "substantially similar" refers to a similarity of at least 90% with respect to the identity of the bases of the sequence.

Target

[0126] The term "target" is used in a variety of different forms throughout this document and is defined by the context in which it is used. "Target mRNA" refers to a messenger RNA to which a given siRNA can be directed against. "Target sequence" and "target site" refer to a sequence within the mRNA to which the sense strand of an siRNA shows varying degrees of homology and the antisense strand exhibits varying degrees of complementarity. The phrase "siRNA target" can refer to the gene, mRNA, or protein against which an siRNA is directed. Similarly, "target silencing" can refer to the state of a gene, or the corresponding mRNA or protein.

Transfection

[0127] The term "transfection" refers to a process by which agents are introduced into a cell. The list of agents that can be transfected is large and includes, but is not limited to, siRNA, sense and/or anti-sense sequences, DNA encoding one or more genes and organized into an expression plasmid, proteins, protein fragments, and more. There are multiple methods for transfecting agents into a cell including, but not limited to, electroporation, calcium phosphate-based transfections, DEAE-dextran-based transfections, lipid-based transfections, molecular conjugate-based transfections (e.g., polylysine-DNA conjugates), microinjection and others.

[0128] The present invention is directed to improving the efficiency of gene silencing by siRNA. Through the inclusion of multiple siRNA sequences that are targeted to a particular gene and/or selecting an siRNA sequence based on certain defined criteria, improved efficiency may be achieved.

[0129] The present invention will now be described in connection with preferred embodiments. These embodiments are presented in order to aid in an understanding of the present invention and are not intended, and should not be construed, to limit the invention in any way. All alternatives, modifications and equivalents that may become apparent to those of ordinary skill upon reading this disclosure are included within the spirit and scope of the present invention.

[0130] Furthermore, this disclosure is not a primer on RNA interference. Basic concepts known to persons skilled in the art have not been set forth in detail.

[0131] The present invention is directed to increasing the efficiency of RNAi, particularly in mammalian systems. Accordingly, the present invention provides kits, siRNAs and methods for increasing siRNA efficacy.

[0132] According to a first embodiment, the present invention provides a kit for gene silencing, wherein said kit is comprised of a pool of at least two siRNA duplexes, each of which is comprised of a sequence that is complementary to a portion of the sequence of one or more target messenger RNA, and each of which is selected using non-target specific criteria. Each of the at least two siRNA duplexes of the kit complementary to a portion of the sequence of one or more target mRNAs is preferably selected using Formula X.

[0133] According to a second embodiment, the present invention provides a method for selecting an siRNA, said method comprising applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; and determining the relative functionality of the at least two siRNAs.

[0134] In one embodiment, the present invention also provides a method wherein said selection criteria are embodied in a formula comprising: (-14)*G.sub.13-13*A.sub.1-12*U.sub.7-11*U.sub.2-10*A.sub.11-10*U.sub.4-10- *C.sub.3-10*C.sub.5-10*C.sub.6-9*A.sub.10-9*U.sub.9-9*C.sub.18-8*G.sub.10-- 7*U.sub.1-7*U.sub.16-7*C.sub.17-7*C.sub.19+7*U.sub.17+8*A.sub.2+8*A.sub.4+- 8*A.sub.5+8*C.sub.4+9*G.sub.8+10*A.sub.7+10*U.sub.18+11*A.sub.19+11*C.sub.- 9+15*G.sub.1+18*A.sub.3+19*U.sub.10-Tm-3*(GC.sub.total)-6*(GC.sub.15-19)-3- 0*X; or Formula VIII: (-8)*A1+(-1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(-4)*A9+(-5- )*A10+(-2)*A11+(-5)*A12+(17)*A13+(-3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18- +(30)*A19+(-13)*U1+(-10)*U2+(2)*U3+(-2)*U4+(-5)*U5+(5)*U6+(-2)*U7+(-10)*U8- +(-5)*U9+(15)*U10+(-1)*U11+(0)*U12+(10)*U13+(-9)*U14+(-13)*U15+(-10)*U16+(- 3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(-21)*C3+(5)*C4+(-9)*C5+(-20)*C6+(-18- )*C7+(-5)*C8+(5)*C9+(1)*C10+(2)*C11+(-5)*C12+(-3)*C13+(-6)*C14+(-2)*C15+(-- 5)*C16+(-3)*C17+(-12)*C18+(-18)*C19+(14)*G1+(8)*G2+(7)*G3+(-10)*G4+(-4)*G5- +(2)*G6+(1)*G7+(9)*G8+(5)*G9+(-1)*G10+(1)*G11+(9)*G12+(-24)*G13+(18)*G14+(- 11)*G15+(13)*G16+(-7)*G17+(-9)*G18+(-22)*G19+6*(number of A+U in position 15-19)-3*(number of G+C in whole siRNA), Formula X wherein position numbering begins at the 5'-most position of a sense strand, and

[0135] A.sub.1=1 if A is the base at position 1 of the sense strand, otherwise its value is 0; [0136] A.sub.2=1 if A is the base at position 2 of the sense strand, otherwise its value is 0; [0137] A.sub.3=1 if A is the base at position 3 of the sense strand, otherwise its value is 0; [0138] A.sub.4=1 if A is the base at position 4 of the sense strand, otherwise its value is 0; [0139] A.sub.5=1 if A is the base at position 5 of the sense strand, otherwise its value is 0; [0140] A.sub.6=1 if A is the base at position 6 of the sense strand, otherwise its value is 0; [0141] A.sub.7=1 if A is the base at position 7 of the sense strand, otherwise its value is 0; [0142] A.sub.10=1 if A is the base at position 10 of the sense strand, otherwise its value is 0; [0143] A.sub.11=1 if A is the base at position 11 of the sense strand, otherwise its value is 0; [0144] A.sub.13=1 if A is the base at position 13 of the sense strand, otherwise its value is 0; [0145] A.sub.19=1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;

[0146] C.sub.3=1 if C is the base at position 3 of the sense strand, otherwise its value is 0; [0147] C.sub.4=1 if C is the base at position 4 of the sense strand, otherwise its value is 0; [0148] C.sub.5=1 if C is the base at position 5 of the sense strand, otherwise its value is 0; [0149] C.sub.6=1 if C is the base at position 6 of the sense strand, otherwise its value is 0; [0150] C.sub.7=1 if C is the base at position 7 of the sense strand, otherwise its value is 0; [0151] C.sub.9=1 if C is the base at position 9 of the sense strand, otherwise its value is 0; [0152] C.sub.17=1 if C is the base at position 17 of the sense strand, otherwise its value is 0; [0153] C.sub.18=1 if C is the base at position 18 of the sense strand, otherwise its value is 0; [0154] C.sub.19=1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;

[0155] G.sub.1=1 if G is the base at position 1 on the sense strand, otherwise its value is 0; [0156] G.sub.2=1 if G is the base at position 2 of the sense strand, otherwise its value is 0; [0157] G.sub.8=1 if G is the base at position 8 on the sense strand, otherwise its value is 0; [0158] G.sub.10=1 if G is the base at position 10 on the sense strand, otherwise its value is 0; [0159] G.sub.13=1 if G is the base at position 13 on the sense strand, otherwise its value is 0; [0160] G.sub.19=1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;

[0161] U.sub.1=1 if U is the base at position 1 on the sense strand, otherwise its value is 0; [0162] U.sub.2=1 if U is the base at position 2 on the sense strand, otherwise its value is 0; [0163] U.sub.3=1 if U is the base at position 3 on the sense strand, otherwise its value is 0; [0164] U.sub.4=1 if U is the base at position 4 on the sense strand, otherwise its value is 0; [0165] U.sub.7=1 if U is the base at position 7 on the sense strand, otherwise its value is 0; [0166] U.sub.9=1 if U is the base at position 9 on the sense strand, otherwise its value is 0; [0167] U.sub.10=1 if U is the base at position 10 on the sense strand, otherwise its value is 0; [0168] U.sub.15=1 if U is the base at position 15 on the sense strand, otherwise its value is 0; [0169] U.sub.16=1 if U is the base at position 16 on the sense strand, otherwise its value is 0; [0170] U.sub.17=1 if U is the base at position 17 on the sense strand, otherwise its value is 0; [0171] U.sub.18=1 if U is the base at position 18 on the sense strand, otherwise its value is 0.

[0172] GC.sub.15-19=the number of G and C bases within positions 15-19 of the sense strand, or within positions 15-18 if the sense strand is only 18 base pairs in length;

[0173] GC.sub.total=the number of G and C bases in the sense strand;

[0174] Tm=100 if the siRNA oligo has the internal repeat longer then 4 base pairs, otherwise its value is 0; and

[0175] X=the number of times that the same nucleotide repeats four or more times in a row.

[0176] Any of the methods of selecting siRNA in accordance with the invention can further comprise comparing the internal stability profiles of the siRNAs to be selected, and selecting those siRNAs with the most favorable internal stability profiles. Any of the methods of selecting siRNA can further comprise selecting either for or against sequences that contain motifs that induce cellular stress. Such motifs include, for example, toxicity motifs. Any of the methods of selecting siRNA can further comprise either selecting for or selecting against sequences that comprise stability motifs.

