DPO4 polymerase variants with improved properties

Abstract

Recombinant DPO4-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and accurate incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.

Description

DPO4 POLYMERASE VARIANTS WITH IMPROVED PROPERTIESSEQUENCE LISTING INCORPORATION BY REFERENCE

[0001] This application contains a sequence listing, which has been filed electronically in xml format and is herein incorporated by reference in its entirety. Said xml copy has a file name of P60131-WO.xml, was created on April 8, 2026, and is 71,680 bytes in size.FIELD OF THE INVENTION

[0002] The disclosure relates generally to polymerase compositions and methods. More particularly, the disclosure relates to modified DPO4 polymerases and their use in biological and biomolecular applications including, for example, high-accuracy nucleotide analog incorporation, primer-extension, and nanopore-based sequencing systems.BACKGROUND OF THE INVENTION

[0003] DNA polymerases replicate the genomes of living organisms. In addition to this central role in biology, DNA polymerases are also ubiquitous tools of biotechnology. They are widely used, e.g., for reverse transcription, amplification, labeling, and sequencing, all central technologies for a variety of applications, such as nucleic acid sequencing, nucleic acid amplification, cloning, protein engineering, diagnostics, molecular medicine, and many other technologies.

[0004] Because of their significance, DNA polymerases have been extensively studied, with a focus, e.g., on phylogenetic relationships among polymerases, structure of polymerases, structure-function features of polymerases, and the role of polymerases in DNA replication and other basic biological processes, as well as ways of using DNA polymerases in biotechnology. Scientists have comprehensively catalogued DNA polymerases from all three kingdoms of life, with the enzymes being classified into six major families (A, B, C, D, X, and Y) according to their sequence homology. For a review of polymerases, see, e.g., Hubscher et al. (2002) "Eukaryotic DNA Polymerases" Annual Review of Biochemistry Vol. 71: 133-163, Alba (2001) "Protein Family Review: Replicative DNA Polymerases" Genome Biology 2(1): reviews 3002.1-3002.4, Steitz (1999) "DNA polymerases: structural diversity and common mechanisms" J Biol Chem 274:17395-17398, and Burgers et al. (2001) "Eukaryotic DNA polymerases: proposal for a revised nomenclature" J Biol. Chem. 276(47): 43487-90. Crystalstructures have been solved for many polymerases, which often share a similar architecture. The basic mechanisms of action for many polymerases have been determined.

[0005] A fundamental application of DNA polymerases is in DNA sequencing technologies. From the classical Sanger sequencing method to recent “next-generation” sequencing (NGS) technologies, the nucleotide substrates used for sequencing have necessarily changed overtime. The series of nucleotide modifications required by these rapidly changing technologies has introduced daunting tasks for DNA polymerase researchers to look for, design, or evolve compatible enzymes for ever-changing DNA sequencing chemistries. DNA polymerase mutants have been identified that have a variety of useful properties, including altered nucleotide analog incorporation abilities relative to wild-type counterpart enzymes. For example, VentA488LDNA polymerase can incorporate certain non-standard nucleotides with a higher efficiency than native Vent DNA polymerase. See Gardner et al. (2004) "Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase" J. Biol. Chem.279(12): 11834-11842 and Gardner and Jack (1999) "Determinants of nucleotide sugar recognition in an archaeon DNA polymerase" Nucleic Acids Research 27(12):2545-2553. The altered residue in this mutant, A488, is predicted to be facing away from the nucleotide binding site of the enzyme. The pattern of relaxed specificity at this position roughly correlates with the size of the substituted amino acid side chain and affects incorporation by the enzyme of a variety of modified nucleotide sugars.

[0006] More recently, NGS technologies have introduced the need to adapt DNA polymerase enzymes to accept nucleotide substrates modified with reversible terminators on the 3’ -OH, such as -ONH2. To this end, Chen and colleagues combined structural analyses with a “reconstructed evolutionary adaptive path” analysis to generate a TAQL616Avariant that is able to efficiently incorporate both reversible and irreversible terminators. See Chen et al. (2010) “Reconstructed Evolutionary Adaptive Paths Give Polymerases Accepting Reversible Terminators for Sequencing and SNP Detection” Proc. Nat. Acad. Sci. 107(5): 1948-1953. Modeling studies suggested that this variant might open space behind Phe-667, allowing it to accommodate the larger 3’ substituents. U.S. Patent No. 8,999,676 to Emig et al. discloses additional modified polymerases that display improved properties useful for single molecule sequencing technologies based on fluorescent detection. In particular, substitution of q>29 DNA polymerase at positions E375 and K512 was found to enhance the ability of the polymerase toutilize non-natural, phosphate-labeled nucleotide analogs incorporating different fluorescent dyes.

[0007] Recently, Kokoris et al. have described a method, termed “Sequencing by Expansion” (SBX®), that uses a DNA polymerase to transcribe the sequence of DNA onto a measurable polymer called an Xpandomer (see, e.g., U.S. Patent No. 8,324,360 to Kokoris et al., herein incorporated by reference in its entirety). The transcribed sequence is encoded along the Xpandomer backbone in high signal -to-noise reporters that are separated by -lOrnn and are designed for high signal-to-noise, well differentiated responses when read by nanopore-based sequencing systems. Xpandomers are generated from non-natural nucleotide analogs, termed XNTPs, characterized by bulky substituents that enable the Xpandomer backbone to be expanded following synthesis. Such XNTP analogs introduce novel challenges as substrates for currently available DNA polymerases. Published PCT applications no. WO2017 / 087281, WO2018 / 2047072, and WO2019 / 118372 to Kokoris et al., each herein incorporated by reference in their entireties, describe engineered DPO4 polymerase variants with enhanced primer extension activity utilizing non-natural, bulky nucleotide analogues as substrates.

[0008] Other challenges facing DNA polymerases are presented by certain nucleotide sequence motifs in the template. Of particular consequence are runs of homopolymers or short repeated DNA sequences that can trigger slipped-strand mispairing, or “replication slippage”. Replication slippage is thought to encompass the following steps: (i) copying of the first repeat by the replication machinery, (ii) replication pausing and dissociation of the polymerase from the newly synthesized end, (iii) unpairing of the newly synthesized strand and its pairing with the second repeat, and (iv) resumption of DNA synthesis. Arrest of the replication machinery within a repeated region thus results in misalignment of primer and template. In vivo, misalignment of two DNA strands during replication can lead to DNA rearrangements such as deletions or duplications of varying lengths. In vitro, replication slippage results in replication errors at the site of the slippage event. Such reduction in polymerase accuracy significantly impairs the particular application or desired genetic manipulation.

[0009] Thus, new modified polymerases, e.g., polymerases engineered for improved properties that find use in Sequencing by Expansion (SBX®) and other applications in biotechnology and biomedicine (e.g., DNA amplification, conventional sequencing, labeling, detection, cloning, etc.), would find value in the art as novel reagents. The present invention provides new recombinant DNA polymerases with such desirable properties, including theability to incorporate nucleotide analogs with bulky substitutions with improved efficiency and replication accuracy and other advantageous properties. Also provided are methods of making and using such polymerases, and many other features that will become apparent upon a complete review of the following.SUMMARY

[0010] Recombinant DNA polymerases and modified DNA polymerases, e.g., modified archaeal DPO4, can find use in such applications as, e.g., single-molecule Sequencing by Expansion (SBX®). Among other aspects, the invention provides recombinant DNA polymerases and modified DNA polymerase variants comprising mutations that confer properties, which can be particularly desirable for these applications. These properties can, e.g., 1) improve the ability of the polymerase to utilize bulky nucleotide analogs (e.g., XNTPs) as substrates during template-dependent polymerization of a daughter strand; 2) increase the accuracy of nucleotide analog incorporation, particularly when the template includes nucleotide repeat sequences that can promote replication errors, 3) increase polymerase thermostability or solubility, 4) increase the processivity of the polymerase on long nucleic acid template, and 5) enable the polymerase to synthesize Xpandomer copies of complex nucleic acid templates, e.g., duplex hairpin constructs. Also provided are compositions comprising such DNA polymerases and modified DPO4-type polymerases, nucleic acids encoding such modified polymerases, methods of generating such modified polymerases and methods in which such polymerases can be used, e.g., to synthesize Xpandomer products for nanopore sequence determination.

