Methods and compositions for selecting sirna of improved functionality

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.

What is claimed is:  
     
         1 . 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 selection criteria that are embodied in a formula comprising: selection criteria are embodied in a formula comprising:  
         (−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(number of A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula X 
 
       wherein position numbering begins at the 5′-most position of a sense strand, and 
 A 1 =1 if A is the base at position 1 of the sense strand, otherwise its value is 0;  
 A 2 =1 if A is the base at position 2 of the sense strand, otherwise its value is 0;  
 A 3 =1 if A is the base at position 3 of the sense strand, otherwise its value is 0;  
 A 4 =1 if A is the base at position 4 of the sense strand, otherwise its value is 0;  
 A 5 =1 if A is the base at position 5 of the sense strand, otherwise its value is 0;  
 A 6 =1 if A is the base at position 6 of the sense strand, otherwise its value is 0;  
 A 7 =1 if A is the base at position 7 of the sense strand, otherwise its value is 0;  
 A 10 =1 if A is the base at position 10 of the sense strand, otherwise its value is 0;  
 A 11 =1 if A is the base at position 11 of the sense strand, otherwise its value is 0;  
 A 13 =1 if A is the base at position 13 of the sense strand, otherwise its value is 0;  
 A 19 =1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;  
 C 3 =1 if C is the base at position 3 of the sense strand, otherwise its value is 0;  
 C 4 =1 if C is the base at position 4 of the sense strand, otherwise its value is 0;  
 C 5 =1 if C is the base at position 5 of the sense strand, otherwise its value is 0;  
 C 6 =1 if C is the base at position 6 of the sense strand, otherwise its value is 0;  
 C 7 =1 if C is the base at position 7 of the sense strand, otherwise its value is 0;  
 C 9 =1 if C is the base at position 9 of the sense strand, otherwise its value is 0;  
 C 17 =1 if C is the base at position 17 of the sense strand, otherwise its value is 0;  
 C 18 =1 if C is the base at position 18 of the sense strand, otherwise its value is 0;  
 C 19 =1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;  
 G 1 =1 if G is the base at position 1 on the sense strand, otherwise its value is 0;  
 G 2 =1 if G is the base at position 2 of the sense strand, otherwise its value is 0;  
 G 8 =1 if G is the base at position 8 on the sense strand, otherwise its value is 0;  
 G 10 =1 if G is the base at position 10 on the sense strand, otherwise its value is 0;  
 G 13 =1 if G is the base at position 13 on the sense strand, otherwise its value is 0;  
 G 19 =1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;  
 U 1 =1 if U is the base at position 1 on the sense strand, otherwise its value is 0;  
 U 2 =1 if U is the base at position 2 on the sense strand, otherwise its value is 0;  
 U 3 =1 if U is the base at position 3 on the sense strand, otherwise its value is 0;  
 U 4 =1 if U is the base at position 4 on the sense strand, otherwise its value is 0;  
 U 7 =1 if U is the base at position 7 on the sense strand, otherwise its value is 0;  
 U 9 =1 if U is the base at position 9 on the sense strand, otherwise its value is 0;  
 U 10 =1 if U is the base at position 10 on the sense strand, otherwise its value is 0;  
 U 15 =1 if U is the base at position 15 on the sense strand, otherwise its value is 0;  
 U 16 =1 if U is the base at position 16 on the sense strand, otherwise its value is 0;  
 U 17 =1 if U is the base at position 17 on the sense strand, otherwise its value is 0;  
 U 18 =1 if U is the base at position 18 on the sense strand, otherwise its value is 0.  
 
