Pol6 polymerase variants

Abstract

The present disclosure provides variant Pol6 polymerase polypeptides, compositions comprising the Pol6 variant polypeptides, and methods for using the variant Pol6 polypeptides for determining the sequencing of nucleic acids, for example, by nanopore sequencing. The variant Pol6 polymerases possess decreased rates of dissociation of template from the polymerase-template complex, which result in increased processivity relative to the parental Pol6 polypeptides from which they are derived.

What is claimed is: 
     
         1 . An isolated polypeptide having a DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity SEQ ID NO: 1, wherein the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 comprises one or more amino acid substitutions relative to a position of SEQ ID NO: 2 selected from the group consisting of Y30A/C/G/V, I42E/L, D44T, E46 W, C48G, G62A/E/T, G64A, W127L/S/R/V/G/C/P/Q/A/E, G146K/L, T162C/R/V, V164Q/R/T, I172A, I178A, S198F/L/Q/W/Y/V, L206S, F209I/R/T/V, A218R, Q221F/M/R/W/G/L/E/Y/K, F222L/M/Q/T, H223L, N224K/R/T, Y225T, V226T, H227E/G/A/Q/T, F233E/T/W/P/S, Y234R, K235S/Q/N/F/W/P, M236K, G237A, D241 W/H, D243P, D263Q/W/L, I264Y, Y265E, M266V/W, R268E, E269I, V270A/S, G278V/E/M/R/L/P, E283G, L284R/Y, P285L/C/Q/R/S/T, M289P, R290V/C, A292S/UT, S293G/Q/R/T/M/W/A, S294Y/T/R/M/L/K/G/F/E, I295A/L, A296M, F297M/T/L, N298K/L/R/W/G, E311N/K/H, E312R/L/K, Y314 W, Y338L, V350R/L/M, C359T, S360A/F, L361A/M/F, Y367F, S369G/F/Y/K/A, Q370A, M371L, A372G/L, Y373T/R/A, K374H/P/N/F/G/W, F376L/V, K380R, V382UI, R388P/S, P390L/V, E396N/F, Y398A/P, L399G, I400P/M/L, E4011, F404L, F406K/Y, K410V, H411P, Y414P/R/l/Q/S/T/K/G, A415P, L416 W, D417E, I418Q/H, S430P, P431G, I432F, G438R/L/P/Q/C, D448L, K456R, L458P/R, K462G, T464P, N468G, V469E/R/S/T/Y, V470L/I/M/Q/H/A/F, L471C/E/H/K/Q/R/S/V/Y, V474G, E475D/F/G/H/L/N/Q/S/T/V, Y476L, E477G, F478Y/H, W479K, K481L, H482T/G/D/R/W, F483G/N/Q/S/T, E502P/A, G505C/R, L506T/G/C, I508M/P, S510P/F/L, Y514 W, K518 W, F521M/L, K522R/Q, P523Y/T/K/G/E/R/M/L/Q/A, Y524A/F/W, V525M, D526Q/E, H527F/M/L/R, F528Q/L/Y, K530T/G/F/E/W/Y/R/Q, M531R/L/A, V533A, E534R, N535Y, K537L/E, L538R/Q/K/A, K541R/G/A/Q/W, L543A/K/T/R, T544A, N545Y, Q546A, L549T/R/A, L551M, G552T, G553T/Q/K/A/Y/R/M, A554Q/T, Y555A, G556R, K557 W/R/E/Q, F558M, T560Y/R/M/G/F/K/A/L, Q562K/R/G, E566Q/Y/G/F, E587K/T, G588E/V/Q, E590R/W, P594A/V, G603Y/L/A, W608R, A610T/C/S/V/M, L621 W, C623A/G/T, D624L, I628M, Y629K/W, C630A/Q, N631L/T/W/A, R632K, V634L, I638A/E/G/L/M/Q/Y/F/K, E639F/T/W/Y, D640A/E/K/Q/T, M641L, N642E/F/K/Q/T/W/Y/M, A643K/R/T/Y/L, I644A, T647A/E/F/G/L/M/Q/W/Y/K, K650Q/R/Y/A, T651M, I652A/E/G/K/M/Q/R/W/L, L653M/A, K655A/F/G/M/W/Y, D657E/R, V658F/M/Y/T, E659K/T, K672R/T, E680A/Q/K/F/Y/W/M, K682F/M/Y/V/I, T683F/W, D684F/K/M/Y/G/W/E/T/Q, L685A, K686R/M/T, C687A, C688A/S, S692F/K/R/Y/D/L, D693P/R/M/A/K/Y/W, A694L/M/T/Y/F, R695K, K696Q, I697A, I698L, L708H/P/Y, V712C, R718K, I722R, G724Y, F732Y, and M738 W/V. 
     