[0011] One general class of embodiments provides an isolated recombinant DNA polymerase, which recombinant DNA polymerase includes an amino acid sequence that is at least 80% identical to amino acids 1-340 of SEQ ID NO:1, which recombinant polymerase includes a mutation at an amino acid position selected from the group consisting of 8, 32, 37, 39, 48, 49, 53, 59, 74, 179, and 272, in which identification of positions is relative to wildtype DPO4 polymerase (SEQ ID NO:1), which recombinant DNA polymerase further includes a deletion to remove the terminal 12 amino acids (the PIP box region) of the SEQ ID NO: land which recombinant DNA polymerase exhibits polymerase activity. Another general class of embodiments provides an isolated recombinant DNA polymerase in which the polymerase includes an amino acid sequence that is at least 85% identical to amino acids 1-340 of SEQ ID NO: 1. In certain embodiments, the mutation at amino acid position 8 is F8Y, the mutation atamino acid position 32 is A32N, the mutation at amino acid position 37 is F37G, F37S, or F37T, the mutation at position 39 is D39L, D39N, or D39F, the mutation at amino acid position 48 is Y48H, the mutation at amino acid position 49 is E49Y, the mutation at amino acid position 53 is F53E, the mutation at position 59 is I59M, the mutation at amino acid position 74 is L74I, the mutation at position 179 is D179N, and the mutation at position 272 is S272C, or S272A. In certain embodiments, the polymerase includes mutations at amino acid positions 37, 39, 59, 179, and 272. In yet other embodiments, the mutation at amino acid position 37 is F37T, the mutation at amino acid position 39 is D39L, the mutation at position 59 is I59M, the mutation at amino acid position 179 is D179N, and the mutation at amino acid position 272 is S272C. In other embodiments, the polymerase further includes at least one mutation at an amino acid position selected from the group consisting of 57, 152, 153, 155, 157, 158, 161, 192, 249, 250, 289, 290, 291, 292, 293, 294, 296, 300, 324, 325, and 327, in which the mutation at position 57 is A57S, the mutation at position 152 is K152H, K152N, or K152W, the mutation at position 153 is I153N or I153L, the mutation at position 155 is A155S, the mutation at position 157 is M157W, M157H, or M157K, the mutation at position 158 is A158C, A158G, A158V, A158Q, orA158M, the mutation at position 161 isN161A,N161S,N161G,N161E,N161M, orN161Q, the mutation at position 192 is E192Q, the mutation at position 249 is V249R, the mutation at position 250 is T250M, the mutation at position 290 is T290H, T290F, T290I, T290S, T290D, T290M, T290H, T290W, T290A, T290Q, T290V, or T290Y, the mutation at position 291 is E291G, E291S, E291T, E291N, E291H, E291D, or E291Q, the mutation at position 292 is D292R, the mutation at position 293 is L293H or L293W, the mutation at position 294 is D294Y or D294N, the mutation at position 296 is V296T or V296Q, the mutation at position 300 is R300K or R300A, and the mutation at position 1 is E327T, E327K, E327P or E327I. In certain embodiments, the polymerase includes mutations T290H and E291T or T290D and E291Q. In some embodiments, the polymerase further includes at least one mutation at an amino acid position selected from the group consisting of 56, 63, 76, 78, 79 82, 83, 86, 152, 153, 155, 156, 184, 187, 188, 189, 248, 289, 295, 297, 299, 301, 321, 324, and 325. In certain embodiments, the mutations at amino acid positions 56, 63, 76, 78, 79 82, 83, 86, 152, 153, 155, 156, 184, 187, 188, 189, 248, 289, 295, 297, 299, 301, 321, 324, and 325 are K56Y, E63R, M76W, K78E, E79P, Q82W, Q83G, S86E, K152A, I153V, A155G, D156S, P184Q, G187P, N188Y, I189F, I248T, V289W, I295S, S297Y, G299W, T301W, E324K, and E325K. In some embodiments, the polymerase includes a deletion of at least one internal amino acid. In certain embodiments, the internal amino acid is selected from the group consisting of amino acid 291,amino acid 292, amino acid 293, and amino acid 294. In some embodiments, the internal amino acid includes two amino acids. In some embodiments, the polymerase includes the amino acid sequence set forth in any one of SEQ ID NOs: 2 - 59.

[0012] In a related aspect, the invention provides compositions containing any of the recombinant DPO4-type DNA polymerase set forth above. In certain embodiments, the compositions may also contain at least one non-natural nucleotide analog substrate. In one embodiment, the non-natural nucleotide analog substrate is an XNTP. In other embodiments, the composition also includes a polymerase enhancing molecule (i.e., a PEM) and / or a manganese salt. In some embodiments, the composition may further include one or more of a buffering agent, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, polyethylenglycol (PEG), dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and urea. In some embodiments, the composition may further include D2O. In certain embodiments, the composition may include between 70% w / v and 75% w / v D2O.

[0013] In a related aspect, the invention provides a kit for performing an Xpandomer synthesis reaction, in which the kit includes one or more reagents selected from the group consisting of a DPO4 polymerase variant as set forth above and one or more of a buffering agent, a salt, a sugar, a polymerase enhancing molecule (PEM), a polyphosphate, imidazole, a pyrazole, a triazole, an alkanediol, acetamide, glycerol, betaine, a polyethylenglycol (PEG), N-methyl-2 -pyrrolidone (NMP), butylated hydroxyanisole (BHA), dimethyl sulfoxide (DMSO), urea, a single stranded binding protein (SSB), XNTPs, and D2O. In one embodiment, the one or more reagents are provided in a single tube . In another embodiment, the one or more reagents are provided in more than one tube. In some embodiments, the kit is suitable for storage at -20 degrees Celsius. In some embodiments, the kit is suitable for storage at -80 degrees Celsius.

[0014] In another related aspect, the invention provides modified nucleic acids encoding any of the modified DPO4-type DNA polymerase set forth above. In related aspects, the invention provides a host cell including a modified nucleic acid encoding a modified DPO4-type DNA polymerase.BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1 shows the amino acid sequence of the DPO4 polymerase protein (SEQ ID NO: 1) with the Mutl through Mutl5 regions outlined and variable amino acids underscored.

[0016] FIG. 2 shows a simplified illustration of one embodiment of an expandable nonnatural nucleotide analog (XNTP). XNTPs are the substrates the polymerase uses to synthesis the Xpandomer copy of a nucleic acid template.DEFINITIONS

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0018] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes mixtures of cells, and the like.

[0019] The term "about" as used herein indicates the value of a given quantity varies by + / -10% of the value, or optionally + / -5% of the value, or in some embodiments, by + / -!% of the value so described.

[0020] "Nucleobase" is a heterocyclic base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof. A nucleobase can be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted at the 8 position with methyl or bromine, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine, 2,6-diaminopurine, N6-ethano-2,6-diaminopurine, 5 -methylcytosine, 5-(C3-C6)-alkynylcytosine, 5 -fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5 -methyl -4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturally occurring nucleobases described in U.S. Pat. Nos. 5,432,272 and 6,150,510 and PCTPublication Nos. WO 92 / 002258, WO 93 / 10820, WO 94 / 22892, and WO 94 / 24144, and Fasman ("Practical Handbook of Biochemistry and Molecular Biology", pp. 385-394, 1989, CRC Press, Boca Raton, La.), all herein incorporated by reference in their entireties.

[0021] "Nucleobase residue" includes nucleotides, nucleosides, fragments thereof, and related molecules having the property of binding to a complementary nucleotide. Deoxynucleotides and ribonucleotides, and their various analogs, are contemplated within the scope of this definition. Nucleobase residues may be members of oligomers and probes. "Nucleobase" and "nucleobase residue" may be used interchangeably herein and are generally synonymous unless context dictates otherwise.

[0022] "Polynucleotides", also called nucleic acids, are covalently linked series of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the next. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are biologically occurring polynucleotides in which the nucleotide residues are linked in a specific sequence by phosphodiester linkages. As used herein, the terms "polynucleotide" or "oligonucleotide" encompass any polymer compound having a linear backbone of nucleotides. Oligonucleotides, also termed oligomers, are generally shorter chained polynucleotides.

[0023] "Nucleic acid" is a polynucleotide or an oligonucleotide. A nucleic acid molecule can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination of both. Nucleic acids are generally referred to as "target nucleic acids" or "target sequence" if targeted for sequencing. Nucleic acids can be mixtures or pools of molecules targeted for sequencing.

[0024] A "polynucleotide sequence" or "nucleotide sequence" is a polymer of nucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.

[0025] A "polypeptide" is a polymer comprising two or more amino acid residues (e.g., a peptide or a protein). The polymer can additionally comprise non-amino acid elements such as labels, quenchers, blocking groups, or the like and can optionally comprise modifications such as glycosylation or the like. The amino acid residues of the polypeptide can be natural or non-natural and can be unsubstituted, unmodified, substituted or modified.