[0177] In another embodiment, the present invention provides a method of gene silencing, comprising introducing into a cell at least one siRNA selected according to any of the methods of the present invention. The siRNA can be introduced by allowing passive uptake of siRNA, or through the use of a vector.

[0178] According to a third embodiment, the invention provides a method for developing an algorithm for selecting siRNA, said method comprising: (a) selecting a set of siRNA; (b) measuring gene silencing ability of each siRNA from said set; (c) determining relative functionality of each siRNA; (d) determining improved functionality by the presence or absence of at least one variable selected from the group consisting of the presence or absence of a particular nucleotide at a particular position, the total number of As and Us in positions 15-19, the number of times that the same nucleotide repeats within a given sequence, and the total number of Gs and Cs; and (e) developing an algorithm using the information of step (d).

[0179] In another embodiment, the invention provides a method for selecting an siRNA with improved functionality, comprising using the above-mentioned algorithm to identify an siRNA of improved functionality.

[0180] According to a fourth embodiment, the present invention provides a kit, wherein said kit is comprised of at least two siRNAs, wherein said at least two siRNAs comprise a first optimized siRNA and a second optimized siRNA, wherein said first optimized siRNA and said second optimized siRNA are optimized according a formula comprising Formula X.

[0181] According to a fifth embodiment, the present invention provides a method for identifying a hyperfunctional siRNA, comprising applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; determining the relative functionality of the at least two siRNAs and assigning each of the at least two siRNAs a functionality score; and selecting siRNAs from the at least two siRNAs that have a functionality score that reflects greater than 80 percent silencing at a concentration in the picomolar range, wherein said greater than 80 percent silencing endures for greater than 120 hours.

[0182] In other embodiments, the invention provides kits and/or methods wherein the siRNA are comprised of two separate polynucleotide strands; wherein the siRNA are comprised of a single contiguous molecule such as, for example, a unimolecular siRNA (comprising, for example, either a nucleotide or non-nucleotide loop); wherein the siRNA are expressed from one or more vectors; and wherein two or more genes are silenced by a single administration of siRNA.

[0183] According to a sixth embodiment, the present invention provides a hyperfunctional siRNA that is capable of silencing Bcl2.

[0184] According to a seventh embodiment, the present invention provides a method for developing an siRNA algorithm for selecting functional and hyperfunctional siRNAs for a given sequence. The method comprises:

[0185] (a) selecting a set of siRNAs;

[0186] (b) measuring the gene silencing ability of each siRNA from said set;

[0187] (c) determining the relative functionality of each siRNA;

[0188] (d) determining the amount of improved functionality by the presence or absence of at least one variable selected from the group consisting of the total GC content, melting temperature of the siRNA, GC content at positions 15-19, the presence or absence of a particular nucleotide at a particular position, relative thermodynamic stability at particular positions in a duplex, and the number of times that the same nucleotide repeats within a given sequence; and

[0189] (e) developing an algorithm using the information of step (d).

[0190] According to this embodiment, preferably the set of siRNAs comprises at least 90 siRNAs from at least one gene, more preferably at least 180 siRNAs from at least two different genes, and most preferably at least 270 and 360 siRNAs from at least three and four different genes, respectively. Additionally, in step (d) the determination is made with preferably at least two, more preferably at least three, even more preferably at least four, and most preferably all of the variables. The resulting algorithm is not target sequence specific.

[0191] In another embodiment, the present invention provides rationally designed siRNAs identified using the formulas above.

[0192] In yet another embodiment, the present invention is directed to hyperfunctional siRNA.

[0193] The ability to use the above algorithms, which are not sequence or species specific, allows for the cost-effective selection of optimized siRNAs for specific target sequences. Accordingly, there will be both greater efficiency and reliability in the use of siRNA technologies.

[0194] The methods disclosed herein can be used in conjunction with comparing internal stability profiles of selected siRNAs, and designing an siRNA with a desirable internal stability profile; and/or in conjunction with a selection either for or against sequences that contain motifs that induce cellular stress, for example, cellular toxicity.

[0195] Any of the methods disclosed herein can be used to silence one or more genes by introducing an siRNA selected, or designed, in accordance with any of the methods disclosed herein. The siRNA(s) can be introduced into the cell by any method known in the art, including passive uptake or through the use of one or more vectors.

[0196] Any of the methods and kits disclosed herein can employ either unimolecular siRNAs, siRNAs comprised of two separate polynucleotide strands, or combinations thereof. Any of the methods disclosed herein can be used in gene silencing, where two or more genes are silenced by a single administration of siRNA(s). The siRNA(s) can be directed against two or more target genes, and administered in a single dose or single transfection, as the case may be.

Optimizing siRNA

[0197] According to one embodiment, the present invention provides a method for improving the effectiveness of gene silencing for use to silence a particular gene through the selection of an optimal siRNA. An siRNA selected according to this method may be used individually, or in conjunction with the first embodiment, i.e., with one or more other siRNAs, each of which may or may not be selected by this criteria in order to maximize their efficiency.

[0198] The degree to which it is possible to select an siRNA for a given mRNA that maximizes these criteria will depend on the sequence of the mRNA itself. However, the selection criteria will be independent of the target sequence. According to this method, an siRNA is selected for a given gene by using a rational design. That said, rational design can be described in a variety of ways. Rational design is, in simplest terms, the application of a proven set of criteria that enhance the probability of identifying a functional or hyperfunctional siRNA. In one method, rationally designed siRNA can be identified by maximizing one or more of the following criteria:

[0199] (1) A low GC content, preferably between about 30-52%.

[0200] (2) At least 2, preferably at least 3 A or U bases at positions 15-19 of the siRNA on the sense strand. [0201] (3) An A base at position 19 of the sense strand. [0202] (4) An A base at position 3 of the sense strand. [0203] (5) A U base at position 10 of the sense strand. [0204] (6) An A base at position 14 of the sense strand. [0205] (7) A base other than C at position 19 of the sense strand. [0206] (8) A base other than G at position 13 of the sense strand.

[0207] (9) A Tm, which refers to the character of the internal repeat that results in inter- or intramolecular structures for one strand of the duplex, that is preferably not stable at greater than 50.degree. C., more preferably not stable at greater than 37.degree. C., even more preferably not stable at greater than 30.degree. C. and most preferably not stable at greater than 20.degree. C. [0208] (10) A base other than U at position 5 of the sense strand. [0209] (11) A base other than A at position 11 of the sense strand. [0210] (12) A base other than an A at position 1 of the sense strand. [0211] (13) A base other than an A at position 2 of the sense strand. [0212] (14) An A base at position 4 of the sense strand. [0213] (15) An A base at position 5 of the sense strand. [0214] (16) An A base at position 6 of the sense strand. [0215] (17) An A base at position 7 of the sense strand. [0216] (18) An A base at position 8 of the sense strand. [0217] (19) A base other than an A at position 9 of the sense strand. [0218] (20) A base other than an A at position 10 of the sense strand. [0219] (21) A base other than an A at position 11 of the sense strand. [0220] (22) A base other than an A at position 12 of the sense strand. [0221] (23) An A base at position 13 of the sense strand. [0222] (24) A base other than an A at position 14 of the sense strand. [0223] (25) An A base at position 15 of the sense strand [0224] (26) An A base at position 16 of the sense strand. [0225] (27) An A base at position 17 of the sense strand. [0226] (28) An A base at position 18 of the sense strand. [0227] (29) A base other than a U at position 1 of the sense strand. [0228] (30) A base other than a U at position 2 of the sense strand. [0229] (31) A U base at position 3 of the sense strand. [0230] (32) A base other than a U at position 4 of the sense strand. [0231] (33) A base other than a U at position 5 of the sense strand. [0232] (34) A U base at position 6 of the sense strand. [0233] (35) A base other than a U at position 7 of the sense strand. [0234] (36) A base other than a U at position 8 of the sense strand. [0235] (37) A base other than a U at position 9 of the sense strand. [0236] (38) A base other than a U at position 11 of the sense strand. [0237] (39) A U base at position 13 of the sense strand. [0238] (40) A base other than a U at position 14 of the sense strand. [0239] (41) A base other than a U at position 15 of the sense strand. [0240] (42) A base other than a U at position 16 of the sense strand. [0241] (43) A U base at position 17 of the sense strand. [0242] (44) A U base at position 18 of the sense strand. [0243] (45) A U base at position 19 of the sense strand. [0244] (46) A C base at position 1 of the sense strand. [0245] (47) A C base at position 2 of the sense strand. [0246] (48) A base other than a C at position 3 of the sense strand. [0247] (49) A C base at position 4 of the sense strand. [0248] (50) A base other than a C at position 5 of the sense strand. [0249] (51) A base other than a C at position 6 of the sense strand. [0250] (52) A base other than a C at position 7 of the sense strand. [0251] (53) A base other than a C at position 8 of the sense strand. [0252] (54) A C base at position 9 of the sense strand. [0253] (55) A C base at position 10 of the sense strand. [0254] (56) A C base at position 11 of the sense strand. [0255] (57) A base other than a C at position 12 of the sense strand. [0256] (58) A base other than a C at position 13 of the sense strand. [0257] (59) A base other than a C at position 14 of the sense strand. [0258] (60) A base other than a C at position 15 of the sense strand. [0259] (61) A base other than a C at position 16 of the sense strand. [0260] (62) A base other than a C at position 17 of the sense strand. [0261] (63) A base other than a C at position 18 of the sense strand. [0262] (64) A G base at position 1 of the sense strand. [0263] (65) A G base at position 2 of the sense strand. [0264] (66) A G base at position 3 of the sense strand. [0265] (67) A base other than a G at position 4 of the sense strand. [0266] (68) A base other than a G at position 5 of the sense strand. [0267] (69) A G base at position 6 of the sense strand. [0268] (70) A G base at position 7 of the sense strand. [0269] (71) A G base at position 8 of the sense strand. [0270] (72) A G base at position 9 of the sense strand. [0271] (73) A base other than a G at position 10 of the sense strand. [0272] (74) A G base at position 11 of the sense strand. [0273] (75) A G base at position 12 of the sense strand. [0274] (76) A G base at position 14 of the sense strand. [0275] (77) A G base at position 15 of the sense strand. [0276] (78) A G base at position 16 of the sense strand. [0277] (79) A base other than a G at position 17 of the sense strand. [0278] (80) A base other than a G at position 18 of the sense strand. [0279] (81) A base other than a G at position 19 of the sense strand.

