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 . A variant polypeptide capable of catalyzing polymerization of deoxynucleotides, said polypeptide comprising an amino acid sequence having at least 70% identity with SEQ ID NO: 2, wherein the amino acid sequence comprises a substitution corresponding to a position of SEQ ID NO: 2 selected from the group consisting of V173, N175, N176, N177, I178, V179, Y180, S211, Y212, I214, T287, G288, M289, R290, T291, A292, S293, S294, 1295, Y338, T339, G340, G341, Y342, T343, H344, A345, D417, I418, F419, K420, I421, G422, G434, A435, V436, S437, G438, Q439, E440, Y441, G559, T560, N563, E565, E566, D568, L569, I570, M571, D572, N574, G575, L576, L577, T578, F579, T580, G581, S582, V583, T584, E587, G588, E590, F591, Y596, Q662, V667, L668, G669, Q670, L685, C687, C688, G689, L690, P691, S692, D693, A694, L708, G709, Q717, R718, V721, L731, F732, T733, I734, I737, M738, and F739. 
     
     
         2 . The polypeptide of  claim 1 , wherein said substitution is a substitution to an amino acid selected from the group consisting of K, R, H, Y, F, W, and T. 
     
     
         3 . The polypeptide of  claim 1 , wherein said substitution is selected from the group consisting of V173K, N175K, N176K, N177K, I178K, V179K, Y180K, S211K, Y212K, I214K, T287K, G288K, M289K, R290K, T291 K, A292K, S293K, S294K, I295K, Y338K, T339K, G340K, G341K, Y342K, T343K, H344K, A345K, D417K, I418K, F419K, K420K, I421K, G422K, G434K, A435K, V436K, S437K, G438K, Q439K, E440K, Y441 K, G559K, T560K, N563K, E566K, E565K, D568K, L569K, I570K, M571 K, D572K, N574K, G575K, L576K, L577K, T578K, F579K, T580K, G581 K, S582K, V583K, T584K, E587K, G588K, E590K, F591 K, Y596K, Q662K, V667K, L668K, G669K, Q670K, L685K, C687K, C688K, G689K, L690K, P691 K, S692K, D693K, A694K, L708K, G709K, Q717K, R718K, V721K, L731K, F732K, T733K , I734K, I737K, M738K, and F739K. 
     
     
         4 . The polypeptide of  claim 1 , wherein said polypeptide comprises at least two substitutions selected from the group consisting of V173K, N175K, N176K, N177K, I178K, V179K, Y180K, S211K, Y212K, I214K, T287K, G288K, M289K, R290K, T291 K, A292K, S293K, S294K, I295K, Y338K, T339K, G340K, G341K, Y342K, T343K, H344K, A345K, D417K, I418K, F419K, K420K, I421 K, G422K, G434K, A435K, V436K, S437K, G438K, Q439K, E440K, Y441 K, G559K, T560K, N563K, E566K, E565K, D568K, L569K, I570K, M571 K, D572K, N574K, G575K, L576K, L577K, T578K, F579K, T580K, G581 K, S582K, V583K, T584K, E587K, G588K, E590K, F591 K, Y596K, Q662K, V667K, L668K, G669K, Q670K, L685K, C687K, C688K, G689K, L690K, P691 K, S692K, D693K, A694K, L708K, G709K, Q717K, R718K, V721 K, L731K, F732K, T733K , I734K, I737K, M738K, and F739K. 
     
     
         5 . The polypeptide of  claim 1 , wherein said substitution is L731 K/R/H. 
     
     
         6 . The polypeptide of  claim 1 , wherein said substitution is M738K/R/H. 
     
     
         7 . The polypeptide of  claim 1 , wherein said amino acid sequence further comprises a D44A substitution relative to SEQ ID NO: 2. 
     
     
         8 . The polypeptide of  claim 1 , wherein said amino acid sequence further comprises a S366A substitution relative to SEQ ID NO: 2. 
     
     
         9 . The polypeptide of  claim 1 , wherein said amino acid sequence further comprises a T529M substitution relative to SEQ ID NO: 2. 
     
     
         10 . The polypeptide of  claim 1 , wherein said amino acid sequence further comprises a A547F substitution relative to SEQ ID NO: 2. 
     
     
         11 . The polypeptide of  claim 1 , wherein said polypeptide has a reduced rate of dissociation in the presence of 500 mM potassium glutamate relative to a polypeptide consisting of SEQ ID NO: 4. 
     
     
         12 . The polypeptide of  claim 1 , further comprising an affinity tag. 
     
     
         13 . The polypeptide of  claim 1 , further comprising a member of an attachment selected from the group consisting of (a) a SpyCatcher/SpyTag peptide system, (b) a native chemical ligation system, (c) a sortase system, (d) a transglutaminase system, (e) a formylglycine linkage system, and (g) a zinc finger system. 
     
     
         14 . A composition comprising the polypeptide of  claim 1  attached to a biological nanopore. 
     
     
         15 . The composition of  claim 14 , wherein the attachment between the polypeptide and the biological nanopore results from an attachment system selected from the group consisting of: (a) a SpyCatcher/SpyTag peptide system, (b) a native chemical ligation system, (c) a sortase system, (d) a transglutaminase systems, (e) a formylglycine linkage system, (f) a system comprising reaction between 6-hydrazino-nicotinic acid and 4-formylbenzoate, and (g) a zinc finger system. 
     
     
         16 . The composition of  claim 14 , further comprising a polynucleotide complexed with the polypeptide. 
     
     
         17 . The composition of  claim 14 , wherein said composition is a part of a nanopore sequencing complex on a biochip, the nanopore sequencing complex comprising:
 the nanopore of the composition inserted in a membrane adjacent to an electrode of a sensing circuit of the biochip, and   a polynucleotide complexed with the polypeptide.   
     
     
         18 . The composition of  claim 17 , wherein the biochip comprises at least 500 nanopore sequencing complexes per 1 mm 2 . 
     
     
         19 . A method for nanopore sequencing a polynucleotide, said method comprising:
 (a) providing a biochip comprising a plurality of nanopore sequencing complexes, each nanopore sequencing complex comprising:
 (a1) a sensing circuit comprising an electrode, 
 (a2) a nanopore adjacent to the electrode, 
 (a3) the polypeptide of  claim 1  attached to the nanopore, and 
 (a4) the polynucleotide complexed with the polypeptide; 
   (b) contacting the nanopore sequencing complexes with a set of tagged nucleotides, wherein the tagged nucleotides of the set contain a tag coupled to a nucleotide, which tag is detectable with the aid of said nanopore;   (c) carrying out a polymerization reaction catalyzed by the polypeptide of the nanopore sequencing complex, thereby incorporating individual tagged nucleotides of the set into a growing strand complementary to a single stranded portion of the polynucleotide; and   (d) detecting, with the aid of said nanopore, the tag associated with the individual tagged nucleotide during incorporation of the individual tagged nucleotide into the growing strand, wherein said tag is detected with the aid of the nanopore while the nucleotide is associated with the polypeptide.   
     
     
         20 . The method of  claim 19 , wherein the nanopore is a biological nanopore inserted into a membrane.