[0026] An "amino acid sequence" is a polymer of amino acid residues (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context.

[0027] Numbering of a given amino acid or nucleotide polymer "corresponds to numbering of or is "relative to" a selected amino acid polymer or nucleic acid when the position of any given polymer component (amino acid residue, incorporated nucleotide, etc.) is designated by reference to the same residue position in the selected amino acid or nucleotide polymer, rather than by the actual position of the component in the given polymer. Similarly, identification of a given position within a given amino acid or nucleotide polymer is "relative to" a selected amino acid or nucleotide polymer when the position of any given polymer component (amino acid residue, incorporated nucleotide, etc.) is designated by reference to the residue name and position in the selected amino acid or nucleotide polymer, rather than by the actual name and position of the component in the given polymer. Correspondence of positions is typically determined by aligning the relevant amino acid or polynucleotide sequences.

[0028] The term "recombinant" indicates that the material (e.g., a nucleic acid or a protein) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is, e.g., a polypeptide or protein which is produced by expression of a recombinant nucleic acid.

[0029] A “DPO4-type DNA polymerase" is a DNA polymerase naturally expressed by the archaea, Sulfolobus solfataricus, or a related Y-family DNA polymerase, which generally function in the replication of damaged DNA by a process known as translesion synthesis (TLS). Y-family DNA polymerases are homologous to the DPO4 polymerase (e.g., as listed in SEQ ID NO: 1); examples include the prokaryotic enzymes, PolII, PolIV, PolV, the archaeal enzyme, Dbh, and the eukaryotic enzymes, Rev3p, Revlp, Pol q, REV3, REV1, Pol J, and Pol K DNA polymerases, as well as chimeras thereof. A modified recombinant DPO4-type DNA polymerase includes one or more mutations relative to naturally-occurring wild-type DPO4-type DNA polymerases, for example, one or more mutations that increase the ability to utilize bulky nucleotide analogs as substrates or another polymerase property, and may include additional alterations or modifications over the wild-type DPO4-type DNA polymerase, suchas one or more deletions, insertions, and / or fusions of additional peptide or protein sequences (e.g., for immobilizing the polymerase on a surface or otherwise tagging the polymerase enzyme).

[0030] "Template-directed synthesis", "template-directed assembly", "template-directed hybridization", "template-directed binding" and any other template-directed processes, e.g., primer extension, refers to a process whereby nucleotide residues or nucleotide analogs bind selectively to a complementary target nucleic acid, and are incorporated into a nascent daughter strand. A daughter strand produced by a template-directed synthesis is complementary to the single-stranded target from which it is synthesized. It should be noted that the corresponding sequence of a target strand can be inferred from the sequence of its daughter strand, if that is known. "Template-directed polymerization" is a special case of template-directed synthesis whereby the resulting daughter strand is polymerized.

[0031] "XNTP" is an expandable, 5' triphosphate modified non-natural nucleotide analog compatible with template dependent enzymatic polymerization. An XNTP has two distinct functional components; namely, a selectively cleavable phophoramidate bonds linking the alpha phosphate and the nucleoside and a symmetrically synthesized reporter tether (SSRT) that is attached within each XNTP at positions that allow for controlled expansion cleavage of the phosphoramidate bond. In certain embodiments, a first end of the tether is attached to the alpha phosphate via a first linker and a second end of the tether is attached to the nucleobase via a second linker.

[0032] "Xpandomer intermediate" is an intermediate product (also referred to herein as a "daughter strand") assembled from XNTPs and is formed by a template-directed assembly of XNTPs using a target nucleic acid template. The Xpandomer intermediate contains two structures: namely, the constrained Xpandomer and the primary backbone. The constrained Xpandomer comprises all the tethers in the daughter strand but may comprise all, a portion or none of the nucleobase 5 ’-triphosphates as required by the method. The primary backbone comprises all the abutted nucleobase 5 ’-triphosphates. Under the process step in which the primary backbone is fragmented or dissociated, the constrained Xpandomer is no longer constrained and is the Xpandomer product which is extended as the tethers are stretched out. "Duplex daughter strand" refers to an Xpandomer intermediate that is hybridized or duplexed to the target template.

[0033] "Xpandomer" or "Xpandomer product" is a synthetic molecular construct produced by expansion of a constrained Xpandomer, which is itself synthesized by template-directed assembly of XNTPs. The Xpandomer is elongated relative to the target template it was produced from. It is composed of a concatenation of XNTPs, each XNTP including a tether comprising one or more reporters encoding sequence information. The Xpandomer is designed to expand to be longer than the target template thereby lowering the linear density of the sequence information of the target template along its length. In addition, the Xpandomer optionally provides a platform for increasing the size and abundance of reporters which in turn improves signal to noise for detection. Lower linear information density and stronger signals increase the resolution and reduce sensitivity requirements to detect and decode the sequence of the template strand.

[0034] "Tether" or "tether member" refers to a polymer or molecular construct having a generally linear dimension and with an end moiety at each of two opposing ends. A tether is attached to a nucleobase 5'-triphosphate with a linkage in at least one end moiety to form an XNTP. The end moieties of the tether may be connected to cleavable linkages to the nucleobase 5 '-triphosphate that serve to constrain the tether in a "constrained configuration". After the daughter strand is synthesized, each end moiety has an end linkage that couples directly or indirectly to other tethers. The coupled tethers comprise the constrained Xpandomer that further comprises the daughter strand. Tethers have a "constrained configuration" and an "expanded configuration". The constrained configuration is found in XNTPs and in the daughter strand. The constrained configuration of the tether is the precursor to the expanded configuration, as found in Xpandomer products. The transition from the constrained configuration to the expanded configuration results cleaving of selectively cleavable bonds that may be within the primary backbone of the daughter strand or intra-tether linkages. A tether in a constrained configuration is also used where a tether is added to form the daughter strand after assembly of the "primary backbone". Tethers can optionally comprise one or more reporters or reporter constructs along its length that can encode sequence information of substrates. The tether provides a means to expand the length of the Xpandomer and thereby lower the sequence information linear density.

[0035] A variety of additional terms are defined or otherwise characterized herein.DETAILED DESCRIPTION

[0036] One aspect of the invention is generally directed to compositions comprising a recombinant polymerase, e.g., a recombinant DPO4-type DNA polymerase that includes one or more mutations as compared to a reference polymerase, e.g., a wildtype DPO4-type polymerase. Depending on the particular mutation or combination of mutations, the polymerase exhibits one or more properties that find use, e.g., in single molecule sequencing applications. Exemplary properties exhibited by various polymerases of the invention include the ability to incorporate “bulky” nucleotide analogs into a growing daughter strand during DNA replication with improved efficiency and accuracy relative to known polymerases. The polymerases can include one or more exogenous or heterologous features at the N- and / or C-terminal regions of the protein for use, e.g., in the purification of the recombinant polymerase. The polymerases can also include one or more deletions that facilitate purification of the protein, e.g., by increasing the solubility of recombinantly produced protein.

[0037] These new polymerases are particularly well suited to DNA replication and / or sequencing applications, particularly sequencing protocols that synthesis of an Xpandomer copy of a duplex template construct for the duplex Sequencing by Expansion (SBX) protocol, as further described below.