[0280] The importance of various criteria can vary greatly. For instance, a C base at position 10 of the sense strand makes a minor contribution to duplex functionality. In contrast, the absence of a C at position 3 of the sense strand is very important. Accordingly, preferably an siRNA will satisfy as many of the aforementioned criteria as possible.

[0281] With respect to the criteria, GC content, as well as a high number of AU in positions 15-19 of the sense strand, may be important for easement of the unwinding of double stranded siRNA duplex. Duplex unwinding has been shown to be crucial for siRNA functionality in vivo.

[0282] With respect to criterion 9, the internal structure is measured in terms of the melting temperature of the single strand of siRNA, which is the temperature at which 50% of the molecules will become denatured. With respect to criteria 2-8 and 10-11, the positions refer to sequence positions on the sense strand, which is the strand that is identical to the mRNA.

[0283] In one preferred embodiment, at least criteria 1 and 8 are satisfied. In another preferred embodiment, at least criteria 7 and 8 are satisfied. In still another preferred embodiment, at least criteria 1, 8 and 9 are satisfied.

[0284] It should be noted that all of the aforementioned criteria regarding sequence position specifics are with respect to the 5' end of the sense strand. Reference is made to the sense strand, because most databases contain information that describes the information of the mRNA. Because according to the present invention a chain can be from 18 to 30 bases in length, and the aforementioned criteria assumes a chain 19 base pairs in length, it is important to keep the aforementioned criteria applicable to the correct bases.

[0285] When there are only 18 bases, the base pair that is not present is the base pair that is located at the 3' of the sense strand. When there are twenty to thirty bases present, then additional bases are added at the 5' end of the sense chain and occupy positions .sup.-1 to .sup.-11. Accordingly, with respect to SEQ. ID NO. 0001 NNANANNNNUCNAANNNNA and SEQ. ID NO. 0028 GUCNNANANNNNUCNAANNNNA, both would have A at position 3, A at position 5, U at position 10, C at position 11, A and position 13, A and position 14 and A at position 19. However, SEQ. ID NO. 0028 would also have C at position -1, U at position -2 and G at position -3.

[0286] For a 19 base pair siRNA, an optimal sequence of one of the strands may be represented below, where N is any base, A, C, G, or U:

Sequence CWU 1

1

498 1 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 1 nnanannnnu cnaannnna 19 2 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 2 nnanannnnu gnaannnna 19 3 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 3 nnanannnnu unaannnna 19 4 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 4 nnanannnnu cncannnna 19 5 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 5 nnanannnnu gncannnna 19 6 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 6 nnanannnnu uncannnna 19 7 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 7 nnanannnnu cnuannnna 19 8 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 8 nnanannnnu gnuannnna 19 9 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 9 nnanannnnu unuannnna 19 10 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 10 nnancnnnnu cnaannnna 19 11 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 11 nnancnnnnu gnaannnna 19 12 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 12 nnancnnnnu unaannnna 19 13 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 13 nnancnnnnu cncannnna 19 14 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 14 nnancnnnnu gncannnna 19 15 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 15 nnancnnnnu uncannnna 19 16 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 16 nnancnnnnu cnuannnna 19 17 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 17 nnancnnnnu gnuannnna 19 18 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 18 nnancnnnnu unuannnna 19 19 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 19 nnangnnnnu cnaannnna 19 20 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 20 nnangnnnnu gnaannnna 19 21 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 21 nnangnnnnu unaannnna 19 22 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 22 nnangnnnnu cncannnna 19 23 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 23 nnangnnnnu gncannnna 19 24 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 24 nnangnnnnu uncannnna 19 25 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 25 nnangnnnnu cnuannnna 19 26 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 26 nnangnnnnu gnuannnna 19 27 19 RNA Artificial Sequence Synthetic misc_feature 1, 2, 4, 6-9, 12, 15-18 n is any nucleotide 27 nnangnnnnu unuannnna 19 28 22 RNA Artificial Sequence Synthetic misc_feature 4, 5, 7, 9-12, 15, 18-21 n is any nucleotide 28 gucnnanann nnucnaannn na 22 29 208 DNA Homo Sapiens misc_feature (1)...(108) Human cyclophilin fragment 29 gttccaaaaa cagtggataa ttttgtggcc ttagctacag gagagaaagg atttggctac 60 aaaaacagca aattccatcg tgtaatcaag gacttcatga tccagggcgg agacttcacc 120 aggggagatg gcacaggagg aaagagcatc tacggtgagc gcttccccga tgagaacttc 180 aaactgaagc actacgggcc tggctggg 208 30 200 DNA Photinus pyralis misc_feature (1)...(200) Firefly luciferase fragment 30 tgaacttccc gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat 60 cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt 120 tgtggacgaa gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat 180 cctcataaag gccaagaagg 200 31 108 DNA Homo sapiens misc_feature (1)...(108) Human DBL fragment 31 acgggcaagg ccaagtggga tgcctggaat gagctgaaag ggacttccaa ggaagatgcc 60 atgaaagctt acatcaacaa agtagaagag ctaaagaaaa aatacggg 108 32 19 RNA Artificial Sequence Synthetic 32 guuccaaaaa caguggaua 19 33 19 RNA Artificial Sequence Synthetic 33 uccaaaaaca guggauaau 19 34 19 RNA Artificial Sequence Synthetic 34 caaaaacagu ggauaauuu 19 35 19 RNA Artificial Sequence Synthetic 35 aaaacagugg auaauuuug 19 36 19 RNA Artificial Sequence Synthetic 36 aacaguggau aauuuugug 19 37 19 RNA Artificial Sequence Synthetic 37 caguggauaa uuuuguggc 19 38 19 RNA Artificial Sequence Synthetic 38 guggauaauu uuguggccu 19 39 19 RNA Artificial Sequence Synthetic 39 ggauaauuuu guggccuua 19 40 19 RNA Artificial Sequence Synthetic 40 auaauuuugu ggccuuagc 19 41 19 RNA Artificial Sequence Synthetic 41 aauuuugugg ccuuagcua 19 42 19 RNA Artificial Sequence Synthetic 42 uuuuguggcc uuagcuaca 19 43 19 RNA Artificial Sequence Synthetic 43 uuguggccuu agcuacagg 19 44 19 RNA Artificial Sequence Synthetic 44 guggccuuag cuacaggag 19 45 19 RNA Artificial Sequence Synthetic 45 ggccuuagcu acaggagag 19 46 19 RNA Artificial Sequence Synthetic 46 ccuuagcuac aggagagaa 19 47 19 RNA Artificial Sequence Synthetic 47 uuagcuacag gagagaaag 19 48 19 RNA Artificial Sequence Synthetic 48 agcuacagga gagaaagga 19 49 19 RNA Artificial Sequence Synthetic 49 cuacaggaga gaaaggauu 19 50 19 RNA Artificial Sequence Synthetic 50 acaggagaga aaggauuug 19 51 19 RNA Artificial Sequence Synthetic 51 aggagagaaa ggauuuggc 19 52 19 RNA Artificial Sequence Synthetic 52 gagagaaagg auuuggcua 19 53 19 RNA Artificial Sequence Synthetic 53 gagaaaggau uuggcuaca 19 54 19 RNA Artificial Sequence Synthetic 54 gaaaggauuu ggcuacaaa 19 55 19 RNA Artificial Sequence Synthetic 55 aaggauuugg cuacaaaaa 19 56 19 RNA Artificial Sequence Synthetic 56 ggauuuggcu acaaaaaca 19 57 19 RNA Artificial Sequence Synthetic 57 auuuggcuac aaaaacagc 19 58 19 RNA Artificial Sequence Synthetic 58 uuggcuacaa aaacagcaa 19 59 19 RNA Artificial Sequence Synthetic 59 ggcuacaaaa acagcaaau 19 60 19 RNA Artificial Sequence Synthetic 60 cuacaaaaac agcaaauuc 19 61 19 RNA Artificial Sequence Synthetic 61 acaaaaacag caaauucca 19 62 19 RNA Artificial Sequence Synthetic 62 aaaaacagca aauuccauc 19 63 19 RNA Artificial Sequence Synthetic 63 aaacagcaaa uuccaucgu 19 64 19 RNA Artificial Sequence Synthetic 64 acagcaaauu ccaucgugu 19 65 19 RNA Artificial Sequence Synthetic 65 agcaaauucc aucguguaa 19 66 19 RNA Artificial Sequence Synthetic 66 caaauuccau cguguaauc 19 67 19 RNA Artificial Sequence Synthetic 67 aauuccaucg uguaaucaa 19 68 19 RNA Artificial Sequence Synthetic 68 uuccaucgug uaaucaagg 19 69 19 RNA Artificial Sequence Synthetic 69 ccaucgugua aucaaggac 19 70 19 RNA Artificial Sequence Synthetic 70 aucguguaau caaggacuu 19 71 19 RNA Artificial Sequence Synthetic 71 cguguaauca aggacuuca 19 72 19 RNA Artificial Sequence Synthetic 72 uguaaucaag gacuucaug 19 73 19 RNA Artificial Sequence Synthetic 73 uaaucaagga cuucaugau 19 74 19 RNA Artificial Sequence Synthetic 74 aucaaggacu ucaugaucc 19 75 19 RNA Artificial Sequence Synthetic 75 caaggacuuc augauccag 19 76 19 RNA Artificial Sequence Synthetic 76 aggacuucau gauccaggg 19 77 19 RNA Artificial Sequence Synthetic 77 gacuucauga uccagggcg 19 78 19 RNA Artificial Sequence Synthetic 78 cuucaugauc cagggcgga 19 79 19 RNA Artificial Sequence Synthetic 79 ucaugaucca gggcggaga 19 80 19 RNA Artificial Sequence Synthetic 80 augauccagg gcggagacu 19 81 19 RNA Artificial Sequence Synthetic 81 gauccagggc ggagacuuc 19 82 19 RNA Artificial Sequence Synthetic 82 uccagggcgg agacuucac 19 83 19 RNA Artificial Sequence Synthetic 83 cagggcggag acuucacca 19 84 19 RNA Artificial Sequence Synthetic 84 gggcggagac uucaccagg 19 85 19 RNA Artificial Sequence Synthetic 85 gcggagacuu caccagggg 19 86 19 RNA Artificial Sequence Synthetic 86 ggagacuuca ccaggggag 19 87 19 RNA Artificial Sequence Synthetic 87 agacuucacc aggggagau 19 88 19 RNA Artificial Sequence Synthetic 88 acuucaccag gggagaugg 19 89 19 RNA Artificial Sequence Synthetic 89 uucaccaggg gagauggca 19 90 19 RNA Artificial Sequence Synthetic 90 caccagggga gauggcaca 19 91 19 RNA Artificial Sequence Synthetic 91 ccaggggaga uggcacagg 19 92 19 RNA Artificial Sequence Synthetic 92 aggggagaug gcacaggag 19 93 19 RNA Artificial Sequence Synthetic 93 gggagauggc acaggagga 19 94 19 RNA Artificial Sequence Synthetic 94 gagauggcac aggaggaaa 19 95 19 RNA Artificial Sequence Synthetic 95 gauggcacag gaggaaaga 19 96 19 RNA Artificial Sequence Synthetic 96 uggcacagga ggaaagagc 19 97 19 RNA Artificial Sequence Synthetic 97 gcacaggagg aaagagcau 19 98 19 RNA Artificial Sequence Synthetic 98 acaggaggaa agagcaucu 19 99 19 RNA Artificial Sequence Synthetic 99 aggaggaaag agcaucuac 19 100 19 RNA Artificial Sequence Synthetic 100 gaggaaagag caucuacgg 19 101 19 RNA Artificial Sequence Synthetic 101 ggaaagagca ucuacggug 19 102 19 RNA Artificial Sequence Synthetic 102 aaagagcauc uacggugag 19 103 19 RNA Artificial Sequence Synthetic 103 agagcaucua cggugagcg 19 104 19 RNA Artificial Sequence Synthetic 104 agcaucuacg gugagcgcu 19 105 19 RNA Artificial Sequence Synthetic 105 caucuacggu gagcgcuuc 19 106 19 RNA Artificial Sequence Synthetic 106 ucuacgguga gcgcuuccc 19 107 19 RNA Artificial Sequence Synthetic 107 uacggugagc gcuuccccg 19 108 19 RNA Artificial Sequence Synthetic 108 cggugagcgc uuccccgau 19 109 19 RNA Artificial Sequence Synthetic 109 gugagcgcuu ccccgauga 19 110 19 RNA Artificial Sequence Synthetic 110 gagcgcuucc ccgaugaga 19 111 19 RNA Artificial Sequence Synthetic 111 gcgcuucccc gaugagaac 19 112 19 RNA Artificial Sequence Synthetic 112 gcuuccccga ugagaacuu 19 113 19 RNA Artificial Sequence Synthetic 113 uuccccgaug agaacuuca 19 114 19 RNA Artificial Sequence Synthetic 114 ccccgaugag aacuucaaa 19 115 19 RNA Artificial Sequence Synthetic 115 ccgaugagaa cuucaaacu 19 116 19 RNA Artificial Sequence Synthetic 116 gaugagaacu ucaaacuga 19 117 19 RNA Artificial Sequence Synthetic 117 ugagaacuuc aaacugaag 19 118 19 RNA Artificial Sequence Synthetic 118 agaacuucaa acugaagca 19 119 19 RNA Artificial Sequence Synthetic 119 aacuucaaac ugaagcacu 19 120 19 RNA Artificial Sequence Synthetic 120 cuucaaacug aagcacuac 19 121 19 RNA Artificial Sequence Synthetic 121 ucaaacugaa gcacuacgg 19 122 19 RNA Artificial Sequence Synthetic 122 acgggcaagg ccaaguggg 19 123 19 RNA Artificial Sequence Synthetic 123 cgggcaaggc caaguggga 19 124 19 RNA Artificial Sequence Synthetic 124 gggcaaggcc aagugggau 19 125 19 RNA Artificial Sequence Synthetic 125 ggcaaggcca agugggaug 19 126 19 RNA Artificial Sequence Synthetic 126 gcaaggccaa gugggaugc 19 127 19 RNA Artificial Sequence Synthetic 127 caaggccaag ugggaugcc 19 128 19 RNA Artificial Sequence Synthetic 128 aaggccaagu gggaugccu 19 129 19 RNA Artificial Sequence Synthetic 129 aggccaagug ggaugccug 19 130 19 RNA Artificial Sequence Synthetic 130 ggccaagugg gaugccugg 19 131 19 RNA Artificial Sequence Synthetic 131 gccaaguggg augccugga 19 132 19 RNA Artificial Sequence Synthetic 132 ccaaguggga ugccuggaa 19 133 19 RNA Artificial Sequence Synthetic 133 caagugggau gccuggaau 19 134 19 RNA Artificial Sequence Synthetic 134 aagugggaug ccuggaaug 19 135 19 RNA Artificial Sequence Synthetic 135 agugggaugc cuggaauga

