Functional and hyperfunctional siRNA

• US 20070031844A1
• Filed: 11/14/2003
• Published: 02/08/2007
• Est. Priority Date: 11/14/2002
• Status: Active Grant

• Current Assignee: Thermo Fisher Scientific Incorporated
• Original Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Inventors: Marshall, William, Khvorova, Anastasia, Reynolds, Angela, Scaringe, Stephen, Leake, Devin
• Granted Patent: US 8,090,542 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
• CPC Class Codes: C12Y 502/01008 Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin
• CPC Class Codes: G16B 20/00 : ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 : Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/50 : Mutagenesis

Abstract

Efficient sequence specific gene silencing is possible through the use of siRNA technology. By selecting particular siRNAs by rationale design, one can maximize the generation of an effective gene silencing reagent, as well as methods for silencing genes.

Claims

1. A method for selecting siRNA comprising selecting an siRNA molecule of 19-25 nucleoside bases, said method comprising:

(a) selecting a target gene;

(b) measuring the functionality of sequences of 19-25 nucleotides in length that are substantially complementary to a stretch of nucleotides of the target sequence, wherein said functionality is dependent upon non-target specific criteria.

2. The method according to claim 1 wherein said functionality is determined by applying one of the following formulas:

Formula I=−

(GC/3)+(AU_15-19)−

(Tm<sub>20°

C.</sub>)*3−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃)+(U₁₀)+(A₁₄)−

(U₅)−

(A₁₁);


Formula II=−

(GC/3)−

(AU_15-19)*3−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃);


Formula III=−

(GC/3)+(AU_15-19)−

(Tm<sub>20°

C.</sub>)*3;


Formula IV=−

(GC/2)+(AU_15-19)/2−

(Tm<sub>20°

C.</sub>)*2−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃)+(U₁₀)+(A₁₄)−

(U₅)−

(A₁₁);


Formula V=−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃)+(U₁₀)+(A₁₄)−

(U₅)−

(A₁₁);


Formula VI=−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃);


Formula VII=−

(GC/2)+(AU_15-19)/2−

(Tm<sub>20°

C.</sub>)*1−

(G₁₃)*3−

(C₁₉)+(A₁₉)*3+(A₃)*3+(U₁₀)/2+(A₁₄)/2−

(U₅)/2−

(A₁₁)/2;

wherein in Formulas I-VII;

AU_15-19=0-5 depending on the number of A or U bases on the sense strand at positions 15-19;

G₁₃=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;

C₁₉=1 if C is the base at position 19 of the sense strand, otherwise its value is 0;

GC=the number of C and C bases in the entire sense strand;

Tm<sub>20°

C.</sub>=1 if the Tm is greater than 20°

C.;

A₃=1 if A is the base at position 3 on the sense strand, otherwise its value is 0;

A₁₁=1 if A is the base at position 11 on the sense strand, otherwise its value is 0;

A₁₄=1 if A is the base at position 14 on the sense strand, otherwise its value is 0;

A₁₉=1 if A is the base at position 19 on the sense strand, otherwise its value is 0;

U₅=1 if U is the base at position 5 on the sense strand, otherwise its value is 0;

U₁₀=1 if U is the base at position 10 on the sense strand, otherwise its value is 0;

or,

(−

14)*G₁₃−

13*A₁−

12*U₇−

11*U₂−

10*A₁₁−

10*U₄−

10*C₃−

10*C₅−

10*C₆−


9*A₁₀−

9*U₉−

9*C₁₈−

8*G₁₀−

7*U₁−

7*U₁₆−

7*C₁₇−

7*C₁₉+7*U₁₇+8*A₂+8*A₄
+8*A₅+8*C₄+9*G₈+10*A₇+10*U₁₈+11*A₁₉+11*C₉+15*G₁+
18*A₃+19*U₁₀−

Tm−

3*(GC_total)−

6*(GC_15-19)−

30*X; and

 

 

Formula VIII;