     
         2 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide further has a substitution corresponding to Y242A. 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide further has a substitution corresponding to E585K. 
     
     
         4 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide further has substitutions corresponding to Y242A and E585K. 
     
     
         5 . The isolated polypeptide of  claim 1 , comprising a set of substitutions selected from the group consisting of:
 A. A547F further comprising one or more mutations selected from
 a. Y30A/C/G/V; 
 b. 142E/L; 
 c. D44T; 
 d. E46 W; 
 e. C48G; 
 f. G62A/E/T; 
 g. G64A; 
 h. W127L/S/R/V/G/C/P/Q/A/E; 
 i. G146K/L; 
 j. T162C/R/V; 
 k. V164Q/R/T; 
 l. I172A; 
 m. I178A; 
 n. S198V; 
 o. F209I/R/T/V; 
 p. A218R; 
 q. Q221 W/R/G/L/E/F; 
 r. F233E/T/W/P/S; 
 s. Y234R; 
 t. K235S/Q/N/F/W/P; 
 u. M236K; 
 v. G237A; 
 w. D241 W/H; 
 x. D243P; 
 y. D263Q/W/L; 
 z. I264Y; 
 aa. Y265E; 
 bb. M266V/W; 
 cc. R268E; 
 dd. E2691; 
 ee. V270A/S; 
 ff. G278V/E/M/R/L/P; 
 gg. L284R/Y; 
 hh. P285L/C/Q/R/S/T; 
 ii. M289P; 
 jj. R290V/C; 
 kk. A292S/L/T; 
 ll. E311N/H; 
 mm. E312R/L/K; 
 nn. Y314 W; 
 oo. V350R/L/M; 
 pp. K374H/P/N/F/G/W; 
 qq. F376L/V; 
 rr. K380R; 
 ss. V382L/I; 
 tt. R388P/S; 
 uu. P390L/V; 
 vv. E396N; 
 ww. Y398A/P; 
 xx. L399G; 
 yy. 1400P/M/L; 
 zz. E4011; 
 aaa. F404L; 
 bbb. K410V; 
 ccc. H411P; 
 ddd. Y414P/R/I/Q/S/T/K/G; 
 eee. A415P; 
 fff. L416 W; 
 ggg. D417E; 
 hhh. I418Q/H; 
 iii. S430P; 
 jjj. P431G; 
 kkk. I432F; 
 lll. G438R/L/P/Q/C; 
 mmm. K456R; 
 nnn. L458P/R; 
 ooo. K462G; 
 qqq. N468G; 
 rrr. V469E/R/S/T/Y; 
 sss. V470L/I/M/Q/H/A/F; 
 ttt. L471C/E/H/K/Q/R/S/V/Y; 
 uuu. E475D/F/G/H/L/N/Q/S/T/V; 
 vvv. Y476L; 
 www. E477G; 
 xxx. F478Y/H; 
 yyy. W479K; 
 zzz. K481L; 
 aaaa. H482T/D/R/W; 
 bbbb. F483G/N/Q/S/T; 
 cccc. E502P/A; 
 dddd. G505C/R; 
 eeee. L506T/G/C; 
 ffff. I508M/P; 
 gggg. S510P/F/L; 
 hhhh. Y514 W; 
 iiii. K518 W; 
 hhhhh. N545Y; 
 iiiii. Q562K/R/G; 
 jjjj. E587K/T; 
 kkkk. G588E/V/Q; 
 llll. E590R/W; 
 mmmm. P594A/V; 
 nnnn. W608R; 
 oooo. A610T/C/S/V/M; 
 pppp. K672R; 
 qqqq. K682F/M/Y/V/I; 
 rrrr. D684G; 
 ssss. L685A; 
 tttt. C688A/S; 
 uuuu. S692F/D/R/L; 
 vvw. D693P/R; 
 wwww. K696Q; 
 xxxx. I698L; 
 yyyy. L708H/P/Y; 
 zzzz. V712C; 
 aaaaa. R718K; 
 bbbbb. I722R; 
 ccccc. G724Y; 
 ddddd. F732Y; and 
 eeeee. M738 W/V; 
   B. A547F+L206S, and further comprising
 a. P285L; 
   C. A547F+F209I, and further comprising
 a. D448L; 
   D. A547F+E283G, and further comprising
 a. L471K; 
   E. A547F+P285L, and further comprising
 a. L206S; 
   F. A547F+E311K, and further comprising
 a. V474G; 
   G. A547F+L361F, further comprising
 a. H482G; 
   H. A547F+L471K, and further comprising
 a. E283G; 
   I. A547F+V474G, and further comprising
 a. E311K; 
   J. A547F+H482G, and further comprising
 a. L361F; 
   K. N545K+T647G+A547F, and further comprising one or more mutations selected from
 a. Q221 W; 
 b. Y225T; 
 c. Y242A; 
 d. F528Q/L; 
 e. K530 W; 
 f. T560K/Y; 
 g. I638A; 
 h. L653A; and 
 i. D657E/R; 
   L. T529M+S366A+A547F+N545L+Y225L+D657R+S692F, and further comprising one or more mutations selected from
 a. S198F/L/Q/W/Y; 
 b. Q221F; 
 c. F222L/T/Q; 
 d. H227E; 
 e. Y242A; 
 f. S293G/Q/R/T; 
 g. F297M; 
 h. S294G/Y; 
 i. L361M; 
 j. S369G; 
 k. Y373A/R; 
 l. F406K; 
 m. F521M/L; 
 n. K522Q; 
 o. P523G/Y; 
 p. D526Q; 
 q. K530T; 
 r. M531R/L/A; 
 s. L538O; 
 t. K541R/G; 
 u. T544A; 
 v. Q546A; 
 w. K557 W/R; 
 x. F558M; 
 y. T560G/F/Y/M; 
 z. E566Q/Y/G/F; 
 aa. G603Y; 
 bb. I628M; 
 cc. Y629 W; 
 dd. I638G/A/L/E/Y/M; 
 ee. E639F/T/W/Y; 
 ff. D640A/E/K; 
 gg. N642E/Q/T; 
 hh. A643Y/T; 
 ii. I644A; 
 jj. T647Y/M; 
 kk. I652K; 
 ll. K655G; 
 mm. V658F/M; and 
 nn. D684F/K/M/Y. 
   