19 136 19 RNA Artificial Sequence Synthetic 136 gugggaugcc uggaaugag 19 137 19 RNA Artificial Sequence Synthetic 137 ugggaugccu ggaaugagc 19 138 19 RNA Artificial Sequence Synthetic 138 gggaugccug gaaugagcu 19 139 19 RNA Artificial Sequence Synthetic 139 ggaugccugg aaugagcug 19 140 19 RNA Artificial Sequence Synthetic 140 gaugccugga augagcuga 19 141 19 RNA Artificial Sequence Synthetic 141 augccuggaa ugagcugaa 19 142 19 RNA Artificial Sequence Synthetic 142 ugccuggaau gagcugaaa 19 143 19 RNA Artificial Sequence Synthetic 143 gccuggaaug agcugaaag 19 144 19 RNA Artificial Sequence Synthetic 144 ccuggaauga gcugaaagg 19 145 19 RNA Artificial Sequence Synthetic 145 cuggaaugag cugaaaggg 19 146 19 RNA Artificial Sequence Synthetic 146 uggaaugagc ugaaaggga 19 147 19 RNA Artificial Sequence Synthetic 147 ggaaugagcu gaaagggac 19 148 19 RNA Artificial Sequence Synthetic 148 gaaugagcug aaagggacu 19 149 19 RNA Artificial Sequence Synthetic 149 aaugagcuga aagggacuu 19 150 19 RNA Artificial Sequence Synthetic 150 augagcugaa agggacuuc 19 151 19 RNA Artificial Sequence Synthetic 151 ugagcugaaa gggacuucc 19 152 19 RNA Artificial Sequence Synthetic 152 gagcugaaag ggacuucca 19 153 19 RNA Artificial Sequence Synthetic 153 agcugaaagg gacuuccaa 19 154 19 RNA Artificial Sequence Synthetic 154 gcugaaaggg acuuccaag 19 155 19 RNA Artificial Sequence Synthetic 155 cugaaaggga cuuccaagg 19 156 19 RNA Artificial Sequence Synthetic 156 ugaaagggac uuccaagga 19 157 19 RNA Artificial Sequence Synthetic 157 gaaagggacu uccaaggaa 19 158 19 RNA Artificial Sequence Synthetic 158 aaagggacuu ccaaggaag 19 159 19 RNA Artificial Sequence Synthetic 159 aagggacuuc caaggaaga 19 160 19 RNA Artificial Sequence Synthetic 160 agggacuucc aaggaagau 19 161 19 RNA Artificial Sequence Synthetic 161 gggacuucca aggaagaug 19 162 19 RNA Artificial Sequence Synthetic 162 ggacuuccaa ggaagaugc 19 163 19 RNA Artificial Sequence Synthetic 163 gacuuccaag gaagaugcc 19 164 19 RNA Artificial Sequence Synthetic 164 acuuccaagg aagaugcca 19 165 19 RNA Artificial Sequence Synthetic 165 cuuccaagga agaugccau 19 166 19 RNA Artificial Sequence Synthetic 166 uuccaaggaa gaugccaug 19 167 19 RNA Artificial Sequence Synthetic 167 uccaaggaag augccauga 19 168 19 RNA Artificial Sequence Synthetic 168 ccaaggaaga ugccaugaa 19 169 19 RNA Artificial Sequence Synthetic 169 caaggaagau gccaugaaa 19 170 19 RNA Artificial Sequence Synthetic 170 aaggaagaug ccaugaaag 19 171 19 RNA Artificial Sequence Synthetic 171 aggaagaugc caugaaagc 19 172 19 RNA Artificial Sequence Synthetic 172 ggaagaugcc augaaagcu 19 173 19 RNA Artificial Sequence Synthetic 173 gaagaugcca ugaaagcuu 19 174 19 RNA Artificial Sequence Synthetic 174 aagaugccau gaaagcuua 19 175 19 RNA Artificial Sequence Synthetic 175 agaugccaug aaagcuuac 19 176 19 RNA Artificial Sequence Synthetic 176 gaugccauga aagcuuaca 19 177 19 RNA Artificial Sequence Synthetic 177 augccaugaa agcuuacau 19 178 19 RNA Artificial Sequence Synthetic 178 ugccaugaaa gcuuacauc 19 179 19 RNA Artificial Sequence Synthetic 179 gccaugaaag cuuacauca 19 180 19 RNA Artificial Sequence Synthetic 180 ccaugaaagc uuacaucaa 19 181 19 RNA Artificial Sequence Synthetic 181 caugaaagcu uacaucaac 19 182 19 RNA Artificial Sequence Synthetic 182 augaaagcuu acaucaaca 19 183 19 RNA Artificial Sequence Synthetic 183 ugaaagcuua caucaacaa 19 184 19 RNA Artificial Sequence Synthetic 184 gaaagcuuac aucaacaaa 19 185 19 RNA Artificial Sequence Synthetic 185 aaagcuuaca ucaacaaag 19 186 19 RNA Artificial Sequence Synthetic 186 aagcuuacau caacaaagu 19 187 19 RNA Artificial Sequence Synthetic 187 agcuuacauc aacaaagua 19 188 19 RNA Artificial Sequence Synthetic 188 gcuuacauca acaaaguag 19 189 19 RNA Artificial Sequence Synthetic 189 cuuacaucaa caaaguaga 19 190 19 RNA Artificial Sequence Synthetic 190 uuacaucaac aaaguagaa 19 191 19 RNA Artificial Sequence Synthetic 191 uacaucaaca aaguagaag 19 192 19 RNA Artificial Sequence Synthetic 192 acaucaacaa aguagaaga 19 193 19 RNA Artificial Sequence Synthetic 193 caucaacaaa guagaagag 19 194 19 RNA Artificial Sequence Synthetic 194 aucaacaaag uagaagagc 19 195 19 RNA Artificial Sequence Synthetic 195 ucaacaaagu agaagagcu 19 196 19 RNA Artificial Sequence Synthetic 196 caacaaagua gaagagcua 19 197 19 RNA Artificial Sequence Synthetic 197 aacaaaguag aagagcuaa 19 198 19 RNA Artificial Sequence Synthetic 198 acaaaguaga agagcuaaa 19 199 19 RNA Artificial Sequence Synthetic 199 caaaguagaa gagcuaaag 19 200 19 RNA Artificial Sequence Synthetic 200 aaaguagaag agcuaaaga 19 201 19 RNA Artificial Sequence Synthetic 201 aaguagaaga gcuaaagaa 19 202 19 RNA Artificial Sequence Synthetic 202 aguagaagag cuaaagaaa 19 203 19 RNA Artificial Sequence Synthetic 203 guagaagagc uaaagaaaa 19 204 19 RNA Artificial Sequence Synthetic 204 uagaagagcu aaagaaaaa 19 205 19 RNA Artificial Sequence Synthetic 205 agaagagcua aagaaaaaa 19 206 19 RNA Artificial Sequence Synthetic 206 gaagagcuaa agaaaaaau 19 207 19 RNA Artificial Sequence Synthetic 207 aagagcuaaa gaaaaaaua 19 208 19 RNA Artificial Sequence Synthetic 208 agagcuaaag aaaaaauac 19 209 19 RNA Artificial Sequence Synthetic 209 gagcuaaaga aaaaauacg 19 210 19 RNA Artificial Sequence Synthetic 210 agcuaaagaa aaaauacgg 19 211 19 RNA Artificial Sequence Synthetic 211 gcuaaagaaa aaauacggg 19 212 19 RNA Artificial Sequence Synthetic 212 auccucauaa aggccaaga 19 213 19 RNA Artificial Sequence Synthetic 213 agauccucau aaaggccaa 19 214 19 RNA Artificial Sequence Synthetic 214 agagauccuc auaaaggcc 19 215 19 RNA Artificial Sequence Synthetic 215 agagagaucc ucauaaagg 19 216 19 RNA Artificial Sequence Synthetic 216 ucagagagau ccucauaaa 19 217 19 RNA Artificial Sequence Synthetic 217 aaucagagag auccucaua 19 218 19 RNA Artificial Sequence Synthetic 218 aaaaucagag agauccuca 19 219 19 RNA Artificial Sequence Synthetic 219 gaaaaaucag agagauccu 19 220 19 RNA Artificial Sequence Synthetic 220 aagaaaaauc agagagauc 19 221 19 RNA Artificial Sequence Synthetic 221 gcaagaaaaa ucagagaga 19 222 19 RNA Artificial Sequence Synthetic 222 acgcaagaaa aaucagaga 19 223 19 RNA Artificial Sequence Synthetic 223 cgacgcaaga aaaaucaga 19 224 19 RNA Artificial Sequence Synthetic 224 cucgacgcaa gaaaaauca 19 225 19 RNA Artificial Sequence Synthetic 225 aacucgacgc aagaaaaau 19 226 19 RNA Artificial Sequence Synthetic 226 aaaacucgac gcaagaaaa 19 227 19 RNA Artificial Sequence Synthetic 227 ggaaaacucg acgcaagaa 19 228 19 RNA Artificial Sequence Synthetic 228 ccggaaaacu cgacgcaag 19 229 19 RNA Artificial Sequence Synthetic 229 uaccggaaaa cucgacgca 19 230 19 RNA Artificial Sequence Synthetic 230 cuuaccggaa aacucgacg 19 231 19 RNA Artificial Sequence Synthetic 231 gucuuaccgg aaaacucga 19 232 19 RNA Artificial Sequence Synthetic 232 aggucuuacc ggaaaacuc 19 233 19 RNA Artificial Sequence Synthetic 233 aaaggucuua ccggaaaac 19 234 19 RNA Artificial Sequence Synthetic 234 cgaaaggucu uaccggaaa 19 235 19 RNA Artificial Sequence Synthetic 235 accgaaaggu cuuaccgga 19 236 19 RNA Artificial Sequence Synthetic 236 guaccgaaag gucuuaccg 19 237 19 RNA Artificial Sequence Synthetic 237 aaguaccgaa aggucuuac 19 238 19 RNA Artificial Sequence Synthetic 238 cgaaguaccg aaaggucuu 19 239 19 RNA Artificial Sequence Synthetic 239 gacgaaguac cgaaagguc 19 240 19 RNA Artificial Sequence Synthetic 240 uggacgaagu accgaaagg 19 241 19 RNA Artificial Sequence Synthetic 241 uguggacgaa guaccgaaa 19 242 19 RNA Artificial Sequence Synthetic 242 uuuguggacg aaguaccga 19 243 19 RNA Artificial Sequence Synthetic 243 uguuugugga cgaaguacc 19 244 19 RNA Artificial Sequence Synthetic 244 uguguuugug gacgaagua 19 245 19 RNA Artificial Sequence Synthetic 245 guuguguuug uggacgaag 19 246 19 RNA Artificial Sequence Synthetic 246 gaguuguguu uguggacga 19 247 19 RNA Artificial Sequence Synthetic 247 aggaguugug uuuguggac 19 248 19 RNA Artificial Sequence Synthetic 248 ggaggaguug uguuugugg 19 249 19 RNA Artificial Sequence Synthetic 249 gcggaggagu uguguuugu 19 250 19 RNA Artificial Sequence Synthetic 250 gcgcggagga guuguguuu 19 251 19 RNA Artificial Sequence Synthetic 251 uugcgcggag gaguugugu 19 252 19 RNA Artificial Sequence Synthetic 252 aguugcgcgg aggaguugu 19 253 19 RNA Artificial Sequence Synthetic 253 aaaguugcgc ggaggaguu 19 254 19 RNA Artificial Sequence Synthetic 254 aaaaaguugc gcggaggag 19 255 19 RNA Artificial Sequence Synthetic 255 cgaaaaaguu gcgcggagg 19 256 19 RNA Artificial Sequence Synthetic 256 cgcgaaaaag uugcgcgga 19 257 19 RNA Artificial Sequence Synthetic 257 accgcgaaaa aguugcgcg 19 258 19 RNA Artificial Sequence Synthetic 258 caaccgcgaa aaaguugcg 19 259 19 RNA Artificial Sequence Synthetic 259 aacaaccgcg aaaaaguug 19 260 19 RNA Artificial Sequence Synthetic 260 guaacaaccg cgaaaaagu 19 261 19 RNA Artificial Sequence Synthetic 261 aaguaacaac cgcgaaaaa 19 262 19 RNA Artificial Sequence Synthetic 262 ucaaguaaca accgcgaaa 19 263 19 RNA Artificial Sequence Synthetic 263 agucaaguaa caaccgcga 19 264 19 RNA Artificial Sequence Synthetic 264 ccagucaagu aacaaccgc 19 265 19 RNA Artificial Sequence Synthetic 265 cgccagucaa guaacaacc 19 266 19 RNA Artificial Sequence Synthetic 266 gucgccaguc aaguaacaa 19 267 19 RNA Artificial Sequence Synthetic 267 acgucgccag ucaaguaac 19 268 19 RNA Artificial Sequence Synthetic 268 uuacgucgcc agucaagua 19 269 19 RNA Artificial Sequence Synthetic 269 gauuacgucg ccagucaag 19 270 19 RNA Artificial Sequence Synthetic 270 uggauuacgu cgccaguca 19 271 19 RNA Artificial Sequence Synthetic 271 cguggauuac gucgccagu 19 272 19 RNA Artificial Sequence Synthetic 272 aucguggauu acgucgcca 19 273 19 RNA Artificial Sequence Synthetic 273 agaucgugga uuacgucgc 19 274 19 RNA Artificial Sequence Synthetic 274 agagaucgug gauuacguc 19 275 19 RNA Artificial Sequence Synthetic 275 aaagagaucg uggauuacg 19 276 19 RNA Artificial Sequence Synthetic 276 aaaaagagau cguggauua 19 277 19 RNA Artificial Sequence Synthetic 277 ggaaaaagag aucguggau 19 278 19 RNA Artificial Sequence Synthetic 278 acggaaaaag agaucgugg 19 279 19 RNA Artificial Sequence Synthetic 279 ugacggaaaa agagaucgu 19 280 19 RNA Artificial Sequence Synthetic 280 gaugacggaa aaagagauc 19 281 19 RNA Artificial Sequence Synthetic 281 acgaugacgg aaaaagaga 19 282 19 RNA Artificial Sequence Synthetic 282 agacgaugac ggaaaaaga 19 283 19 RNA Artificial Sequence Synthetic 283 aaagacgaug acggaaaaa 19 284 19 RNA Artificial Sequence Synthetic 284 ggaaagacga ugacggaaa 19 285 19 RNA Artificial Sequence Synthetic 285 acggaaagac gaugacgga 19 286 19 RNA Artificial Sequence Synthetic 286 gcacggaaag

