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 method of synthesizing a messenger ribonucleic acid (mRNA), 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 RNA, 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;
c) contacting the provided cDNA and the selected nucleotide with an RNA polymerase under conditions such that an RNA transcript is synthesized, and
d) 5′-capping the RNA transcript concomitantly or post-transcriptionally such that the mRNA is synthesized.
2. The method of claim 1 , wherein the 5′-capping is performed concomitantly.
3. The method of claim 1 , wherein the nucleobase comprising a modification on the major groove face is a pyrimidine nucleobase.
4. The method of claim 3 , wherein the pyrimidine nucleobase is selected from cytosine and uracil.
5. The method of claim 3 , wherein the pyrimidine nucleobase is uracil.
6. The method of claim 3 , wherein the pyrimidine nucleobase is cytosine.
7. The method of claim 5 , wherein the nucleotide comprises 1-methyl-pseudouridine or 5-methyl-uridine.
8. The method of claim 7 , wherein the nucleotide comprises 1-methyl-pseudouridine.
9. The method of claim 6 , wherein the nucleotide comprises 5-methyl-cytidine.
10. The method of claim 1 , wherein the major groove binding partner is TLR3, TLR7, or TLR8.
11. The method of claim 1 , wherein the major groove binding partner is RIG-I, MDA5, or LGP2.
12. The method of claim 2 , wherein the nucleobase comprising a modification on the major groove face is a pyrimidine nucleobase.
13. The method of claim 12 , wherein the pyrimidine nucleobase is selected from cytosine and uracil.
14. The method of claim 12 , wherein the pyrimidine nucleobase is uracil.
15. The method of claim 12 , wherein the pyrimidine nucleobase is cytosine.
16. The method of claim 14 , wherein the nucleotide comprises 1-methyl-pseudouridine or 5-methyl-uridine.
17. The method of claim 16 , wherein the nucleotide comprises 1-methyl-pseudouridine.
18. The method of claim 15 , wherein the nucleotide comprises 5-methyl-cytidine.
19. The method of claim 2 , wherein the major groove binding partner is TLR3, TLR7, or TLR8.
20. The method of claim 2 , wherein the major groove binding partner is RIG-I, MDA5, or LGP2.