     
     
         6 . The isolated polypeptide of  claim 1 , wherein the amino acid sequence of the isolated polypeptide has at least 95% sequence identity to SEQ ID NO: 2 when aligned using CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix. 
     
     
         7 . The isolated polypeptide of  claim 1 , wherein the amino acid sequence of the isolated polypeptide has at least 98% sequence identity to SEQ ID NO: 2 when aligned using a CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix. 
     
     
         8 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide is capable of catalyzing template-directed synthesis of a DNA polynucleotide. 
     
     
         9 . A composition comprising the isolated polypeptide of  claim 1  covalently linked to an alpha-hemolysin monomer. 
     
     
         10 . The composition of  claim 9 , wherein the alpha-hemolysin monomer to which the isolated polypeptide is covalently linked is a constituent of a heptamer nanopore. 
     
     
         11 . The composition of  claim 10 , wherein the heptamer pore comprises 6 alpha-hemolysin monomers that are not covalently attached to an isolated polypeptide and one alpha-hemolysin monomer that is attached to the isolated polypeptide. 
     
     
         12 . A method for sequencing a nucleic acid sample with the aid of an alpha-hemolysin nanopore in a membrane adjacent to a sensing electrode, the method comprising:
 a. providing tagged nucleotides into a reaction chamber comprising said nanopore, wherein an individual tagged nucleotide of said tagged nucleotides contains a tag coupled to a nucleotide, which tag is detectable with the aid of said nanopore;   b. carrying out a polymerization reaction with the aid of a single isolated polypeptide according to  claim 1 , said isolated polypeptide being coupled to the nanopore, thereby incorporating an individual tagged nucleotide of said tagged nucleotides into a growing strand complementary to a single stranded nucleic acid molecule from said nucleic acid sample; and   c. detecting, with the aid of said nanopore, a tag associated with said individual tagged nucleotide during incorporation of said individual tagged nucleotide, wherein said tag is detected with the aid of said nanopore while said nucleotide is associated with said variant polymerase.