(14.1)*A₃+(14.9)*A₆+(17.6)*A₁₃+(24.7)*A₁₉+(14.2)*U₁₀+(10.5)*
C₉+(23.9)*G₁+(16.3)*G₂+(−

12.3)*A₁₁+(−

19.3)*U₁+(−

12.1)*U₂+
(−

11)*U₃+(−

15.2)*U₁₅+(−

11.3)*U₁₆+(−

11.8)*C₃+(−

17.4)*C₆+(−

10.5)*C₇+
(−

13.7)*G₁₃+(−

25.9)*G₁₉−

Tm−

3*(GC_total)−

6*(GC_15-19)−

30*X 

 

Formula IX;

wherein

A₁=1 if A is the base at position 1 of the sense strand, otherwise its value is 0;

A₂=1 if A is the base at position 2 of the sense strand, otherwise its value is 0;

A₃=1 if A is the base at position 3 of the sense strand, otherwise its value is 0;

A₄=1 if A is the base at position 4 of the sense strand, otherwise its value is 0;

A₅=1 if A is the base at position 5 of the sense strand, otherwise its value is 0;

A₆=1 if A is the base at position 6 of the sense strand, otherwise its value is 0;

A₇=1 if A is the base at position 7 of the sense strand, otherwise its value is 0;

A₁₀=1 if A is the base at position 10 of the sense strand, otherwise its value is 0;

A₁₁=1 if A is the base at position 11 of the sense strand, otherwise its value is 0;

A₁₃=1 if A is the base at position 13 of the sense strand, otherwise its value is 0;

A₁₉=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;

C₃=1 if C is the base at position 3 of the sense strand, otherwise its value is 0;

C₄=1 if C is the base at position 4 of the sense strand, otherwise its value is 0;

C₅=1 if C is the base at position 5 of the sense strand, otherwise its value is 0;

C₆=1 if C is the base at position 6 of the sense strand, otherwise its value is 0;

C₇=1 if C is the base at position 7 of the sense strand, otherwise its value is 0;

C₉=1 if C is the base at position 9 of the sense strand, otherwise its value is 0;

C₁₇=1 if C is the base at position 17 of the sense strand, otherwise its value is 0;

C₁₈=1 if C is the base at position 18 of the sense strand, otherwise its value is 0;

C₁₉=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;

G₁=1 if G is the base at position 1 on the sense strand, otherwise its value is 0;

G₂=1 if G is the hare at position 2 of the sense strand, otherwise its valise is 0;

G₈=1 if G is the base at position 8 on the sense strand, otherwise its value is 0;

G₁₀=1 if 0 is the base at position 10 on the sense strand, otherwise its value is 0;

G₁₃=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;

G₁₉=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;

U₁=1 if U is the base at position 1 on the sense strand, otherwise its value is 0;

U₂=1 if U is the base at position 2 on the sense strand, otherwise its value is 0;

U₃=1 if U is the base at position 3 on the sense strand, otherwise its value is 0;

U₄=1 if U is the base at position 4 on the sense strand, otherwise its value is 0;

U₇=1 if U is the base at position 7 on the sense strand, otherwise its value is 0;

U₉=1 if U is the base at position 9 on the sense strand, otherwise its value is 0;

U₁₀=1 if U is the base at position 10 on the sense strand, otherwise its value is 0;

U₁₅=1 if U is the base at position 15 on the sense strand, otherwise its value is 0;

U₁₆=1 if U is the base at position 16 on the sense strand, otherwise its value is 0;

U₁₇=1 if U is the base at position 17 on the sense strand, otherwise its value is 0;

U₁₈=1 if U is the base at position 18 on the sense strand, otherwise its value is 0;

GC_15-19=the number of G and C bases within positions 15-19 of the sense strand or within positions 15-18 if the sense strand is only 18 base pairs in length;

GC_total=the number of G and C bases in the sense strand;

Tm=100 if the targeting site contains an inverted repeat longer then 4 base pairs, otherwise its value is 0; and

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

3. A method of gene-silencing comprising selecting an siRNA according to claim 2 and introducing it into a cell.

4. The method according to claim 3 wherein said introducing is by allowing passive uptake of the siRNA.

5. The method according to claim 3, wherein said introducing is through the use of a vector.

6. A method for developing an siRNA algorithm for selecting siRNA, said method comprising:

(a) selecting a set of siRNA;

(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 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 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).

