Synthetic reverse transcriptases and uses thereof
The present disclosure provides non-natural reverse transcriptases for conducting reverse transcription. The non-natural reverse transcriptases herein may have increased thermostability and can conduct reverse transcription more efficiently than natural reverse transcriptases.
1. A non-natural reverse transcriptase comprising a first domain and a second domain wherein:
(a) the first domain comprises an enzyme, and
(b) the second domain comprises a modified ribonuclease polypeptide;
wherein the first and second domains are not derived from the same organism and wherein the modified ribonuclease polypeptide comprises an amino acid sequence designed to reduce activity of the modified ribonuclease polypeptide when compared to a naturally-occurring version of the modified ribonuclease polypeptide, wherein the modified ribonuclease polypeptide is derived from an organism of the genus Thermus or the genus Thermococcus and comprises an RNase H II polypeptide,
wherein the non-natural reverse transcriptase produces at least a same amount of cDNA at an elevated reaction temperature of at least 25° C. as is produced by a natural reverse transcriptase under a lower temperature, and wherein the non-natural reverse transcriptase has at between 25% and 75% of the ribonuclease activity of a wild type version of the modified ribonuclease polypeptide.
2. The non-natural reverse transcriptase of claim 1 , wherein the RNase H II polypeptide comprises a mutated RNase H II domain.
3. The non-natural reverse transcriptase of claim 2 , wherein the mutated RNase H II domain comprises at least one mutation in an active site.
4. The non-natural reverse transcriptase of claim 3 , wherein the at least one mutation in the active site decreases RNase H II activity of the RNase H II domain compared to an un-mutated version of the RNase H II domain.
5. The non-natural reverse transcriptase of claim 4 , wherein the at least one mutation is an amino acid substitution, insertion, or deletion.
6. The non-natural reverse transcriptase of claim 1 , wherein the first domain is linked to a N-terminus of the modified ribonuclease polypeptide.
7. The non-natural reverse transcriptase of claim 1 , further comprising a third polypeptide, wherein the third polypeptide comprises a peptide tag that confers thermal stability to the non-natural reverse transcriptase.
8. The non-natural reverse transcriptase of claim 7 , wherein the third polypeptide is positioned at the N-terminus of the non-natural reverse transcriptase.
9. The non-natural reverse transcriptase of claim 7 , wherein the third polypeptide is positioned at the C-terminus of the non-natural reverse transcriptase.
10. The non-natural reverse transcriptase of claim 1 , wherein the enzyme comprises a polymerase domain.
11. The non-natural reverse transcriptase of claim 10 , wherein the enzyme comprises a polymerase and the polymerase is derived from a virus, an avian virus, a human virus, a murine virus, a retrovirus, a lentivirus, or a gammaretrovirus.
12. The non-natural reverse transcriptase of claim 10 , wherein the polymerase domain is derived from a viral reverse transcriptase selected from the group consisting of: Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Myeloblastosis Associated Virus (MAV) reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Human Immunodeficiency Virus 1 (HIV-1) reverse transcriptase.
13. The non-natural reverse transcriptase of claim 10 , wherein the enzyme is derived from a polymerase and the polymerase is a DNA polymerase.
14. The non-natural reverse transcriptase of claim 10 , wherein the enzyme is derived from a polymerase and the polymerase is an RNA-dependent DNA polymerase.
15. The non-natural reverse transcriptase of claim 10 , wherein the enzyme is derived from a polymerase and the polymerase is a DNA-dependent DNA polymerase.
16. The non-natural reverse transcriptase of claim 12 , wherein the polymerase is derived from a M-MLV reverse transcriptase.
17. The non-natural reverse transcriptase of claim 1 , wherein the modified ribonuclease polypeptide is derived from a polypeptide selected from the group consisting of: Thermococcus litoralis RNase H II, and Thermus thermophilus RNase H II.
18. The non-natural reverse transcriptase of claim 1 , wherein the modified ribonuclease polypeptide comprises an amino acid sequence at least 85% identical to SEQ ID NO: 22.
19. The non-natural reverse transcriptase of claim 18 , wherein the modified ribonuclease polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 22.
20. The non-natural reverse transcriptase of claim 1 , wherein the elevated reaction temperature is at least 55° C.
21. The non-natural reverse transcriptase of claim 20 , wherein the elevated reaction temperature is at least 60° C.
22. The non-natural reverse transcriptase of claim 21 , wherein the elevated reaction temperature is at least 65° C.
23. The non-natural reverse transcriptase of claim 1 , wherein the non-natural reverse transcriptase comprises an amino acid sequence at least 85% identical to SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 17.
24. The non-natural reverse transcriptase of claim 1 , wherein the non-natural reverse transcriptase has a specific activity of at least 30,000 U/μg.
25. The non-natural reverse transcriptase of claim 1 , wherein the modified ribonuclease polypeptide comprises an active site comprising a mutation at least one of residues 7 (D), 8 (E), 105(D), and 135 (D) of SEQ ID NO:8.
26. The non-natural reverse transcriptase of claim 1 , wherein the non-natural reverse transcriptase has at least 50% of the ribonuclease activity of a wild type version of the modified ribonuclease polypeptide.