acgaugacg 19 287 19 RNA Artificial Sequence Synthetic 287 gagcacggaa agacgauga 19 288 19 RNA Artificial Sequence Synthetic 288 uggagcacgg aaagacgau 19 289 19 RNA Artificial Sequence Synthetic 289 uuuggagcac ggaaagacg 19 290 19 RNA Artificial Sequence Synthetic 290 guuuuggagc acggaaaga 19 291 19 RNA Artificial Sequence Synthetic 291 uuguuuugga gcacggaaa 19 292 19 RNA Artificial Sequence Synthetic 292 uguuguuuug gagcacgga 19 293 19 RNA Artificial Sequence Synthetic 293 guuguuguuu uggagcacg 19 294 19 RNA Artificial Sequence Synthetic 294 ccguuguugu uuuggagca 19 295 19 RNA Artificial Sequence Synthetic 295 cgccguuguu guuuuggag 19 296 19 RNA Artificial Sequence Synthetic 296 gccgccguug uuguuuugg 19 297 19 RNA Artificial Sequence Synthetic 297 ccgccgccgu uguuguuuu 19 298 19 RNA Artificial Sequence Synthetic 298 ucccgccgcc guuguuguu 19 299 19 RNA Artificial Sequence Synthetic 299 cuucccgccg ccguuguug 19 300 19 RNA Artificial Sequence Synthetic 300 aacuucccgc cgccguugu 19 301 19 RNA Artificial Sequence Synthetic 301 ugaacuuccc gccgccguu 19 302 19 RNA Artificial Sequence Synthetic 302 gggagauagu gaugaagua 19 303 19 RNA Artificial Sequence Synthetic 303 gaaguacauc cauuauaag 19 304 19 RNA Artificial Sequence Synthetic 304 guacgacaac cgggagaua 19 305 19 RNA Artificial Sequence Synthetic 305 agauagugau gaaguacau 19 306 19 RNA Artificial Sequence Synthetic 306 ugaagacucu gcucaguuu 19 307 19 RNA Artificial Sequence Synthetic 307 gcaugcggcc ucuguuuga 19 308 19 RNA Artificial Sequence Synthetic 308 ugcggccucu guuugauuu 19 309 19 RNA Artificial Sequence Synthetic 309 gagauaguga ugaaguaca 19 310 19 RNA Artificial Sequence Synthetic 310 ggagauagug augaaguac 19 311 19 RNA Artificial Sequence Synthetic 311 gaagacucug cucaguuug 19 312 19 DNA Artificial Sequence Synthetic 312 gaaagaatct gtagagaaa 19 313 19 DNA Artificial Sequence Synthetic 313 gcaatgagct gtttgaaga 19 314 19 DNA Artificial Sequence Synthetic 314 tgacaaaggt ggataaatt 19 315 19 DNA Artificial Sequence Synthetic 315 ggaaatggat ctctttgaa 19 316 19 DNA Artificial Sequence Synthetic 316 ggaaagtaat ggtccaaca 19 317 19 DNA Artificial Sequence Synthetic 317 agacagttat gcagctatt 19 318 19 DNA Artificial Sequence Synthetic 318 ccaattctcg gaagcaaga 19 319 19 DNA Artificial Sequence Synthetic 319 gaaagtaatg gtccaacag 19 320 19 DNA Artificial Sequence Synthetic 320 gcgccagagt gaacaagta 19 321 19 DNA Artificial Sequence Synthetic 321 gaaggtggcc cagctatgt 19 322 19 DNA Artificial Sequence Synthetic 322 ggaaccagcg ccagagtga 19 323 19 DNA Artificial Sequence Synthetic 323 gagcgagatt gcaggcata 19 324 19 DNA Artificial Sequence Synthetic 324 gttagtatct gatgacttg 19 325 19 DNA Artificial Sequence Synthetic 325 gaaatggaac cactaagaa 19 326 19 DNA Artificial Sequence Synthetic 326 ggaaatggaa ccactaaga 19 327 19 DNA Artificial Sequence Synthetic 327 caactacact ttccaatgc 19 328 19 DNA Artificial Sequence Synthetic 328 ccaccaagat ttcatgata 19 329 19 DNA Artificial Sequence Synthetic 329 gatcggaact ccaacaaga 19 330 19 DNA Artificial Sequence Synthetic 330 aaacggagct acagattat 19 331 19 DNA Artificial Sequence Synthetic 331 ccacacagca ttcttgtaa 19 332 19 DNA Artificial Sequence Synthetic 332 gaagttacct tgagcaatc 19 333 19 DNA Artificial Sequence Synthetic 333 ggacttggcc gatccagaa 19 334 19 DNA Artificial Sequence Synthetic 334 gcacttggat cgagatgag 19 335 19 DNA Artificial Sequence Synthetic 335 caaagaccaa ttcgcgtta 19 336 19 DNA Artificial Sequence Synthetic 336 ccgaatcaat cgcatcttc 19 337 19 DNA Artificial Sequence Synthetic 337 gacatgatcc tgcagttca 19 338 19 DNA Artificial Sequence Synthetic 338 gagcgaatcg tcaccactt 19 339 19 DNA Artificial Sequence Synthetic 339 cctccgagct ggcgtctac 19 340 19 DNA Artificial Sequence Synthetic 340 tcacatggtt aacctctaa 19 341 19 DNA Artificial Sequence Synthetic 341 gatgagggac gccataatc 19 342 19 DNA Artificial Sequence Synthetic 342 cctctaacta caaatctta 19 343 19 DNA Artificial Sequence Synthetic 343 ggaaggtgct atccaaaat 19 344 19 DNA Artificial Sequence Synthetic 344 gcaagcaagt cctaacatt 19 345 19 DNA Artificial Sequence Synthetic 345 ggaagaggag tagacctta 19 346 19 DNA Artificial Sequence Synthetic 346 aggaatcagt gttgtagta 19 347 19 DNA Artificial Sequence Synthetic 347 gaagaggagt agaccttac 19 348 19 DNA Artificial Sequence Synthetic 348 gaaagtcaag cctggtatt 19 349 19 DNA Artificial Sequence Synthetic 349 aaagtcaagc ctggtatta 19 350 19 DNA Artificial Sequence Synthetic 350 gctatgaacg tgaatgatc 19 351 19 DNA Artificial Sequence Synthetic 351 caagcctggt attacgttt 19 352 19 DNA Artificial Sequence Synthetic 352 ggaacaagat ctgtcaatt 19 353 19 DNA Artificial Sequence Synthetic 353 gcaatgaacg tgaacgaaa 19 354 19 DNA Artificial Sequence Synthetic 354 caatgaacgt gaacgaaat 19 355 19 DNA Artificial Sequence Synthetic 355 ggacaggagc ggtatcaca 19 356 19 DNA Artificial Sequence Synthetic 356 agacagagct tgagaataa 19 357 19 DNA Artificial Sequence Synthetic 357 gagaagatct ttatgcaaa 19 358 19 DNA Artificial Sequence Synthetic 358 gaagagaaat cagcagata 19 359 19 DNA Artificial Sequence Synthetic 359 gcaagtaact caactaaca 19 360 19 DNA Artificial Sequence Synthetic 360 gagctaatct gccacattg 19 361 19 DNA Artificial Sequence Synthetic 361 gcagatgagt tactagaaa 19 362 19 DNA Artificial Sequence Synthetic 362 caacttaatt gtccagaaa 19 363 19 DNA Artificial Sequence Synthetic 363 caacacagga ttctgataa 19 364 19 DNA Artificial Sequence Synthetic 364 agattgtgcc taagtctct 19 365 19 DNA Artificial Sequence Synthetic 365 atgaagatct ggaggtgaa 19 366 19 DNA Artificial Sequence Synthetic 366 tttgagactt cttgcctaa 19 367 19 DNA Artificial Sequence Synthetic 367 agatcaccct ccttaaata 19 368 19 DNA Artificial Sequence Synthetic 368 caacggattt ggtcgtatt 19 369 19 DNA Artificial Sequence Synthetic 369 gaaatcccat caccatctt 19 370 19 DNA Artificial Sequence Synthetic 370 gacctcaact acatggttt 19 371 19 DNA Artificial Sequence Synthetic 371 tggtttacat gttccaata 19 372 19 DNA Artificial Sequence Synthetic 372 gaagaaatcg atgttgttt 19 373 19 DNA Artificial Sequence Synthetic 373 acacaaactt gaacagcta 19 374 19 DNA Artificial Sequence Synthetic 374 ggaagaaatc gatgttgtt 19 375 19 DNA Artificial Sequence Synthetic 375 gaaacgacga gaacagttg 19 376 19 DNA Artificial Sequence Synthetic 376 gcacatggat ggaggttct 19 377 19 DNA Artificial Sequence Synthetic 377 gcagagagag cagatttga 19 378 19 DNA Artificial Sequence Synthetic 378 gaggttctct ggatcaagt 19 379 19 DNA Artificial Sequence Synthetic 379 gagcagattt gaagcaact 19 380 19 DNA Artificial Sequence Synthetic 380 caaagacgat gacttcgaa 19 381 19 DNA Artificial Sequence Synthetic 381 gatcagcatt tgcatggaa 19 382 19 DNA Artificial Sequence Synthetic 382 tccaggagtt tgtcaataa 19 383 19 DNA Artificial Sequence Synthetic 383 ggaagctgat ccaccttga 19 384 19 DNA Artificial Sequence Synthetic 384 gcagaaatct aaggatata 19 385 19 DNA Artificial Sequence Synthetic 385 caacaaggat gaagtctat 19 386 19 DNA Artificial Sequence Synthetic 386 cagcagaaat ctaaggata 19 387 19 DNA Artificial Sequence Synthetic 387 ctagatggct ttctcagta 19 388 19 DNA Artificial Sequence Synthetic 388 agacaaggtc ccaaagaca 19 389 19 DNA Artificial Sequence Synthetic 389 ggaatggcaa gaccagcaa 19 390 19 DNA Artificial Sequence Synthetic 390 agaattattc cagggttta 19 391 19 DNA Artificial Sequence Synthetic 391 gcagacaagg tcccaaaga 19 392 19 DNA Artificial Sequence Synthetic 392 agaagcagct tcaggatga 19 393 19 DNA Artificial Sequence Synthetic 393 gagcttgact tccagaaga 19 394 19 DNA Artificial Sequence Synthetic 394 ccaccgaagt tcaccctaa 19 395 19 DNA Artificial Sequence Synthetic 395 gagaagagct cctccatca 19 396 19 DNA Artificial Sequence Synthetic 396 gaaagagcat ctacggtga 19 397 19 DNA Artificial Sequence Synthetic 397 gaaaggattt ggctacaaa 19 398 19 DNA Artificial Sequence Synthetic 398 acagcaaatt ccatcgtgt 19 399 19 DNA Artificial Sequence Synthetic 399 ggaaagactg ttccaaaaa 19 400 19 DNA Artificial Sequence Synthetic 400 caacacgcct catcctcta 19 401 19 DNA Artificial Sequence Synthetic 401 catgaaagct tacatcaac 19 402 19 DNA Artificial Sequence Synthetic 402 aagatgccat gaaagctta 19 403 19 DNA Artificial Sequence Synthetic 403 gcacataccg cctgagtct 19 404 19 DNA Artificial Sequence Synthetic 404 gatcaaatct gaagaagga 19 405 19 DNA Artificial Sequence Synthetic 405 gccaagaagt ttcctaata 19 406 19 DNA Artificial Sequence Synthetic 406 cagcatatct tgaaccatt 19 407 19 DNA Artificial Sequence Synthetic 407 gaacaaagga aacggatga 19 408 19 DNA Artificial Sequence Synthetic 408 cggaaacggt ccaggctat 19 409 19 DNA Artificial Sequence Synthetic 409 gcttcgagca gacatgata 19 410 19 DNA Artificial Sequence Synthetic 410 cctacacggt cctcctata 19 411 19 DNA Artificial Sequence Synthetic 411 gccaagaacc tcatcatct 19 412 19 DNA Artificial Sequence Synthetic 412 gatatgggct gaatacaaa 19 413 19 DNA Artificial Sequence Synthetic 413 gcactctgat tgacaaata 19 414 19 DNA Artificial Sequence Synthetic 414 tgaagtctct gattaagta 19 415 19 DNA Artificial Sequence Synthetic 415 tcagagagat cctcataaa 19 416 19 DNA Artificial Sequence Synthetic 416 gcaagaagat caccatttc 19 417 19 DNA Artificial Sequence Synthetic 417 gagagaaatt tgaggatga 19 418 19 DNA Artificial Sequence Synthetic 418 gaaaggattt ggctataag 19 419 19 DNA Artificial Sequence Synthetic 419 gaaagaaggc atgaacatt 19 420 19 DNA Artificial Sequence Synthetic 420 gggagatagt gatgaagta 19 421 19 DNA Artificial Sequence Synthetic 421 gaagtacatc cattataag 19 422 19 DNA Artificial Sequence Synthetic 422 gtacgacaac cgggagata 19 423 19 DNA Artificial Sequence Synthetic 423 agatagtgat gaagtacat 19 424 19 DNA Artificial Sequence Synthetic 424 tgaagactct gctcagttt 19 425 19 DNA Artificial Sequence Synthetic 425 gcatgcggcc tctgtttga 19 426 19 RNA Artificial Sequence Synthetic 426 gcacacagcu uacuacauc 19 427 19 RNA Artificial Sequence Synthetic 427 gaaaugcccu gguaucuca 19 428 19 RNA Artificial Sequence Synthetic 428 gaaggaacgu gaugugauc 19 429 19 RNA Artificial Sequence Synthetic 429 gcacuacucc uguguguga 19 430 19 RNA Artificial Sequence Synthetic 430 gaacccagcu ggagaacuu 19 431 19 RNA Artificial Sequence Synthetic 431 gauauacagu gugaucuua 19 432 19 RNA Artificial Sequence Synthetic 432 guacuacgau ccugauuau 19 433 19 RNA Artificial Sequence Synthetic 433 gugccgaccu uuacaauuu 19 434 19 DNA Artificial Sequence Synthetic 434 gaaggaaact gaattcaaa 19 435 19 DNA Artificial Sequence Synthetic 435 ggaaatatgt actacgaaa 19 436 19 DNA Artificial Sequence Synthetic 436 ccacaaagca gtgaattta 19 437 19 DNA