7. A method of selecting an siRNA with improved functionality, said method comprising using the algorithm of claim 6.

8. A method of selecting hyperfunctional siRNA, said method comprising using at least one functional siRNA, wherein at least one said functional siRNA has been selected according to the method of claim 7 and measuring the silencing ability of said at least one functional siRNA, wherein silencing ability is measured at a concentration of less than 1 nanomolar siRNA.

9-18. -18. (canceled)

19. A kit, wherein said kit is comprised of at least two siRNA, wherein said at least two siRNA comprise a first optimized siRNA and a second optimized siRNA, wherein said first optimized siRNA and said second optimized siRNA are optimized according to one of the following formulas:

Formula I=−

(GC/3)+(AU_15-19)−

(Tm<sub>20°

C.</sub>)*3−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃)+(U₁₀)+(A₁₄)−

(U₅)−

(A₁₁);


Formula II=−

(GC/3)−

(AU_15-19)*3−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃);


Formula III=−

(GC/3)+(AU_15-19)−

(Tm<sub>20°

C.</sub>)*3;


Formula IV=−

(GC/2)+(AU_15-19)/2−

(Tm<sub>20°

C.</sub>)*2−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃)+(U₁₀)+(A₁₄)−

(U₅)−

(A₁₁);


Formula V=−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃)+(U₁₀)+(A₁₄)−

(U₅)−

(A₁₁);


Formula VI=−

(G₁₃)*3−

(C₁₉)+(A₁₉)*2+(A₃);


Formula VII=−

(GC/2)+(AU_15-19)/2−

(Tm<sub>20°

C.</sub>)*1−

(G₁₃)*3−

(C₁₉)+(A₁₉)*3+(A₃)*3+(U₁₀)/2+(A₁₄)/2−

(U₅)/2−

(A₁₁)/2;

wherein in Formulas I-VII;

AU_15-19=0-5 depending on the number of A or U bases on the sense strand at positions 15-19;

G₁₅=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;

C₁₉=1 if C is the base at position 19 of the sense strand, otherwise its value is 0;

GC=the number of G and C bases in the entire sense strand;

Tm<sub>20°

C.</sub>=1 if the Tm is greater than 20°

C.;

A₃=1 if A is the base at position 3 on the sense strand, otherwise its value is 0;

A₁₁=1 if A is the base at position 11 on the sense strand, otherwise its value is 0;

A₁₄=1 if A is the base at position 14 on the sense strand, otherwise its value is 0;

A₁₉=1 if A is the base at position 19 on the sense strand, otherwise its value is 0;

U₅=1 if U is the base at position 5 on the sense strand, otherwise its value is 0;

U₁₀=1 if U is the base at position 10 on the sense strand, otherwise its value is 0;

or,

(−

14)*G₁₃−

13*A₁−

12*U₇−

11*U₂−

10*A₁₁−

10*U₄−

10*C₃−

10*C₅−

10*C₆−

9*A₁₀−


9*U₉−

9*C₁₈−

8*G₁₀−

7*U₁−

7*U₁₆−

7*C₁₇−

7*C₁₉+7*U₁₇+8*A₂+8*A₄
+8*A₅+8*C₄+9*G₈+10*A₇+10*U₁₈+11*A₁₉+11*C₉+15*G₁+
18*A₃+19*U₁₀−

Tm−

3*(GC_total)−

6*(GC_15-19)−

30*X; and

 

 

Formula VIII;


