RNA-interference by single-stranded RNA molecules

• US 8,101,348 B2
• Filed: 07/10/2003
• Issued: 01/24/2012
• Est. Priority Date: 07/10/2002
• Status: Expired due to Fees

• Current Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Original Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Inventors: Tuschl, Thomas, Martinez, Javier, Patkaniowska, Agnieszka, Urlaub, Henning, Luehrmann, Reinhard
• Primary Examiner(s): SHIN, DANA H
• Application Number:
  US10/520,470
• Publication Number:
  US 20060166910A1
• Time in Patent Office: 3,120 Days
• Field of Search: 514/44, 536/24.5, 435/6, 435/366, 435/375
• US Class Current: 435/6.1
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/557 the modifying agent being biotin
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

The present invention relates to sequence and structural features of single-stranded (ss) RNA molecules required to mediate target-specific nucleic acid modifications by RNA-interference (RNAi), such as target mRNA degradation and/or DNA methylation.

Claims

1. A method of cleaving a nucleic acid target molecule in a mammalian cell in vitro comprising contacting the cell with a single-stranded siRNA molecule, wherein the single-stranded siRNA molecule:

is complementary to the nucleic acid target molecule;

is from 19 to 29 nucleotides in length; and

comprises a phosphate analog at the 5′

-terminus; and

thereby cleaving the nucleic acid target molecule in the cell.

2. The method of claim 1, wherein the phosphate analog at the 5′

-terminus of the single-stranded siRNA molecule is selected from the group consisting of;

a 5′

-guanosine cap, a 5′

-adenosine cap, a 5′

-monothiophosphate, a 5′

-monodithiophosphate, a 5′

-phosphorothiolate, a 5′

-phosphoramidate, a 5′

-alkylphosphonate, and a 5′

-alkyletherphosphonate.

3. The method of claim 1, wherein the phosphate analog at the 5′

-terminus of the single-stranded siRNA molecule is selected from the group consisting of;

a 5′

-monophosphate, a 5′

-diphosphate, and a 5′

-triphosphate.

4. The method of claim 1, wherein the phosphate analog at the 5′

-terminus of the single-stranded siRNA molecule is a 5′

-triphosphate.

5. The method of claim 1, wherein the phosphate analog at the 5′

-terminus of the single-stranded siRNA molecule comprises a monophosphate, a diphosphate, or a triphosphate in which at least one oxygen atom of the monophosphate, diphosphate, or triphosphate is replaced with a sulfur atom.

6. The method of claim 5, wherein the phosphate analog is selected from the group consisting of 5′

-alpha-thiotriphosphate and 5′

-gamma-thiotriphosphate.

7. The method of claim 1, wherein the phosphate analog at the 5′

-terminus of the single-stranded siRNA molecule is an alkylphosphonate.

8. The method of claim 7, wherein the alkylphosphonate has the formula:

RP(OH)(O)—

O-5′

or (OH)₂(O)P-5′

-CH₂—

, wherein R is a C₁-C₃ alkyl.

9. The method of claim 1, wherein the phosphate analog at the 5′

-terminus of the single-stranded siRNA molecule is an alkyletherphosphonate.

10. The method of claim 9, wherein the alkyletherphosphonate has the formula:

RP(OH)(O)—

O-5′

, wherein R is an alkylether.

11. The method of claim 1, wherein the single-stranded siRNA molecule comprises at least one sugar or backbone modified nucleoside at the 3′

terminus, wherein at least 15 consecutive nucleotides at the 5′

terminus are unmodified.

12. The method of claim 11, wherein at least one modified nucleoside comprises a sugar modification wherein the 2′

OH group is replaced by a group selected from the group consisting of H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, and CN, wherein R is selected from the group consisting of C₁-C₆ alkyl, alkenyl, alkynyl, and methoxyethoxy.

13. The method of claim 11, wherein in said backbone modified nucleoside a phosphoester group connecting adjacent ribonucleotides is replaced by a modified group selected from the group consisting of a phosphorothioate, a phosphorodithioate, a N3′

-O5′

phosphoramidate group and a N5′

-O3′

phosphoramidate group.

14. The method of claim 1, wherein the single-stranded siRNA molecule comprises at least one phosphorothioate linkage.

15. The method of claim 1, wherein the single-stranded siRNA molecule comprises a region at the 3′

-terminus comprising at least one adenosine, guanosine or combination thereof.