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 having the structure
wherein each is a subunit of RNA;
wherein each of the subunits is identical;
wherein and • and ∘ are distinct functional groups mediating a bond between the subunits; and
wherein the bond is mediated by a bivalent cross-linking agent comprising maleimide, NHS (N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl azide, isocyanate, pyridyldisulfide, hydrazide, or hydroxyphenyl azide.
2. The purified single-stranded homodimeric RNA of claim 1 , wherein the cross-linking agent has a molecular weight in the range of 100 Daltons to 10,000 Daltons.
3. The purified single-stranded homodimeric RNA of claim 1 , wherein the cross-linking agent comprises DTME (dithiobismaleimidoethane), sulfo-SMCC (sulfosuccinimidyl-4 [N-maleimidomethyl]cyclohexane-1-carboxylate), or BM(PEG) 2 (1,8-bis(maleimido)diethyleneglycol).
4. The purified single-stranded homodimeric RNA of claim 1 , wherein the subunits are bonded 3′ end to 3′ end, or 5′ end to 5′ end.
5. The purified single-stranded homodimeric RNA of claim 1 , wherein the subunits are bonded 3′ end to 5′ end.
6. The purified single-stranded homodimeric RNA of claim 1 , wherein subunits are sense or antisense siRNAs.
7. The purified single-stranded homodimeric RNA of claim 1 , wherein the subunits are complementary to c-myc, c-myb, c-fos, c-jun, bcl-2, VEGF, VEGF-B, VEGF-C, VEGF-D, or PIGF mRNA.
8. The purified single-stranded homodimeric RNA of claim 1 , wherein each of the subunits has 15-50 nucleotides or 15-29 nucleotides.
9. A purified single-stranded homodimeric RNA having the structure
wherein each is a subunit of RNA;
wherein each of the subunits is identical;
wherein and • and ∘ are distinct functional groups mediating a bond between the subunits; and
wherein the bond is a reductant cleavable bond, a bio-cleavable bond, or an enzyme cleavable bond.
10. The purified single-stranded homodimeric RNA of claim 9 , wherein the subunits are bonded 3′ end to 3′ end.
11. The purified single-stranded homodimeric RNA of claim 9 , wherein the subunits are bonded 5′ end to 5′ end.
12. The purified single-stranded homodimeric RNA of claim 9 , wherein the subunits are bonded 3′ end to 5′ end.
13. The purified single-stranded homodimeric RNA of claim 9 , wherein subunits are sense or antisense siRNAs.
14. The purified single-stranded homodimeric RNA of claim 9 , wherein the subunits are complementary to c-myc, c-myb, c-fos, c-jun, bcl-2, VEGF, VEGF-B, VEGF-C, VEGF-D, or PIGF mRNA.
15. The purified single-stranded homodimeric RNA of claim 9 , wherein each of the subunits has 15-50 nucleotides or 15-29 nucleotides.
16. The purified single-stranded homodimeric RNA of claim 9 , wherein the bond is a reductant cleavable bond.
17. The purified single-stranded homodimeric RNA of claim 16 , wherein reductant cleavable bond is disulfide.
18. The purified single-stranded homodimeric RNA of claim 17 , wherein the subunits are bonded 3′ end to 3′ end, or 5′ end to 5′ end.
19. A method of synthesizing a purified single-stranded homodimeric RNA according to claim 1 , the method comprising covalently bonding
to a bivalent cross-linking agent comprising maleimide, NHS (N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl azide, isocyanate, pyridyldisulfide, hydrazide, or hydroxyphenyl azide, thereby synthesizing a single-stranded homodimeric RNA.
20. A method of synthesizing a purified single-stranded homodimeric RNA according to claim 9 , the method comprising covalently bonding
thereby synthesizing the purified single stranded homodimeric RNA, wherein the bond is a reductant cleavable bond, bio-cleavable bond, or enzyme cleavable bond.