Argonaute protein-double stranded RNA complexes and uses related thereto

1. A RNA protein complex (RNP) comprising:

(a) a non-native, short double-stranded RNA (dsRNA), or non-native analog thereof; and

(b) a recombinant Argonaute 2 protein (Ago2), wherein said Ago2 comprises:

(i) a polypeptide having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, or

(ii) a polypeptide having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and

(c) a polyamine comprising a repeat unit represented by the following structural formula:

wherein Rn=

2. The RNP of claim 1 , wherein said polypeptide of (ii) has 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

3. The RNP of claim 1 , wherein said dsRNA is selected from the group consisting of:

(a) small interfering RNA (siRNA or ds siRNA);

(b) small hairpin RNA (shRNA);

(c) microRNA (miRNA);

(d) Piwi-interacting RNA (piRNA);

(e) heterochromatin-related guide RNAs (hc-gRNA); and

(f) antigene RNAs (agRNA).

4. The RNP of claim 3 , wherein the dsRNA is miRNA.

5. The RNP of claim 4 , wherein the miRNA is microRNA34a.

6. The RNP of claim 1 , wherein said dsRNA is 45-50 nucleotides in length.

7. The RNP of claim 1 , wherein said dsRNA comprises at least one phosphate-sugar backbone modification.

8. The RNP of claim 7 , wherein the at least one phosphate-sugar backbone modification is selected from the group consisting of:

(a) phosphorothioate linkage;

(b) phosphoramidate linkage;

(c) phosphodithioate linkage;

(d) chimeric methylphosphonate-phosphodiester linkage; and

(e) 5′-N-phosphoramidite linkage.

9. The RNP of claim 1 , wherein the dsRNA comprises a silencing sequence.

10. The RNP of claim 9 , wherein said silencing sequence comprises one, two, three, four, or five nucleotide basepair mismatch.

11. The RNP of claim 10 , wherein said analog comprises at least one nucleoside selected from the group consisting of:

(a) 2-aminoadenosine;

(b) 2-thiothymidine;

(c) inosine;

(d) pyrrolo-pyrimidine;

(e) 3-methyl adenosine;

(f) C5-propynylcytidine;

(g) C5-propynyluridine;

(h) C5-bromouridine;

(i) C5-fluorouridine;

(j) C5-iodouridine;

(k) C5-methylcytidine;

(1) 7-deazaadenosine;

(m) 7-deazaguanosine;

(n) 8-oxoadenosine;

(o) 8-oxoguanosine;

(p) O(6)-methylguanine; and

(q) 2-thiocytidine.

12. The RNP of claim 1 , for use in silencing the expression of a target gene, wherein the use comprises introducing the RNP into a cell.

13. The RNP of claim 1 , for use in gene silencing in a subject, wherein the use comprises administering a therapeutically effective amount of the RNP to the subject.

14. The RNP of claim 1 , wherein said dsRNA is 40-45 nucleotides in length.

15. The RNP of claim 1 , wherein said dsRNA is 35-40 nucleotides in length.

16. The RNP of claim 1 , wherein said dsRNA is 30-35 nucleotides in length.

17. The RNP of claim 1 , wherein said dsRNA is 25-30 nucleotides in length.

18. The RNP of claim 1 , wherein said dsRNA is 20-25 nucleotides in length.

19. The RNP of claim 1 , wherein said dsRNA is 15-20 nucleotides in length.

20. The RNP of claim 1 , wherein said dsRNA is 10-15 nucleotides in length.

21. The RNP of claim 1 , wherein the RNP has a N/P ratio (number of protonatable amines in the polyamine relative to number of phosphates in siRNA) is 20:1.

22. A method for silencing the expression of a target gene in a cell, comprising:

(i) providing a non-native, short double-stranded RNA (dsRNA), or non-native analog thereof;

(ii) complexing the dsRNA to a recombinant Argonaute 2 protein (Ago2); wherein said Ago2 comprises: (a) a polypeptide having 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, or (b) a polypeptide having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and a polyamine comprising a repeat unit represented by the following structural formula:

wherein Rn=

(iii) forming an Ago2 protein-dsRNA complex (RNP) comprising the dsRNA and the Ago2; and

(iv) introducing the RNP into the cell;

thereby inducing gene silencing of the target gene.

23. The method of claim 22 , wherein the RNP comprises a 1:0.5 or 1:1 ratio of dsRNA to Ago2.

24. The method of claim 22 , wherein the cell is a stem cell or an embryonic stem cell.

25. The method of claim 22 , wherein the cell is in culture.