[0038] Polymerases of the invention include, for example, a recombinant DPO4-type DNA polymerase that has at least one mutation at an amino acid selected from the group consisting 8, 32, 37, 39, 48, 49, 53, 59, 74, 179, and 272, in which identification of positions is relative to wild-type DPO4 polymerase (SEQ ID NO: 1). The polymerase may also have at least one mutation at an amino acid position selected from the group consisting of 57, 152, 153, 155, 157, 158, 161, 192, 249, 250, 289, 290, 291, 292, 293, 294, 296, 300, 324, 325, and 327. The polymerase may also have at least one mutation at an amino acid position selected from the group consisting of 56, 63, 76, 78, 79 82, 83, 86, 156, 184, 187, 188, 189, 248, 295, 297, 299, 301, 321. The polymerase may comprise mutations at 16 or more, up to 20 or more, up to 30 or more, or up to 40 or more of these positions. The polymerases of the invention may also possess a deletion of amino acids 341-352 of the wildtype protein, corresponding to the “PIP box”. In some embodiments, the polymerases of the invention may include mutations at additional residues not cited herein, provided that such mutations provide functional advantages as discussed further herein. In some embodiments, the polymerases of the invention may include one or more internal amino acid deletions. In certain embodiments thepolymerases of the invention are at least 80% identical to SEQ ID NO: 1 (amino acids 1-340 of wildtype DPO4 DNA polymerase. In other embodiments, the polymerases of the invention may be less than 80% identical to SEQ ID NO: 1, provided that such polymerases demonstrate enhanced abilities to utilize XNTPs as polymerization substrates. In some embodiments, the polymerases of the invention are at least around 85%, at least around 86%, at least around 87%, at least around 88%, at least around 89%, or at least around 90% identical to SEQ ID NO: 1. A number of exemplary substitutions at these (and other) positions are described herein.DNA Polymerases

[0039] DNA polymerases that can be modified to increase the ability to incorporate bulky nucleotide analog substrates into a growing daughter nucleic acid strand and / or other desirable properties, such as increased thermostability and solubility, as described herein are generally available. DNA polymerases are sometimes classified into six main groups, or families, based upon various phylogenetic relationships, e.g., with E. coli Pol I (class A), E. coli Pol II (class B), E. coli Pol III (class C), Euryarchaeotic Pol II (class D), human Pol beta (class X), and E. coli UmuC / DinB and eukaryotic RAD30 / xeroderma pigmentosum variant (class Y). For a review of recent nomenclature, see, e.g., Burgers et al. (2001) "Eukaryotic DNA polymerases: proposal for a revised nomenclature" J Biol. Chem. 276(47):43487-90. For a review of polymerases, see, e.g., Hubscher et al. (2002) "Eukaryotic DNA Polymerases" Annual Review of Biochemistry Vol. 71: 133-163; Alba (2001) "Protein Family Review: Replicative DNA Polymerases" Genome Biology 2(1): reviews 3002.1-3002.4; and Steitz (1999) "DNA polymerases: structural diversity and common mechanisms" J Biol Chem 274: 17395-17398. DNA polymerase have been extensively studied and the basic mechanisms of action for many have been determined. In addition, the sequences of literally hundreds of polymerases are publicly available, and the crystal structures for many of these have been determined or can be inferred based upon similarity to solved crystal structures for homologous polymerases. For example, the crystal structure of DPO4, a preferred type of parental enzyme to be modified according to the present invention, is available see, e.g., Ling et al. (2001) “Crystal Structure of a Y-Family DNA Polymerase in Action: A Mechanism for Error-Prone and Lesion-Bypass Replication” Cell 107:91-102, wherein is herein incorporated by reference in its entirety.

[0040] DNA polymerases that are preferred substrates for mutation to increase the use of bulky nucleotide analog as substrates for incorporation into growing nucleic acid daughter strands, and / or to alter one or more other property described herein include DPO4 polymerases and other members of the Y family of translesional DNA polymerases, such as Dbh, and derivatives of such polymerases.

[0041] In one aspect, the polymerase that is modified is a DPO4-type DNA polymerase. For example, the modified recombinant DNA polymerase can be homologous to a wildtype DPO4 DNA polymerase. Alternately, the modified recombinant DNA polymerase can be homologous to other Class Y DNA polymerases, also known as “translesion” DNA polymerases, such as Sulfolohus acidocaldcirius Dbh polymerase. For a review, see Goodwin and Woodgate (2013) “Translesion DNA Polymerases” Cold Spring Harb Perspect in Biol doi: 10.1101 / cshperspect.a010363. See, e.g., SEQ ID NO:1 for the amino acid sequence of wildtype DPO4 polymerase.

[0042] In other aspects, the polymerase that is modified is a DNA Pol Kappa-type polymerase, a DNA Pol Eta-type polymerase, a PrimPol-type polymerase, or a Therminator Gamma-type polymerase derived from any suitable species, such as yeast, human, .S'. islandicus, or T. thermophilus. The polymerase that is modified may be full length or truncated versions that include or lack various features of the protein. Certain desired features of these polymerases may also be combined with any of the DPO4 variants disclosed herein.

[0043] Many polymerases that are suitable for modification, e.g., for use in sequencing technologies, are commercially available. For example, DPO4 polymerase is available from TREVEGAN® and New England Biolabs®.

[0044] In addition to wildtype polymerases, chimeric polymerases made from a mosaic of different sources can be used. For example, DPO4-type polymerases made by taking sequences from more than one parental polymerase into account can be used as a starting point for mutation to produce the polymerases of the invention. Chimeras can be produced, e.g., using consideration of similarity regions between the polymerases to define consensus sequences that are used in the chimera, or using gene shuffling technologies in which multiple DPO4-related polymerases are randomly or semi-randomly shuffled via available gene shuffling techniques (e.g., via "family gene shuffling"; see Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391:288-291; Clackson et al. (1991) "Making antibody fragments using phage displaylibraries" Nature 352:624-628; Gibbs et al. (2001) "Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling" Gene 271:13-20; and Hiraga and Arnold (2003) "General method for sequence -independent site-directed chimeragenesis: J. Mol. Biol. 330:287-296). In these methods, the recombination points can be predetermined such that the gene fragments assemble in the correct order. However, the combinations, e.g., chimeras, can be formed at random. Appropriate mutations to improve incorporation of bulky nucleotide analog substrates or another desirable property can be introduced into the chimeras.Nucleotide Analogs

[0045] As discussed, various polymerases of the invention can incorporate one or more nucleotide analogs into a growing daughter strand chain. Upon incorporation, the analog can leave a residue that is the same as or different than a natural nucleotide in the growing daughter strand (the polymerase can incorporate any non-standard moiety of the analog, or can cleave it off during incorporation into the oligonucleotide). A "nucleotide analog" herein is a compound, that, in a particular application, functions in a manner similar or analogous to a naturally occurring nucleoside triphosphate (a "nucleotide"), and does not otherwise denote any particular structure. A nucleotide analog is an analog other than a standard naturally occurring nucleotide, i.e., other than A, G, C, T, or U, though upon incorporation into the oligonucleotide, the resulting residue in the oligonucleotide can be the same as (or different from) an A, G, C, T, or U residue.

[0046] Many nucleotide analogs are available and can be incorporated by the polymerases of the invention. These include analog structures with core similarity to naturally occurring nucleotides, such as those that comprise one or more substituent on a phosphate, sugar, or base moiety of the nucleoside or nucleotide relative to a naturally occurring nucleoside or nucleotide.

[0047] In one useful aspect of the invention, various tethers, linkers, or other substituents can be incorporated into analogs to create a “bulky” nucleotide analog, wherein the term “bulky” is understood to mean that the size of the analog is substantially larger than a natural nucleotide, while not denoting any particular dimension. For example, the analog caninclude a substituted compound (i.e., a “XNTP”, as disclosed in U.S. Patent No. 7,939,259 and PCT Publication No. WO 2016 / 081871 to Kokoris et al.) of the formula:

[0048] As shown in the above formula, the monomeric XNTP construct has a nucleobase residue, N, that has two moieties separated by a selectively cleavable bond (V), each moiety attaching to one end of a tether (T). The tether ends can attach to the linker group modifications on the heterocycle, the ribose group, or the phosphate backbone. The monomer substrate also has an intra-substrate cleavage site positioned within the phosphororibosyl backbone such that cleavage will provide expansion of the constrained tether. For example, to synthesize a XATP monomer, the amino linker on 8-[(6-Amino)hexyl]-amino-ATP or N6-(6-Amino)hexyl-ATP can be used as a first tether attachment point, and, a mixed backbone linker, such as the non-bridging modification (N-l -aminoalkyl) phosphoramidate or (2 -aminoethyl) phosphonate, can be used as a second tether attachment point. Further, a bridging backbone modification such as a phosphoramidate (3' O— P— N 5') or a phosphorothiolate (3' O— P— S 5'), for example, can be used for selective chemical cleavage of the primary backbone. R1and R2are end groups configured as appropriate for the synthesis protocol in which the substrate construct is used. For example, R1=5 '-triphosphate and R2=3'-OH for a polymerase protocol. The R15' triphosphate may include mixed backbone modifications, such as an aminoethyl phosphonate or 3'-O— P— S-5' phosphorothiolate, to enable tether linkage and backbone cleavage, respectively. Optionally, R2can be configured with a reversible blocking group for cyclical single-substrate addition. Alternatively, R1and R2can be configured with linker end groups for chemical coupling. R1and R2can be of the general type XR, wherein X is a linking group and R is a functional group. Detailed atomic structures of suitable substrates for polymerase variants of the present invention may be found, e.g., in Vaghefi, M. (2005) “Nucleoside Triphosphates and their Analogs” CRC Press Taylor & Francis Group.Applications for Enhanced Ability to Accurately Incorporate Bulky Nucleotide Analog Substrates

[0049] Polymerases of the invention, e.g., modified recombinant polymerases, or variants, may be used in combination with nucleotides and / or nucleotide analogs and nucleic acid templates (DNA or RNA) to copy the template nucleic acid. That is, a mixture of the polymerase, nucleotides / analogs, and optionally other appropriate reagents, the template and a replication initiating moiety (e.g., primer) is reacted such that the polymerase synthesizes a daughter strand (e.g., extends the primer) in a template-dependent manner. The replication initiating moiety can be a standard oligonucleotide primer, or, alternatively, a component of the template, e.g., the template can be a self-priming single stranded DNA, a nicked double stranded DNA, or the like. Similarly, a terminal protein can serve as an initiating moiety. At least one nucleotide analog can be incorporated into the DNA. The template DNA can be a linear or circular DNA, and in certain applications, is desirably a circular template (e.g., for rolling circle replication or for sequencing of circular templates). Optionally, the composition can be present in an automated DNA replication and / or sequencing system.