     
     
         2 . A method for selecting an siRNA, said method comprising: 
 applying selection criteria to a set of potential siRNA that comprise 18-30 base pairs; and    determining the relative functionality of the at least two siRNAs, wherein said section criteria are non-target specific criteria, 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 said selection criteria are embodied in a formula comprising:      (−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(number of A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula X 
   wherein position numbering begins at the 5′-most position of a sense strand, and    A 1 =1 if A is the base at position 1 of the sense strand, otherwise its value is 0;    A 2 =1 if A is the base at position 2 of the sense strand, otherwise its value is 0;    A 3 =1 if A is the base at position 3 of the sense strand, otherwise its value is 0;    A 4 =1 if A is the base at position 4 of the sense strand, otherwise its value is 0;    A 5 =1 if A is the base at position 5 of the sense strand, otherwise its value is 0;    A 6 =1 if A is the base at position 6 of the sense strand, otherwise its value is 0;    A 7 =1 if A is the base at position 7 of the sense strand, otherwise its value is 0;    A 10 =1 if A is the base at position 10 of the sense strand, otherwise its value is 0;    A 11 =1 if A is the base at position 11 of the sense strand, otherwise its value is 0;    A 13 =1 if A is the base at position 13 of the sense strand, otherwise its value is 0;    A 19 =1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;    C 3 =1 if C is the base at position 3 of the sense strand, otherwise its value is 0;    C 4 =1 if C is the base at position 4 of the sense strand, otherwise its value is 0;    C 5 =1 if C is the base at position 5 of the sense strand, otherwise its value is 0;    C 6 =1 if C is the base at position 6 of the sense strand, otherwise its value is 0;    C 7 =1 if C is the base at position 7 of the sense strand, otherwise its value is 0;    C 9 =1 if C is the base at position 9 of the sense strand, otherwise its value is 0;    C 17 =1 if C is the base at position 17 of the sense strand, otherwise its value is 0;    C 18 =1 if C is the base at position 18 of the sense strand, otherwise its value is 0;    C 19 =1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;    G 1 =1 if G is the base at position 1 on the sense strand, otherwise its value is 0;    G 2 =1 if G is the base at position 2 of the sense strand, otherwise its value is 0;    G 8 =1 if G is the base at position 8 on the sense strand, otherwise its value is 0;    G 10 =1 if G is the base at position 10 on the sense strand, otherwise its value is 0;    G 13 =1 if G is the base at position 13 on the sense strand, otherwise its value is 0;    G 19 =1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;    U 1 =1 if U is the base at position 1 on the sense strand, otherwise its value is 0;    U 2 =1 if U is the base at position 2 on the sense strand, otherwise its value is 0;    U 3 =1 if U is the base at position 3 on the sense strand, otherwise its value is 0;    U 4 =1 if U is the base at position 4 on the sense strand, otherwise its value is 0;    U 7 =1 if U is the base at position 7 on the sense strand, otherwise its value is 0;    U 9 =1 if U is the base at position 9 on the sense strand, otherwise its value is 0;    U 10 =1 if U is the base at position 10 on the sense strand, otherwise its value is 0;    U 15 =1 if U is the base at position 15 on the sense strand, otherwise its value is 0;    U 16 =1 if U is the base at position 16 on the sense strand, otherwise its value is 0;    U 17 =1 if U is the base at position 17 on the sense strand, otherwise its value is 0;    U 18 =1 if U is the base at position 18 on the sense strand, otherwise its value is 0.    
     
     
         3 . A method according to  claim 1 , further comprising comparing the internal stability profiles of said at least two siRNAs.  
     
     
         4 . A method according to  claim 2 , further comprising comparing the internal stability profiles of said at least two siRNAs.  
     
     
         5 . A method according to  claim 1 , further comprising selecting either for or against sequences that contain motifs that induce cellular stress.  
     
     
         6 . A method according to  claim 2 , further comprising selecting either for or against sequences that contain motifs that induce cellular stress.  
     
     
         7 . A method according to  claim 1 , further comprising selecting either for or against sequences that comprise stability motifs.  
     
     
         8 . A method according to  claim 2 , further comprising selecting either for or against sequences that comprise stability motifs.  
     
     
         9 . A method of gene silencing, comprising introducing into a cell at least one siRNA selected according to a method of  claim 1 .  
     
     
         10 . A method of gene silencing, comprising introducing into a cell at least one siRNA selected according to a method of  claim 2 .  
     
     
         11 . A method according to  claim 1 , wherein said introducing is by allowing passive uptake of the at least one siRNA.  
     
     
         12 . A method according to  claim 2 , wherein said introducing is by allowing passive uptake of the at least one siRNA.  
     