Artificial Sequence Synthetic 437 gtaacaagct cacgcagtt 19 438 19 RNA Artificial Sequence Synthetic 438 aaagaugacu gagaagacu 19 439 19 RNA Artificial Sequence Synthetic 439 accuaaagcu acuagaaag 19 440 19 RNA Artificial Sequence Synthetic 440 acucugaucu auguugaua 19 441 19 RNA Artificial Sequence Synthetic 441 agaagacugu uaaagcaaa 19 442 19 RNA Artificial Sequence Synthetic 442 agaugaaugc ggcuguuaa 19 443 19 RNA Artificial Sequence Synthetic 443 auaaagcauu cuucaacag 19 444 19 RNA Artificial Sequence Synthetic 444 caacagagcu acagaaaag 19 445 19 RNA Artificial Sequence Synthetic 445 cagaguuugu guguauuug 19 446 19 RNA Artificial Sequence Synthetic 446 cagaugaaug cggcuguua 19 447 19 RNA Artificial Sequence Synthetic 447 ccaccagccu uaccuaaag 19 448 19 RNA Artificial Sequence Synthetic 448 ccaccuguuu gcugugaca 19 449 19 RNA Artificial Sequence Synthetic 449 ccaugggaau ccaaucugu 19 450 19 RNA Artificial Sequence Synthetic 450 ccuguuugcu gugacauag 19 451 19 RNA Artificial Sequence Synthetic 451 cuacucugau cuauguuga 19 452 19 RNA Artificial Sequence Synthetic 452 cugcaauaau ccagaaugg 19 453 19 RNA Artificial Sequence Synthetic 453 cuguuaagac cugcaauaa 19 454 19 RNA Artificial Sequence Synthetic 454 cuucaauccu cuagacuuu 19 455 19 RNA Artificial Sequence Synthetic 455 cuucagaguu uguguguau 19 456 19 RNA Artificial Sequence Synthetic 456 gaagacuguu aaagcaaaa 19 457 19 RNA Artificial Sequence Synthetic 457 gaauggcuac ucugaucua 19 458 19 RNA Artificial Sequence Synthetic 458 gaccugcaau aauccagaa 19 459 19 RNA Artificial Sequence Synthetic 459 gagaagacug uuaaagcaa 19 460 19 RNA Artificial Sequence Synthetic 460 gaggagagag agcuugaaa 19 461 19 RNA Artificial Sequence Synthetic 461 gaguggagug ccucucaug 19 462 19 RNA Artificial Sequence Synthetic 462 gaguuugugu guauuugua 19 463 19 RNA Artificial Sequence Synthetic 463 gaucuauguu gauaaggaa 19 464 19 RNA Artificial Sequence Synthetic 464 gaugaaugcg gcuguuaag 19 465 19 RNA Artificial Sequence Synthetic 465 gaugaugccu auccagaaa 19 466 19 RNA Artificial Sequence Synthetic 466 gaugggagau cucaaguuu 19 467 19 RNA Artificial Sequence Synthetic 467 gcacccgugu gguugcuaa 19 468 19 RNA Artificial Sequence Synthetic 468 gcagucuccu ucaagcauu 19 469 19 RNA Artificial Sequence Synthetic 469 gccugaagag caccagauu 19 470 19 RNA Artificial Sequence Synthetic 470 gcuacucuga ucuauguug 19 471 19 RNA Artificial Sequence Synthetic 471 gcugugacau agauauuua 19 472 19 RNA Artificial Sequence Synthetic 472 gcuguuaaga ccugcaaua 19 473 19 RNA Artificial Sequence Synthetic 473 ggagagagag cuugaaaag 19 474 19 RNA Artificial Sequence Synthetic 474 ggagaucuca aguuucaac 19 475 19 RNA Artificial Sequence Synthetic 475 gucuggaccu ucaaucaaa 19 476 19 RNA Artificial Sequence Synthetic 476 guuaagaccu gcaauaauc 19 477 19 RNA Artificial Sequence Synthetic 477 guuccugccu cagaugaug 19 478 19 RNA Artificial Sequence Synthetic 478 guugaauugc caccuguuu 19 479 19 RNA Artificial Sequence Synthetic 479 uaaagcauuc uucaacaga 19 480 19 RNA Artificial Sequence Synthetic 480 uaaggaaaau ggagaacca 19 481 19 RNA Artificial Sequence Synthetic 481 uaccuaaagc uacuagaaa 19 482 19 RNA Artificial Sequence Synthetic 482 ucaaucaaag ccuuagaug 19 483 19 RNA Artificial Sequence Synthetic 483 ucagaguuug uguguauuu 19 484 19 RNA Artificial Sequence Synthetic 484 ucagaugaau gcggcuguu 19 485 19 RNA Artificial Sequence Synthetic 485 ucugaucuau guugauaag 19 486 19 RNA Artificial Sequence Synthetic 486 ucuuagugcu ucagaguuu 19 487 19 RNA Artificial Sequence Synthetic 487 ugaaugcggc uguuaagac 19 488 19 RNA Artificial Sequence Synthetic 488 ugacgaggag agagagcuu 19 489 19 RNA Artificial Sequence Synthetic 489 ugacugagaa gacuguuaa 19 490 19 RNA Artificial Sequence Synthetic 490 ugagaagacu guuaaagca 19 491 19 RNA Artificial Sequence Synthetic 491 ugauaaggaa aauggagaa 19 492 19 RNA Artificial Sequence Synthetic 492 ugaucuaugu ugauaagga 19 493 19 RNA Artificial Sequence Synthetic 493 ugaugccuau ccagaaaua 19 494 19 RNA Artificial Sequence Synthetic 494 ugccucagau gaugccuau 19 495 19 RNA Artificial Sequence Synthetic 495 uggagugccu cucaugauc 19 496 19 RNA Artificial Sequence Synthetic 496 uggcuacucu gaucuaugu 19 497 19 RNA Artificial Sequence Synthetic 497 ugggagaucu caaguuuca 19 498 19 RNA Artificial Sequence Synthetic 498 uuaccuaaag cuacuagaa 19

