Multi-conjugate of siRNA and preparing method thereof
The present invention relates to a multi-conjugate of small interfering RNA (siRNA) and a preparing method of the same, more precisely a multi-conjugate of siRNA prepared by direct binding of double stranded sense/antisense siRNA monomers or indirect covalent bonding mediated by a cross-linking agent or a polymer, and a preparing method of the same. The preparing method of a siRNA multi-conjugate of the present invention is characterized by simple and efficient reaction and thereby the prepared siRNA multi-conjugate of the present invention has high molecular weight multiple times the conventional siRNA, so that it has high negative charge density, suggesting that it has excellent ionic interaction with a cationic gene carrier and high gene delivery efficiency.
1. A purified single stranded homodimeric RNA of structure
,
,
, or
,
wherein is a single-stranded sense RNA,
is a single-stranded antisense RNA, and
• and ∘ are distinct functional groups mediating a bond between ends of the sense or antisense RNA, wherein the bond is mediated by a cross-linking agent comprising DIME (dithiobismaleimidoethane) or BM(PEG)2 (1,8-bis(maleimido)diethylene glycol).
2. The single stranded homodimeric RNA of claim 1 , comprising , or .
3. The single stranded homodimeric RNA of claim 2 , wherein the , or is complementary to a vascular endothelial growth factor (VEGF) mRNA.
4. The single stranded homodimeric RNA of claim 1 , wherein sense or antisense RNA has 15-29 nucleotides.
5. A purified single stranded hoinodimeric RNA of structure
,
,
, or
,
wherein is a single-stranded sense RNA,
is a single-stranded antisense RNA, and
• and ∘ are distinct functional groups mediating a bond between ends of the sense or antisense RNA,
wherein the bond is a reductant cleavable bond, bio-cleavable bond, or enzyme cleavable bond.
6. The single stranded homodimeric RNA of claim 5 , wherein the bond is a reductant cleavable bond.
7. The single stranded homodimeric RNA of claim 6 , wherein the reductant cleavable bond is a disulfide bond.
8. The single stranded homodimeric RNA of claim 5 , wherein the bond is a bio-cleavable bond.
9. The single stranded homodimeric RNA of claim 1 , wherein the sense or antisense RNA are bonded 3′ end to 3′ end.
10. The single stranded homodimeric RNA of claim 1 , wherein the sense or antisense RNA are bonded 5′ end to 5′ end.
11. The single stranded homodimeric RNA of claim 1 , wherein the sense or antisense RNA are bonded 3′ end to 5′ end.
12. A method of synthesizing a purified single stranded homodimeric RNA according to claim 1 , the method comprising covalently bonding
and ,
and ,
and , or
and ,
to a cross-linking agent comprising DTME (dithiobismaleimidoethane) or BM(PEG) 2 (1,8-bis(maleimido)diethylene glycol), thereby synthesizing a single stranded homodimeric RNA according to claim 1 .
13. The single stranded homodimeric RNA of claim 1 , comprising DTME.
14. The single stranded homodimeric RNA of claim 1 , comprising BM(PEG) 2 .
15. The single stranded homodimeric RNA of claim 5 , wherein the bond is an enzyme cleavable bond.
16. The single stranded homodimeric RNA of claim 5 , wherein the sense or antisense RNA are bonded 3′ end to 3′ end.
17. The single stranded homodimeric RNA of claim 5 , wherein the sense or antisense RNA are bonded 5′ end to 5′ end.
18. The single stranded homodimeric RNA of claim 5 , wherein the sense or antisense RNA are bonded 3′ end to 5′ end.
19. A method of synthesizing a purified single stranded homodimeric RNA according to claim 5 , the method comprising covalently bonding
and ,
and ,
and , or
and ,
thereby synthesizing the purified single stranded homodimeric RNA according to claim 5 , wherein the bond is a reductant cleavable bond, bio-cleavable bond, or enzyme cleavable bond.