[0050] In one embodiment, the daughter strand is an Xpandomer intermediate comprised ofXNTPs, as disclosed in U.S. Patent No. 7,939,259, and PCT Publication No. WO 2016 / 081871, which are herein incorporated by reference in their entirety. Stratos Genomics has developed a method called Sequencing by Expansion (“SBX”) that uses a DNA polymerase to transcribe the sequence of DNA onto a measurable polymer called an “Xpandomer”. In general terms, an Xpandomer encodes (parses) the nucleotide sequence data of the target nucleic acid in a linearly expanded format, thereby improving spatial resolution, optionally with amplification of signal strength. The transcribed sequence is encoded along the Xpandomer backbone in high signal -to-noise reporters that are separated by ~I0 nm and are designed for high-signal-to-noise, well-differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA. Xpandomers can enable several next generation DNA sequencing technologies and are well suited to nanopore sequencing. As discussed above, one method of Xpandomer synthesis uses XNTPs as nucleic acid analogs to extend the templatedependent synthesis and uses a DNA polymerase variant as a catalyst.

[0051] Recently, the inventors have developed next-generation SBX chemistry that enables, amongst other things, improved control of the translocation rate of the Xpandomer molecule as it passes through a nanopore sensor. Exemplary means of improved translocation control are disclosed, e.g., in Applicant’s published PCT publication no. W02020 / 0236526, entitled “Translocation Control Elements, Reporter Codes, and further Means for TranslocationControl for use in Nanopore Sequencing”, which is herein incorporation by reference in its entirety.

[0052] In certain embodiments, improved translocation control is enabled by new features incorporated into the SSRT polymeric structure of the XNTP. One example of a nextgeneration XNTP is depicted in generalized form in FIG. 2. Here, XNTP 200 includes nucleoside triphosphoramidate 210 with linker arm moieties 220A and 220B separated by selectively cleavable phosphoramidate bond 230. The nucleoside triphosphoramidate is capable of being recognized as a substrate by the polymerase. Symmetrically synthesized reporter tether (SSRT) 240 is joined to the nucleoside triphosphoramidate at the ends of the linger arm moieties, such that a first SSRT end is linked to the heterocycle and a second SSRT end is linked to the alpha phosphate of the nucleobase backbone

[0053] In this embodiment, SSRT 240 includes several functional elements, or “features” such as polymerase enhancement regions 250A and 250B, reporter codes 260A and 260B, and translation control element (TCE) 270. Each of these features performs a unique function during translocation of the Xpandomer through a nanopore to produce a series of unique and reproducible electronic signal. SSRT 240 is designed for modulating Xpandomer movement through the nanopore by the TCE based on a combination of sferics and / or electrorepulsion. Four specific reporter codes are provided that each pair with the corresponding nucleobase. Different reporter codes are sized to block ion flow through a nanopore at different measureable levels. Specific SSRT polymeric sequences can be efficiently synthesized using phosphoramidite chemistry typically used for oligonucleotide synthesis. Reporter codes and other features can be designed by selecting a sequence of specific phosphoramidites from commercially available and / or proprietary libraries. Such libraries include, but are not limited to, polyethylene glycol with lengths of 1 to 12 or more ethylene glycol units and aliphatic polymers with lengths of 1 to 12 or more carbon units. In this embodiment, the “polymerase enhancement regions” at the ends of the SSRTs proximal to the nucleotide triphosphoramidate diester may include positively charged polyamine spacers (e.g., primary, secondary, tertiary, or quarternary amines) or triamine spacers (three secondary amines each separated by three carbons) that facilitate incorporation of XNTP structures by the DNA polymerase variant, e.g., a variant of DPO4. In certain embodiments, the polymerase enhancement region includes two repeat units of spermine.

[0054] In certain embodiments, a non-limiting Xpandomer synthesis reaction mixture may include the following reagents: a buffer / salt system, polymerase cofactors, polymeraseenhancing moieties (PEMs), a variant of DPO4 DNA polymerase, XNTP substrates, a phosphate shield molecule, a solvent, a crowding agent, and optionally, additional additives.

[0055] In some embodiments, an Xpandomer synthesis reaction may be run at from around 35 to around 45 degrees Celsius. In some embodiments, an Xpandomer synthesis reaction may be run at from around 37 to around 42 degrees Celsius.

[0056] The product of the Xpandomer synthesis reaction is an Xpandomer in the “constrained” configuration, e.g., the selectively cleavable backbone bonds are intact. In one embodiment, the product of the Xpandomer synthesis reaction may be treated with acid to cleave the selectively cleavable bonds to transition the Xpandomer molecule into the expanded, or processed configuration compatible with nanopore translocation.

[0057] As the final processed Xpandomer product of XNTP polymerization translocates through a nanopore sensor, a reporter enters the stem until its associated translocation control element arrests at the stem entrance. The reporter is held in the stem until the TCE is enabled to pass into and through the stem, whereupon translocation proceeds to the next reporter. Advantageously, the inventors have discovered that TCEs constructed from a novel class of pendant-PEG phorphoramidites provide significantly improved translocation control based on their intrinsic physicochemical and steric properties and thus obviate reliance on association and dissociation of translocation control moieties that act in trans.Compositions Comprising DPO4 Polymerase Variants

[0058] In some embodiments, the disclosure relates to an aqueous composition for Xpandomer synthesis, the composition comprising a variant of DPO4 polymerase, as described herein, a manganese or magnesium salt, XNTP substrates, and D2O.

[0059] In some embodiments, the Dpo4 polymerase variant is selected from the group consisting of SEQ ID NOs: 2-59.

[0060] In some embodiments, a variant of Dpo4 polymerase suitable for the practice of the present invention may be a variant that has at least 85% or at least 95%, such as at least 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NO:2-59. Such a polymerase is capable of using XNTPs as polymerization substrate.

[0061] In some embodiments, the composition comprises at least 30%, such as at least 40% D2O or at least 50% D2O. In some embodiments, the composition comprises 99% or lessD2O, such as 95% or less D2O, 90% or less D2O, 80% or less D2O, 70% or less D2O or 60% or less D2O. For example, the composition may comprise between 40% and 99% D2O, between 40% and 95% D2O, between 40% and 90% D2O, between 40% and 80% D2O, between 40% and 75% D2O, between 40% and 60% D2O. For example, the composition may comprise between 50% and 99% D2O, such as between 50% and 95% D2O, between 50% and 90% D2O, between 50% and 80% D2O, between 50% and 75% D2O, between 50% and 60% D2O, or between 70% and 75% D2O.

[0062] In some embodiments, the composition has a total salt concentration of 100-400 mM, such as 100-300 mM. In some embodiments, the composition has a total inorganic salt concentration of 150 mM or less, such as 100-150 mM.

[0063] In some embodiments, the aqueous composition comprises a manganese salt. In some embodiments, the manganese salt is MnCh.

[0064] In some embodiments, the polymerase is present at a concentration of 0.02-0.2 Rg / pl

[0065] In some embodiments, the XNTPs are present at a concentration of 20-150 pM.

[0066] In some embodiments, the aqueous composition further comprises additional components, and in particular components that are suitable for replicating a nucleic acid template. For example, the composition may comprise one or more component selected from a buffering agent, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, polyethylenglycol (PEG), dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, urea, and a polymerase enhancing molecule (PEM).

[0067] In some embodiments, the buffering agent is selected from the group consisting of TrisCi, TrisOAc, NH4OAc, MES, and HEPES.

