Reverse transcriptase variants
The present invention provides MML V reverse transcriptase enzymes with increased thermal stability as compared with wild type MML V and AMV reverse transcriptases. The improved thermal stability allows for reverse transcription of RNA to cDNA at temperatures above 37° C., thereby reducing error rates introduced during cDNA synthesis. As a result, the reverse transcriptases of the invention allow for increased accuracy in the determination of transcriptomes of living organisms.
1 . A reverse transcriptase comprising a polypeptide comprising a sequence of amino acids having at least about 95% sequence identity with the reverse transcriptase of SEQ ID NO: 2, and at least one amino acid substitution selected from:
S at position 89 substituted with G, A, V, L, or I;
G at position 171 substituted with S, C, T, or M;
S at position 265 substituted with C, T, or M;
L at position 267 substituted with D, E, N, or Q;
G at position 323 substituted with H, K, or R;
Y at position 377 substituted with F or M;
T at position 453 substituted with G, A, V, L, or I;
G at position 457 substituted with D, E, N, or Q;
V at position 477 substituted with G, A, L, or I; and
D at position 648 substituted with E, N, or Q.
2 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase comprises the sequence of SEQ ID NO: 4.
3 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase comprises the sequence of SEQ ID NO: 8.
4 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase comprises the sequence of SEQ ID NO: 14.
5 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase exhibits an increased thermal stability relative to the reverse transcriptase shown in SEQ ID NO: 2, such that the activity of the reverse transcriptase at temperatures of about 50° C. or greater is at least 90% of the reverse transcriptase activity of the reverse transcriptase shown in SEQ ID NO: 2 at 42° C.
6 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase exhibits an increased thermal stability relative to the reverse transcriptase shown in SEQ ID NO: 2, such that the activity of the reverse transcriptase at temperatures of about 65° C. or greater is at least 50% of the reverse transcriptase activity of the reverse transcriptase shown in SEQ ID NO: 2 at 42° C.
7 . The reverse transcriptase of claim 1 , further comprising at least one additional amino acid substitution selected from:
D at position 557 substituted with G, A, V, L, or I;
D at position 616 substituted with E, N, or Q; and
E at position 595 substituted with D, N, or Q.
8 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase has the following substitutions relative to the reverse transcriptase of SEQ ID NO: 2: S89A, L105Q, G171S, T196K, H237R, S265T, L267E, N282E, G323K, T339K, Y377F, T453V, G457D, V477I, D648E, and H675D.
9 . The reverse transcriptase of claim 1 , wherein the activity of the reverse transcriptase is improved compared to the reverse transcriptase shown in SEQ ID NO: 2 in the presence of inhibitors of reverse transcription.
10 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase comprises the sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 15.