Claims

1. An siRNA comprising a sense region and an antisense region, wherein said sense region and said antisense region together form a duplex region, said antisense region and said sense region are each 18-30 nucleotides in length and said antisense region comprises a sequence that is at least 90% complementary to a sequence selected from the group consisting of SEQ. ID NOs. 438-498.

2. An siRNA comprising a sense region and an antisense region, wherein said sense region and said antisense region together form a duplex region and said sense region and said antisense region are each 18-30 nucleotides in length, and said antisense region comprises a sequence that is 100% complementary to a contiguous stretch of at least 18 bases of a sequence selected from the group consisting of SEQ. ID NOs. 438-498.

3. The siRNA of claim 2, wherein each of said antisense region and said sense region are 19-30 nucleotides in length, and said antisense region comprises a sequence that is 100% complementary to said sequence selected from the group consisting of: SEQ. ID NOs. 438-498.

4. A pool of at least two siRNAs, wherein said pool comprises a first siRNA and a second siRNA, said first siRNA comprises a first antisense region and a first sense region that together form a first duplex region and each of said first antisense region and said first sense region are 18-30 nucleotides in length and said first antisense region is at least 90% complementary to 18 bases of a first sequence selected from the group consisting of: SEQ. ID NOs. 438-498 and said second siRNA comprises a second antisense region and a second sense region that together form a second duplex region and each of said second antisense region and said second sense region are 18-30 nucleotides in length and said second antisense region is at least 90% complementary to 18 bases of a second sequence selected from the group consisting of: SEQ. ID NOs. 438-498, wherein said first antisense region and said second antisense region are not identical.

5. The pool of claim 4, wherein said first antisense region comprises a sequence that is 100% complementary to at least 18 bases of said first sequence, and said second antisense region comprises a sequence that is 100% complementary to at least 18 bases of said second sequence.

6. The pool of claim 4, wherein said first siRNA is 19-30 nucleotides in length and said first antisense region comprises a sequence that is at least 90% complementary to said first sequence, and second siRNA is 19-30 nucleotides in length and said second antisense region comprises a sequence that is at least 90% complementary to said second sequence.

7. The pool of claim 4, wherein said first antisense region is 19-30 nucleotides in length and said first antisense region comprises a sequence that is 100% complementary to at least 18 bases of said first sequence, and said second antisense region is 19-30 nucleotides in length and said second antisense region comprises a sequence that is 100% complementary to said second sequence.

8. The siRNA of claim 1, wherein said antisense region and said sense region are each 19-25 nucleotides in length.

9. The siRNA of claim 4, wherein said first antisense region, said first sense region, said second sense region and said second antisense region are each 19-25 nucleotides in length.