     
         13 . A method according  claim 9 , wherein said introducing in through the use of a vector.  
     
     
         14 . A method for developing an siRNA 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 based on the following variables: 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).    
     
     
         15 . A method of selecting an siRNA with improved functionality, said method comprising using the algorithm of  claim 14 .  
     
     
         16 . 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:  
       (−8)*A1+(−1)*A2+(12)*A3+(7)*A4+(18)*A5+(12)*A6+(19)*A7+(6)*A8+(−4)*A9+(−5)*A10+(−2)*A11+(−5)*A12+(17)*A13+(−3)*A14+(4)*A15+(2)*A16+(8)*A17+(11)*A18+(30)*A19+(−13)*U1+(−10)*U2+(2)*U3+(−2)*U4+(−5)*U5+(5)*U6+(−2)*U7+(−10)*U8+(−5)*U9+(15)*U10+(−1)*U11+(0)*U12+(10)*U13+(−9)*U14+(−13)*U15+(−10)*U16+(3)*U17+(9)*U18+(9)*U19+(7)*C1+(3)*C2+(−21)*C3+(5)*C4+(−9)*C5+(−20)*C6+(−18)*C7+(−5)*C8+(5)*C9+(1)*C10+(2)*C11+(−5)*C12+(−3)*C13+(−6)*C14+(−2)*C15+(−5)*C16+(−3)*C17+(−12)*C18+(−18)*C19+(14)*G1+(8)*G2+(7)*G3+(−10)*G4+(−4)*G5+(2)*G6+(1)*G7+(9)*G8+(5)*G9+(−11)*G10+(1)*G11+(9)*G12+(−24)*G13+(18)*G14+(11)*G15+(13)*G16+(−7)*G17+(−9)*G18+(−22)*G19+6*(number of A+U in position 15-19)−3*(number of G+C in whole siRNA),   Formula X  
       wherein position numbering begins at the 5′-most position of a sense strand, and 
 A 1 =1 if A is the base at position 1 of the sense strand, otherwise its value is 0;  
 A 2 =1 if A is the base at position 2 of the sense strand, otherwise its value is 0;  
 A 3 =1 if A is the base at position 3 of the sense strand, otherwise its value is 0;  
 A 4 =1 if A is the base at position 4 of the sense strand, otherwise its value is 0;  
 A 5 =1 if A is the base at position 5 of the sense strand, otherwise its value is 0;  
 A 6 =1 if A is the base at position 6 of the sense strand, otherwise its value is 0;  
 A 7 =1 if A is the base at position 7 of the sense strand, otherwise its value is 0;  
 A 10 =1 if A is the base at position 10 of the sense strand, otherwise its value is 0;  
 A 11 =1 if A is the base at position 11 of the sense strand, otherwise its value is 0;  
 A 13 =1 if A is the base at position 13 of the sense strand, otherwise its value is 0;  
 A 19 =1 if A is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;  
 C 3 =1 if C is the base at position 3 of the sense strand, otherwise its value is 0;  
 C 4 =1 if C is the base at position 4 of the sense strand, otherwise its value is 0;  
 C 5 =1 if C is the base at position 5 of the sense strand, otherwise its value is 0;  
 C 6 =1 if C is the base at position 6 of the sense strand, otherwise its value is 0;  
 C 7 =1 if C is the base at position 7 of the sense strand, otherwise its value is 0;  
 C 9 =1 if C is the base at position 9 of the sense strand, otherwise its value is 0;  
 C 17 =1 if C is the base at position 17 of the sense strand, otherwise its value is 0;  
 C 18 =1 if C is the base at position 18 of the sense strand, otherwise its value is 0;  
 C 19 =1 if C is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;  
 G 1 =1 if G is the base at position 1 on the sense strand, otherwise its value is 0;  
 G 2 =1 if G is the base at position 2 of the sense strand, otherwise its value is 0;  
 G 8 =1 if G is the base at position 8 on the sense strand, otherwise its value is 0;  
 G 10 =1 if G is the base at position 10 on the sense strand, otherwise its value is 0;  
 G 13 =1 if G is the base at position 13 on the sense strand, otherwise its value is 0;  
 G 19 =1 if G is the base at position 19 of the sense strand, otherwise if another base is present or the sense strand is only 18 base pairs in length, its value is 0;  
 U 1 =1 if U is the base at position 1 on the sense strand, otherwise its value is 0;  
 U 2 =1 if U is the base at position 2 on the sense strand, otherwise its value is 0;  
 U 3 =1 if U is the base at position 3 on the sense strand, otherwise its value is 0;  
 U 4 =1 if U is the base at position 4 on the sense strand, otherwise its value is 0;  
 U 7 =1 if U is the base at position 7 on the sense strand, otherwise its value is 0;  
 U 9 =1 if U is the base at position 9 on the sense strand, otherwise its value is 0;  
 U 10 =1 if U is the base at position 10 on the sense strand, otherwise its value is 0;  
 U 15 =1 if U is the base at position 15 on the sense strand, otherwise its value is 0;  
 U 16 =1 if U is the base at position 16 on the sense strand, otherwise its value is 0;  
 U 17 =1 if U is the base at position 17 on the sense strand, otherwise its value is 0;  
 U 18 =1 if U is the base at position 18 on the sense strand, otherwise its value is 0.  
 
