Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using thereof.
1. A synthetic messenger ribonucleic acid (mRNA) that is synthesized according to a method comprising the steps of:
a) providing a complementary deoxyribonucleic acid (cDNA) that encodes a pharmaceutical protein of interest;
b) selecting a nucleotide that disrupts a binding of a major groove binding partner with the mRNA, wherein the nucleotide has decreased binding affinity to the major groove binding partner selected from the group consisting of toll-like receptor (TLR) 3, TLR7, TLR8, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and wherein the nucleotide comprises a modification on the major groove face of the nucleobase where an atom of the major groove face of the nucleobase is replaced or substituted with an alkyl group; and
c) contacting the provided cDNA and the selected nucleotide with an RNA polymerase under conditions such that an mRNA transcript is synthesized.
2. The synthetic mRNA of claim 1 , wherein the method further comprises after c), the step of:
d) 5′-capping the mRNA transcript concomitantly or post-transcriptionally such that the mRNA is synthesized.
3. The synthetic mRNA of claim 1 , wherein the mRNA is at least 300 nucleotides in length.
4. The synthetic mRNA of claim 1 , wherein the nucleotide comprises a modification on the major groove face of a pyrimidine nucleobase.
5. The synthetic mRNA of claim 4 , wherein the pyrimidine nucleobase is selected from cytosine and uracil.
6. The synthetic mRNA of claim 5 , wherein the pyrimidine nucleobase is uracil.
7. The synthetic mRNA of claim 5 , wherein the pyrimidine nucleobase is cytosine.
8. The synthetic mRNA of claim 5 , wherein the nucleotide comprises 1-methyl-pseudouridine or 5-methyl-uridine.
9. The synthetic mRNA of claim 8 , wherein the nucleotide comprises 1-methyl-pseudouridine.
10. The synthetic mRNA of claim 7 , wherein the nucleotide comprises 5-methyl-cytidine.
11. The synthetic mRNA of claim 4 , wherein the major groove binding partner is TLR3, TLR7, or TLR8.
12. The synthetic mRNA of claim 4 , wherein the major groove binding partner is RIG-I, MDA5, or LGP2.
13. A pharmaceutical composition comprising the synthetic mRNA of claim 1 and a pharmaceutically acceptable carrier.
14. A pharmaceutical composition comprising the synthetic mRNA of claim 3 and a pharmaceutically acceptable carrier.
15. A pharmaceutical composition comprising the synthetic mRNA of claim 4 and a pharmaceutically acceptable carrier.
16. A pharmaceutical composition comprising the synthetic mRNA of claim 6 and a pharmaceutically acceptable carrier.
17. A pharmaceutical composition comprising the synthetic mRNA of claim 7 and a pharmaceutically acceptable carrier.
18. A pharmaceutical composition comprising the synthetic mRNA of claim 8 and a pharmaceutically acceptable carrier.
19. A pharmaceutical composition comprising the synthetic mRNA of claim 9 and a pharmaceutically acceptable carrier.
20. A pharmaceutical composition comprising the synthetic mRNA of claim 10 and a pharmaceutically acceptable carrier.
21. The synthetic mRNA of claim 2 , wherein the 5′-capping is performed concomitantly.
22. The synthetic mRNA of claim 2 , wherein the mRNA is at least 300 nucleotides in length.
23. The synthetic mRNA of claim 2 , wherein the nucleotide comprises a modification on the major groove face of a pyrimidine nucleobase.
24. The synthetic mRNA of claim 23 , wherein the pyrimidine nucleobase is selected from cytosine and uracil.
25. The synthetic mRNA of claim 24 , wherein the nucleotide comprises 1-methyl-pseudouridine or 5-methyl-uridine.
26. The synthetic mRNA of claim 23 , wherein the major groove binding partner is TLR3, TLR7, or TLR8.
27. The synthetic mRNA of claim 23 , wherein the major groove binding partner is RIG-I, MDA5, or LGP2.
28. A pharmaceutical composition comprising the synthetic mRNA of claim 2 and a pharmaceutically acceptable carrier.
29. A pharmaceutical composition comprising the synthetic mRNA of claim 22 and a pharmaceutically acceptable carrier.
30. A pharmaceutical composition comprising the synthetic mRNA of claim 23 and a pharmaceutically acceptable carrier.