(14.1)*A₃+(14.9)*A₆+(17.6)*A₁₃+(24.7)*A₁₉+(14.2)*U₁₀+(10.5)*
C₉+(23.9)*G₁+(16.3)*G₂+(−

12.3)*A₁₁+(−

19.3)*U₁+(−

12.1)*U₂+
(−

11)*U₃+(−

15.2)*U₁₅+(−

11.3)*U₁₆+(−

11.8)*C₃+(−

17.4)*C₆+(−

10.5)*C₇+(−


13.7)*G₁₃+(−

25.9)*G₁₉−

Tm−

3*(GC_total)−

6*(GC_15-19)−

30*X 

 

Formula IX;

wherein

A₁=1 if A is the base at position 1 of the sense strand, otherwise its value is 0;

A₂=1 if A is the base at position 2 of the sense strand, otherwise its value is 0;

A₃=1 if A is the base at position 3 of the sense strand, otherwise its value is 0;

A₄=1 if A is the base at position 4 of the sense strand, otherwise its value is 0;

A₅=1 if A is the base at position 5 of the sense strand, otherwise its value is 0;

A₆=1 if A is the base at position 6 of the sense strand, otherwise its value is 0;

A₇=1 if A is the base at position 7 of the sense strand, otherwise its value is 0;

A₁₀=1 if A is the base at position 10 of the sense strand, otherwise its value is 0;

A₁₁=1 if A is the base at position 11 of the sense strand, otherwise its value is 0;

A₁₃=1 if A is the base at position 13 of the sense strand, otherwise its value is 0;

A₁₉=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;

C₃=1 if C is the base at position 3 of the sense strand, otherwise its value is 0;

C₄=1 if C is the base at position 4 of the sense strand, otherwise its value is 0;

C₅=1 if C is the base at position 5 of the sense strand, otherwise its value is 0;

C₆=1 if C is the base at position 6 of the sense strand, otherwise its value is 0;

C₇=1 if C is the base at position 7 of the sense strand, otherwise its value is 0;

C₉=1 if C is the base at position 9 of the sense strand, otherwise its value is 0;

C₁₇=1 if C is the base at position 17 of the sense strand, otherwise its value is 0;

C₁₈=1 if C is the base at position 18 of the sense strand, otherwise its value is 0;

C₁₉=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;

G₁=1 if G is the base at position 1 on the sense strand, otherwise its value is 0;

G₂=1 if G is the base at position 2 of the sense strand, otherwise its value is 0;

G₈=1 if G is the base at position 8 on the sense strand, otherwise its value is 0;

G₁₀=1 if G is the base at position 10 on the sense strand, otherwise its value is 0;

G₁₃=1 if G is the base at position 13 on the sense strand, otherwise its value is 0;

G₁₉=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;

U₁=1 if U is the base at position 1 on the sense strand, otherwise its value is 0;

U₂=1 if U is the base at position 2 on the sense strand, otherwise its value is 0;

U₃=1 if U is the base at position 3 on the sense strand, otherwise its value is 0;

U₄=1 if U is the base at position 4 on the sense strand, otherwise its value is 0;

U₇=1 if U is the base at position 7 on the sense strand, otherwise its value is 0;

U₉=1 if U is the base at position 9 on the sense strand, otherwise its value is 0;

U₁₀=1 if U is the base at position 10 on the sense strand, otherwise its value is 0;

U₁₅=1 if U is the base at position 15 on the sense strand, otherwise its value is 0;

U₁₆=1 if U is the base at position 16 on the sense strand, otherwise its value is 0;

U₁₇=1 if U is the base at position 17 on the sense strand, otherwise its value is 0;

U₁₈=1 if U is the base at position 18 on the sense strand, otherwise its value is 0;

GC_15-19=the number of G and C bases within positions 15-19 of the sense strand or within positions 15-18 if the sense strand is only 18 base pairs in length;

GC_total=the number of G and C bases in the sense strand;

Tm=100 if the targeting site contains an inverted repeat longer then 4 base pairs, otherwise its value is 0; and

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