[0068] In some embodiments, the salt is salt is selected from the group consisting of NaCl, NaBr, NaOAc, NaF, sodium formate, sodium phosphate monobasic, sodium phosphate dibasic, NaSO4. sodium carbonate, sodium bicarbonate, sodium hexanoate, sodium glutamate, sodium perchlorate, CsCl, LiCl, LiOAc, LiF, lithium carbonate, LiPCh. KC1, KOAc, KF, KSO4, potassium phosphate monobasic, potassium phosphate dibasic, potassium carbonate, potassium bicarbonate, potassium glutamate, NH4CI, NH4F, NFUOAc, NH4SO4, NH4Br. ammonium citrate, ammonium carbonate, ammonium bicarbonate, ammonium sulfite, ammonium glutamate, ammonium phosphate monobasic, tetramethylammonium chloride (TMAC1), trimethylamine N-oxide (TMAO), tetraethylammonium chloride (TEACI),guanidinium chloride, guanidinium thiocyanate, guadinium carbonate. In some embodiments, the salt is an inorganic salt. In some embodiments, the salt is NaCl or KC1, such as NaCl.

[0069] In some embodiments, the sugar is maltose, trehalose, cellobiose or sucrose.

[0070] In some embodiments, the SSB is selected from the group consisting of KOD, Gp32, TTH, SSB1, RecA, RPA, andNCp7.

[0071] In some embodiments, the imidazole includes (apart from imidazole as such) imidazole derivatives, such as imidazole chloride, imidazole acetate, 1 -methylimidazole, 2-methylimidazole, I -ethylimidazole, l-ethyl-3-methylimidazolium chloride, 2-methyl-2-imidazoline, l-butyl-3-methylimidazolium chloride, 1-methylimidazolium chloride, 1 -hexyl -3-methylimidazolium, 3 -octyl- 1-methylimidazolium, or 1 -decyl-3 -methylimidazolium.

[0072] In some embodiments, the alkanediol is ethylenglycol, a propanediol, a butanediol, a pentanediol, or a hexanediol.

[0073] In some embodiments, the propanediol is 1,2-propanediol or 1,3-propanediol, such as 1,2 propanediol. In some embodiments, the butanediol is 1,2 -butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 2,4-butanediol, or 3,4-butanediol, such as 1,2-butanediol. In some embodiments, the pentanediol is 1,5 -pentanediol, 1,2-pentanediol, 2,4-pentanediol, or 1,3 -pentanediol, such as 1,5 -pentanediol. In some embodiments, the hexanediol is 1,6-hexanediol or 2,5 -hexanediol, such as 1,6-hexanediol.

[0074] In some embodiments, PEG is PEG5k to PEG25k, such as PEG5k, PEG8k, PEG 10k, PEG 15k or PEG20k.

[0075] In some embodiments, the polyphosphate is tripolyphosphate, tetrapolyphosphate, pentapolyphosphate, hexapolyphosphate, trimetaphosphate (TMP), hexametaphosphate (HMP), or polyphosphate 60.

[0076] In some embodiments, the PEM compound is selected from the group consisting of PEM 1-15, the structures of which are given in Table 1:Table 1

[0077] Suitable PEMs for the practice of the present invention are disclosure further in Applicant’s published PCT applications, WO2019 / 135975, W02020 / 263703, and WO2025 / 137293, the contents of which are herein incorporated by reference in their entirety.

[0078] In some embodiments, the composition may be used as a reaction solution comprising a nucleic acid template. Thus, in some embodiments, the aqueous composition further comprises a nucleic acid template. In some embodiments, the nucleic acid template is a duplex template construct, i.e., a duplex nucleic acid comprising two complementary strands that are joined on one end to a hairpin adapter.

[0079] Regardless of whether the composition is to be used as premix or reaction solution, the composition may also comprise primers for replicating the nucleic acid template. In some embodiments, the composition comprises primers for replicating the nucleic acid template. In some embodiments, the primers are bound to a solid support.

[0080] Examples of suitable concentrations for the further components, when present, are as follows:20-100 mM buffering agent,100-200 mM salt,0.05-0.7 % (w / v) sugar,0.1-50 mM PEM,0.05-0.5 mM polyphosphate,140-300 mM imidazole,50-200 mM pyrazole,50-200 mM triazole,2-8% (w / v) alkanediol,2-10% (w / v) acetamide,0.1-1% (w / v) glycerol200-400 mM betaine,10-30% (w / v) PEG,3-10 % (v / v) NMP,0.5-1.5 mM BHA,1-5 % (v / v) DMSO,0.5-2 mM urea,0.02-1 pg / pl SSB.

[0081] Suitable compositions for Xpandomer synthesis are further disclosed in Applicant’s published PCT application no. W02026 / 050121, the contents of which are herein disclosed by reference in their entirety.Mutating Polymerases

[0082] Various types of mutagenesis are optionally used in the present invention, e.g., to modify polymerases to produce variants, e.g., in accordance with polymerase models and model predictions as discussed above, or using random or semi -random mutational approaches. In general, any available mutagenesis procedure can be used for making polymerase mutants. Such mutagenesis procedures optionally include selection of mutant nucleic acids andpolypeptides for one or more activity of interest (e.g., the ability to incorporate bulky nucleotide analogs into a daughter nucleic acid strand). Procedures that can be used include, but are not limited to: site-directed point mutagenesis, random point mutagenesis, in vitro or in vivo homologous recombination (DNA shuffling and combinatorial overlap PCR), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restrictionpurification, deletion mutagenesis, mutagenesis by total gene synthesis, degenerate PCR, double-strand break repair, and many others known to persons of skill. The starting polymerase for mutation can be any of those noted herein, including wildtype DPO4 polymerase.

[0083] Optionally, mutagenesis can be guided by known information (e.g., “rational” or “semi-rational” design) from a naturally occurring polymerase molecule, or of a known altered or mutated polymerase (e.g., using an existing mutant polymerase as noted in the preceding references), e.g., sequence, sequence comparisons, physical properties, crystal structure and / or the like as discussed above. However, in another class of embodiments, modification can be essentially random (e.g., as in classical or "family" DNA shuffling, see, e.g., Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391:288-291.

[0084] Additional information on mutation formats is found in: Sambrook et al., Molecular Cloning— A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook"); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2011) ("Ausubel")) and PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) ("Innis"). The following publications and references cited within provide additional detail on mutation formats: Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology 4:450-455 (1993); Bass et al., Mutant Trp repressors with new DNA-binding specificities, Science 242:240-245 (1988); Bordo and Argos (1991) Suggestions for "Safe" Residue Substitutions in Site-directed Mutagenesis 217:721-729; Botstein & Shortle, Strategies and applications of in vitro mutagenesis, Science 229: 1193-1201 (1985); Carter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Site-directed mutagenesis, Biochem. J. 237:1-7 (1986); Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymol. 154: 382-403 (1987); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol. Biol. 57:369-374 (1996); Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115 (1986); Fritz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl. Acids Res. 16: 6987-6999 (1988); Grundstrom et al., Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Hayes (2002) Combining Computational and Experimental Screening for rapid Optimization of Protein Properties PNAS 99(25) 15926-15931; Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag, Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol. 154, 367-382 (1987); Kramer et al., The gapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucl. Acids Res. 12: 9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367 (1987); Kramer et al., Point Mismatch Repair, Cell 38:879-887 (1984); Kramer et al., Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucl. Acids Res. 16: 7207 (1988); Ling et al., Approaches to DNA mutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997); Lorimer and Pastan Nucleic Acids Res. 23, 3067-8 (1995); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: amethod for site-specific mutagenesis, Proc. Natl. Acad. Sci. USA, 83:7177-7181(1986); Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698 (1986); Nambiar et al., Total synthesis and cloning of a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Sayers et al., Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidiumbromide, (1988) Nucl. Acids Res. 16: 803-814; Sieber, et al., Nature Biotechnology, 19:456-460 (2001); Smith, In vitro mutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Stemmer, Nature 370, 389-91(1994); Taylor et al., The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., The rapid generation of oligonucleotide -directed mutations at high frequency using phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787 (1985); Wells et al., Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Wells et al., Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34:315-323 (1985); Zoller & Smith, Oligonucleotide-directed mutagenesis using M 13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment, Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol. 154:329-350 (1987); Clackson et al. (1991) "Making antibody fragments using phage display libraries" Nature 352:624-628; Gibbs et al. (2001) "Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling" Gene 271:13-20; and Hiraga and Arnold (2003) "General method for sequence-independent site-directed chimeragenesis: J. Mol. Biol.330:287-296. Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.Screening Polymerases

[0085] Screening or other protocols can be used to determine whether a polymerase displays a modified activity, e.g., for a nucleotide analog, as compared to a parental DNA polymerase. For example, the ability to bind and incorporate bulky nucleotide analogs into a daughter strand during template-dependent DNA synthesis. Assays for such properties, and the like, are described herein. Performance of a recombinant polymerase in a primer extension reaction can be examined to assay properties such as nucleotide analog incorporations etc., as described herein.