     
     
         17 . A method for identifying 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; and    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.    
     
     
         18 . A method according to  claim 1 , wherein said siRNA are unimolecular.  
     
     
         19 . A method according to  claim 2 , wherein said siRNA are unimolecular.  
     
     
         20 . A method according to  claim 14 , wherein said siRNA are unimolecular.  
     
     
         21 . A method according to  claim 16 , wherein said siRNA are unimolecular.  
     
     
         22 . A method according to  claim 17 , wherein said siRNA are unimolecular.  
     
     
         23 . A method according to  claim 1 , wherein said siRNA are comprised of two separate polynucleotide strands.  
     
     
         24 . A method according to  claim 2 , wherein said siRNA are comprised of two separate polynucleotide strands.  
     
     
         25 . A method according to  claim 14 , wherein said siRNA are comprised of two separate polynucleotide strands.  
     
     
         26 . A method according to  claim 16 , wherein said siRNA are comprised of two separate polynucleotide strands.  
     
     
         27 . A method according to  claim 17 , wherein said siRNA are comprised of two separate polynucleotide strands.  
     
     
         28 . A method according to  claim 1 , wherein said siRNA are expressed from one or more vectors.  
     
     
         29 . A method according to  claim 2 , wherein said siRNA are expressed from one or more vectors.  
     
     
         30 . A method according to  claim 14 , wherein said siRNA are expressed from one or more vectors.  
     
     
         31 . A method according to  claim 16 , wherein said siRNA are expressed from one or more vectors.  
     
     
         32 . A method according to  claim 17 , wherein said siRNA are expressed from one or more vectors.  
     
     
         33 . A method according to  claim 1 , wherein two or more genes are silenced by a single administration of siRNA.  
     
     
         34 . A method according to  claim 2 , wherein two or more genes are silenced by a single administration of siRNA.  
     
     
         35 . A method according to  claim 14 , wherein two or more genes are silenced by a single administration of siRNA.  
     
     
         36 . A method according to  claim 16 , wherein two or more genes are silenced by a single administration of siRNA.  
     
     
         37 . A method according to  claim 17 , wherein two or more genes are silenced by a single administration of siRNA.  
     
     
         38 . A kit according to  claim 13 , wherein one or more of said siRNA are unimolecular.  
     
     
         39 . A kit according to  claim 13 , wherein one or more of said siRNA are comprised of two separate polynucleotide strands.  
     
     
         40 . A kit according to  claim 13 , wherein one or more of said siRNA are capable of silencing the Bcl2 gene.  
     
     
         41 . A method for developing an siRNA algorithm for selecting functional and hyperfunctional siRNAs for a given sequence, comprising: 
 (a) selecting a set of siRNAs;    (b) measuring the gene silencing ability of each siRNA from said set;    (c) determining the relative functionality of each siRNA;    (d) determining the amount of improved functionality based on the following variables: 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    (e) developing an algorithm using the information of step (d).