Engineered polymerases for improved sequencing by binding

Abstract

Provided herein are engineered DNA polymerases comprising modifications improving accuracy and processivity of the polymerase including modifications in the Motif A region, optionally, along with additional modifications in the palm and/or exonuclease domains of the polymerase. Also provided are nucleic acids encoding the engineered DNA polymerases comprising modifications in motif A of the polymerase, optionally, with additional modifications. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.

1 . An engineered nucleic acid polymerase comprising a Motif A region having an FLV tripeptide motif. 
     
     
         2 . The engineered polymerase of  claim 1 , wherein the engineered polymerase is at least 90% identical to SEQ ID NO:1, and wherein the Motif A region FLV tripeptide comprises substitutions L408F, Y409L and P410V numbered relative to SEQ ID NO:1. 
     
     
         3 . The engineered polymerase of  claim 2 , wherein the engineered polymerase comprises at least one additional modification relative to SEQ ID NO:1 selected from the group consisting of G165, S166, E167, K501, T514, M523, V524, L544, H545 and combinations thereof. 
     
     
         4 . The engineered polymerase of  claim 2 , wherein the at least one additional modification is selected from the group consisting of G165E, S166E, E167G, K501R, T514S, T514A, M523T, V524T, L544F, H545F and combinations thereof. 
     
     
         5 . The engineered polymerase of  claim 2 , wherein the engineered polymerase comprises at least one additional modification relative to SEQ ID NO:1 selected from the group consisting M523, and V524 numbered relative to SEQ ID NO:1. 
     
     
         6 . The engineered polymerase of  claim 4 , wherein the engineered polymerase comprises at least one additional modification relative to SEQ ID NO:1 selected from the group consisting M523T, and V524T numbered relative to SEQ ID NO:1. 
     
     
         7 . The engineered polymerase of  claim 2 , wherein the engineered polymerase comprises substitutions L544 and H545 numbered relative to SEQ ID NO:1. 
     
     
         8 . The engineered polymerase of  claim 6 , wherein the engineered polymerase comprises substitutions L544F and H545F numbered relative to SEQ ID NO:1. 
     
     
         9 . The engineered polymerase of  claim 6 , wherein the engineered polymerase comprises substitutions M523T, V524T, L544F and H545F numbered relative to SEQ ID NO:1. 
     
     
         10 . The engineered polymerase of  claim 2 , wherein the variant is at least 95% identical to SEQ ID NO:1. 
     
     
         11 . The engineered polymerase of  claim 2 , wherein the variant is at least 99% identical to SEQ ID NO:1. 
     
     
         12 . The engineered polymerase of  claim 2 , wherein the engineered polymerase comprises a segment at least 80% identical to SEQ ID NO:12. 
     
     
         13 . The engineered polymerase of  claim 11 , wherein the engineered polymerase comprises a segment at least 92% identical to SEQ ID NO:12. 
     
     
         14 . The engineered polymerase of  claim 12 , wherein the engineered polymerase comprises a segment at least 96% identical to SEQ ID NO:12. 
     
     
         15 . The engineered polymerase of  claim 1 , wherein the engineered polymerase comprises a segment at least 80% identical to SEQ ID NO:12. 
     
     
         16 . The engineered polymerase of  claim 14 , wherein the engineered polymerase comprises a segment at least 92% identical to SEQ ID NO:12. 
     
     
         17 . The engineered polymerase of  claim 15 , wherein the engineered polymerase comprises a segment at least 96% identical to SEQ ID NO:12. 
     
     
         18 . The engineered polymerase of  claim 14 , wherein the engineered polymerase comprises substitutions M523T, and V524T numbered relative to SEQ ID NO:1. 
     
     
         19 . The engineered polymerase of  claim 14 , wherein the engineered polymerase comprises substitutions L544F and H545F numbered relative to SEQ ID NO:1. 
     
     
         20 . The engineered polymerase of  claim 14 , wherein the engineered polymerase comprises at least one substitution selected from the list consisting of G165, S166, E167, K501, T514, M523, V524, L544, H545 numbered relative to SEQ ID NO: 1. 
     
     
         21 . The engineered polymerase of  claim 19 , wherein the engineered polymerase comprises at least one substitution selected from the list consisting of G165E, S166E, E167G, K501R, T514S, T514A, M523T, V524T, L544F, H545F numbered relative to SEQ ID NO: 1. 
     
     
         22 . The engineered polymerase of  claim 1 , wherein the engineered polymerase comprises SEQ ID NO:11. 
     
     
         23 . The engineered polymerase of  claim 1 , wherein the polymerase is a B-type family polymerase. 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . The engineered polymerase of  claim 1 , wherein the polymerase has increased processivity compared to a control polymerase without the substitutions L408F, Y409L and P410V. 
     
     
         28 . The engineered polymerase of  claim 20 , wherein the control polymerase comprises SEQ ID NO:1, 2, 3, 4 or 5. 
     
     
         29 . The engineered polymerase of  claim 1 , wherein the engineered polymerase further comprises a modification at residues R484, L485, I486, and combinations thereof in motif B of the polymerase numbered relative to SEQ ID NO: 1. 
     
     
         30 . The engineered polymerase of  claim 1 , wherein the polymerase is a DNA Polymerase.