[0086] In one desirable aspect, a library of recombinant DNA polymerases can be made and screened for these properties. For example, a plurality of members of the library can be made to include one or more mutation that alters incorporations and / or randomly generated mutations (e.g., where different members include different mutations or different combinations of mutations), and the library can then be screened for the properties of interest (e.g., incorporations, etc.). In general, the library can be screened to identify at least one member comprising a modified activity of interest.

[0087] Libraries of polymerases can be either physical or logical in nature. Moreover, any of a wide variety of library formats can be used. For example, polymerases can be fixed to solid surfaces in arrays of proteins. Similarly, liquid phase arrays of polymerases (e.g., in microwell plates) can be constructed for convenient high-throughput fluid manipulations of solutions comprising polymerases. Liquid, emulsion, or gel-phase libraries of cells that express recombinant polymerases can also be constructed, e.g., in microwell plates, or on agar plates. Phage display libraries of polymerases or polymerase domains (e.g., including the active site region or interdomain stability regions) can be produced. Likewise, yeast display libraries can be used. Instructions in making and using libraries can be found, e.g., in Sambrook, Ausubel and Berger, referenced herein.

[0088] For the generation of libraries involving fluid transfer to or from microtiter plates, a fluid handling station is optionally used. Several "off the shelf fluid handling stations for performing such transfers are commercially available, including e.g., the Zymate systems from Caliper Life Sciences (Hopkinton, Mass.) and other stations which utilize automatic pipettors, e.g., in conjunction with the robotics for plate movement (e.g., the ORCA® robot, which is used in a variety of laboratory systems available, e.g., from Beckman Coulter, Inc. (Fullerton, Calif.).

[0089] In an alternate embodiment, fluid handling is performed in microchips, e.g., involving transfer of materials from microwell plates or other wells through microchannels on the chips to destination sites (microchannel regions, wells, chambers or the like). Commercially available microfluidic systems include those from Hewlett-Packard / Agilent Technologies (e.g., the HP2100 bioanalyzer) and the Caliper High Throughput Screening System. The Caliper High Throughput Screening System provides one example interface between standard microwell library formats and Labchip technologies. RainDance Technologies' nanodroplet platform provides another method for handling large numbers ofspatially separated reactions. Furthermore, the patent and technical literature includes many examples of microfluidic systems which can interface directly with microwell plates for fluid handling.Tags and Other Optional Polymerase Features

[0090] The recombinant DNA polymerase optionally includes additional features exogenous or heterologous to the polymerase. For example, the recombinant polymerase optionally includes one or more tags, e.g., purification, substrate binding, or other tags, such as a polyhistidine tag, a HislO tag, a His6 tag, an alanine tag, an Alal6 tag, an Alal6 tag, a biotin tag, a biotin ligase recognition sequence or other biotin attachment site (e.g., a BiTag or a Btag or variant thereof, e.g., BtagVl-11), a GST tag, an S Tag, a SNAP-tag, an HA tag, a DSB (Sso7D) tag, a lysine tag, a NanoTag, a Cmyc tag, a tag or linker comprising the amino acids glycine and serine, a tag or linker comprising the amino acids glycine, serine, alanine and histidine, a tag or linker comprising the amino acids glycine, arginine, lysine, glutamine and proline, a plurality of polyhistidine tags, a plurality of HislO tags, a plurality of His6 tags, a plurality of alanine tags, a plurality of AlalO tags, a plurality of Alai 6 tags, a plurality of biotin tags, a plurality of GST tags, a plurality of BiTags, a plurality of S Tags, a plurality of SNAP- tags, a plurality of HA tags, a plurality of DSB (Sso7D) tags, a plurality of lysine tags, a plurality of NanoTags, a plurality of Cmyc tags, a plurality of tags or linkers comprising the amino acids glycine and serine, a plurality of tags or linkers comprising the amino acids glycine, serine, alanine and histidine, a plurality of tags or linkers comprising the amino acids glycine, arginine, lysine, glutamine and proline, biotin, avidin, an antibody or antibody domain, antibody fragment, antigen, receptor, receptor domain, receptor fragment, or ligand, one or more protease site (e.g., Factor Xa, enterokinase, or thrombin site), a dye, an acceptor, a quencher, a DNA binding domain (e.g., a helix-hairpin-helix domain from topoisomerase V), or combination thereof. The one or more exogenous or heterologous features at the N- and / or C-terminal regions of the polymerase can find use not only for purification purposes, immobilization of the polymerase to a substrate, and the like, but can also be useful for altering one or more properties of the polymerase.

[0091] One or more exogenous or heterologous features can be included internal to the polymerase, at the N-terminal region of the polymerase, at the C-terminal region of the polymerase, or both the N-terminal and C-terminal regions of the polymerase. Where thepolymerase includes an exogenous or heterologous feature at both the N-terminal and C-terminal regions, the exogenous or heterologous features can be the same (e.g., a polyhistidine tag, e.g., a HislO tag, at both the N- and C-terminal regions) or different (e.g., a biotin ligase recognition sequence at the N-terminal region and a polyhistidine tag, e.g., HislO tag, at the C-terminal region). Optionally, a terminal region (e.g., the N- or C-terminal region) of a polymerase of the invention can comprise two or more exogenous or heterologous features which can be the same or different (e.g., a biotin ligase recognition sequence and a polyhistidine tag at the N-terminal region, a biotin ligase recognition sequence, a polyhistidine tag, and a Factor Xa recognition site at the N-terminal region, and the like). As a few examples, the polymerase can include a polyhistidine tag at the C-terminal region, a biotin ligase recognition sequence and a polyhistidine tag at the N-terminal region, a biotin ligase recognition sequence and a polyhistidine tag at the N-terminal region and a polyhistidine tag at the C-terminal region, or a polyhistidine tag and a biotin ligase recognition sequence at the C-terminal region.Making and Isolating Recombinant Polymerases

[0092] Generally, nucleic acids encoding a polymerase of the invention can be made by cloning, recombination, in vitro synthesis, in vitro amplification and / or other available methods. A variety of recombinant methods can be used for expressing an expression vector that encodes a polymerase of the invention. Methods for making recombinant nucleic acids, expression and isolation of expressed products are well known and described in the art. A number of exemplary mutations and combinations of mutations, as well as strategies for design of desirable mutations, are described herein. Methods for making and selecting mutations in the active site of polymerases, including for modifying steric features in or near the active site to permit improved access by nucleotide analogs are found hereinabove and, e.g., in PCT Publication Nos. WO 2007 / 076057 and WO 2008 / 051530.

[0093] Additional useful references for mutation, recombinant and in vitro nucleic acid manipulation methods (including cloning, expression, PCR, and the like) include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Kaufinan et al. (2003) Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edition Ceske (ed) CRC Press (Kaufinan); and The Nucleic Acid Protocols Handbook Ralph Rapley (ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley); Chen et al. (ed) PCR Cloning Protocols, SecondEdition (Methods in Molecular Biology, volume 192) Humana Press; and in Viljoen et al. (2005)Molecular Diagnostic PCR Handbook Springer, ISBN 1402034032.

[0094] In addition, a plethora of kits are commercially available for the purification of plasmids or other relevant nucleic acids from cells, (see, e.g., EasyPrep™ FlexiPrep™ both from Pharmacia Biotech; StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). Any isolated and / or purified nucleic acid can be further manipulated to produce other nucleic acids, used to transfect cells, incorporated into related vectors to infect organisms for expression, and / or the like. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both.

[0095] Other useful references, e.g. for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and the references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

[0096] Nucleic acids encoding the recombinant polymerases of the invention are also a feature of the invention. A particular amino acid can be encoded by multiple codons, and certain translation systems (e.g., prokaryotic or eukaryotic cells) often exhibit codon bias, e.g., different organisms often prefer one of the several synonymous codons that encode the same amino acid. As such, nucleic acids of the invention are optionally "codon optimized," meaning that the nucleic acids are synthesized to include codons that are preferred by the particular translation system being employed to express the polymerase. For example, when it is desirable to express the polymerase in a bacterial cell (or even a particular strain of bacteria), the nucleic acid can be synthesized to include codons most frequently found in the genome of that bacterial cell, for efficient expression of the polymerase. A similar strategy can beemployed when it is desirable to express the polymerase in a eukaryotic cell, e.g., the nucleic acid can include codons preferred by that eukaryotic cell.

[0097] A variety of protein isolation and detection methods are known and can be used to isolate polymerases, e.g., from recombinant cultures of cells expressing the recombinant polymerases of the invention. A variety of protein isolation and detection methods are well known in the art, including, e.g., those set forth in R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2. sup .nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and the references cited therein. Additional details regarding protein purification and detection methods can be found in Satinder Ahuja ed., Handbook of Bioseparations, Academic Press (2000).Nucleic Acid and Polypeptide Sequences and Variants

[0098] As described herein, the invention also features polynucleotide sequences encoding, e.g., a polymerase as described herein. Examples of polymerase sequences that include features found herein, e.g., as in Table 2 are provided. However, one of skill in the art will immediately appreciate that the invention is not limited to the specifically exemplified sequences. For example, one of skill will appreciate that the invention also provides, e.g., many related sequences with the functions described herein, e.g., polynucleotides and polypeptides encoding conservative variants of a polymerase of Table 2 and or any other specifically listed polymerase herein. Combinations of any of the mutations noted herein are also features of the invention.

[0099] Accordingly, the invention provides a variety of polypeptides (polymerases) and polynucleotides (nucleic acids that encode polymerases). Exemplary polynucleotides of the invention include, e.g., any polynucleotide that encodes a polymerase of Table 2 orotherwise described herein. Because of the degeneracy of the genetic code, many polynucleotides equivalently encode a given polymerase sequence. Similarly, an artificial or recombinant nucleic acid that hybridizes to a polynucleotide indicated above under highly stringent conditions over substantially the entire length of the nucleic acid (and is other than a naturally occurring polynucleotide) is a polynucleotide of the invention. In one embodiment, a composition includes a polypeptide of the invention and an excipient (e.g., buffer, water, pharmaceutically acceptable excipient, etc.). The invention also provides an antibody or antisera specifically immunoreactive with a polypeptide of the invention (e.g., that specifically recognizes a feature of the polymerase that confers decreased branching or increased complex stability.

[0100] In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, a virus, etc.) comprises a polynucleotide of the invention. In one embodiment, the vector is an expression vector. In another embodiment, the expression vector includes a promoter operably linked to one or more of the polynucleotides of the invention. In another embodiment, a cell comprises a vector that includes a polynucleotide of the invention.

[0101] One of skill will also appreciate that many variants of the disclosed sequences are included in the invention. For example, conservative variations of the disclosed sequences that yield a functionally similar sequence are included in the invention. Variants of the nucleic acid polynucleotide sequences, wherein the variants hybridize to at least one disclosed sequence, are considered to be included in the invention. Unique subsequences of the sequences disclosed herein, as determined by, e.g., standard sequence comparison techniques, are also included in the invention.Conservative Variations

[0102] Owing to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence that encodes an amino acid sequence. Similarly, "conservative amino acid substitutions," where one or a limited number of amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the present invention.

[0103] "Conservative variations" of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. One of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are "conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid, while retaining the relevant mutational feature (for example, the conservative substitution can be of a residue distal to the active site region, or distal to an interdomain stability region). Thus, "conservative variations" of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence, with an amino acid of the same conservative substitution group. Finally, the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or tagging sequence (introns in the nucleic acid, poly His or similar sequences in the encoded polypeptide, etc.), is a conservative variation of the basic nucleic acid or polypeptide.

[0104] Conservative substitution tables providing functionally similar amino acids are well known in the art, where one amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., aromatic side chains or positively charged side chains), and therefore does not substantially change the functional properties of the polypeptide molecule. The following sets forth example groups that contain natural amino acids of like chemical properties, where substitutions within a group is a "conservative substitution" . Table 2Conservative Amino Acid SubstitutionsNucleic Acid Hybridization

[0105] Comparative hybridization can be used to identify nucleic acids of the invention, including conservative variations of nucleic acids of the invention. In addition, target nucleic acids which hybridize to a nucleic acid of the invention under high, ultra-high and ultra-ultra high stringency conditions, where the nucleic acids encode mutants corresponding to those noted in Table 2 or other listed polymerases, are a feature of the invention. Examples of such nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence encoding a polymerase of Table 2 (or other exemplified polymerase), where any conservative substitutions are for residues other than those noted in Table 2 or elsewhere as being relevant to a feature of interest (improved nucleotide analog incorporations, etc.).

[0106] A test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least 50% as well to the probe as to the perfectly matched complementary target, 1.e., with a signal to noise ratio at least half as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids.

[0107] Nucleic acids "hybridize" when they associate, typically in solution. Nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes part I chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (Elsevier, N.Y.), as well as in Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2011); Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England (Hamesand Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.

[0108] An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C with the hybridization being carried out overnight. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, supra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40°C for 15 minutes. In general, a signal to noise ratio of 5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

[0109] "Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and / or increasing the concentration of organic solvents such as formalin in the hybridization or wash), until a selected set of criteria are met. For example, in highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased until a probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least 5x as high as that observed for hybridization of the probe to an unmatched target

[0110] "Very stringent" conditions are selected to be equal to the thermal melting point (Tm) for a particular probe. The Tmis the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. For the purposes of the present invention, generally, "highly stringent" hybridization and wash conditions are selected to be about 5°C lower than the Tmfor the specific sequence at a defined ionic strength and pH.

[0111] "Ultra high-stringency" hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratiofor binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx as high as that observed for hybridization to any of the unmatched target nucleic acids. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least 1 / 2 that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.

[0112] Similarly, even higher levels of stringency can be determined by gradually increasing the hybridization and / or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx, 20x, 50x, lOOx, or 500x or more as high as that observed for hybridization to any of the unmatched target nucleic acids. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least 1 / 2 that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-ultra-high stringency conditions.

[0113] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.Sequence Comparison. Identity, and Homology

[0114] The terms "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.

[0115] The phrase "substantially identical," in the context of two nucleic acids or polypeptides (e.g., DNAs encoding a polymerase, or the amino acid sequence of a polymerase) refers to two or more sequences or subsequences that have at least about 60%, about 70%, about 75%, about 80-85%, about 86-87%, about 88%, about 89%, about 90-95%, about 98%, about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visualinspection. Such "substantially identical" sequences are typically considered to be "homologous," without reference to actual ancestry. Preferably, the "substantial identity" exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared.

[0116] Proteins and / or protein sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and / or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity over 50, 100, 150 or more residues is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more identity, can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.

[0117] For sequence comparison and homology determination, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0118] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Current Protocols inMolecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented through 2011).

[0119] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0120] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0121] For reference, the amino acid sequence of a wild-type DPO4 polymerase is presented in Table 2.Exemplary Mutation Combinations

[0122] A list of exemplary polymerase mutation combinations and the amino acid sequences of recombinant DPO4 polymerases harboring the exemplary mutation combinations are provided in Table 3. Positions of amino acid substitutions are identified relative to a wildtype DPO4 DNA polymerase (SEQ ID NO:1). Polymerases of the invention (including those provided in Table 3) can include any exogenous or heterologous feature (or combination of such features) at the N- and / or C-terminal region. For example, it will be understood that polymerase mutants in Table 3 that do not include, e.g., a N-terminal polyhistidine tag can be modified to include a polyhistidine tag at the N-terminal region, alone or in combination with any of the exogenous or heterologous features described herein. The variants set forth herein include a deletion of the last 12 amino acids of the protein (i.e., amino acids 341-352) so as to, e.g., increase protein solubility in bacterial expression systems.Table 3DPO4 Variants Identified through Rational DesignKits

[0123] In another aspect, kits comprising the reagents necessary for performing the methods described herein are provided. In certain embodiments, the kits may include a composition for performing an Xpandomer synthesis reaction comprising a DPO4 polymerase variant, as described herein.

[0124] In some instances, the kit may include one or more reagents, e.g., buffers and / orreaction components for performing an Xpandomer synthesis reaction. For example, the kit may include a DPO4 polymerase variant, as described herein, and one or more of XNTPs, a buffering agent, a salt, a sugar, a PEM, a polyphosphate, imidazole, a pyrazole, a triazole, an alkanediol, acetamide, glycerol, betaine, a PEG, NMP, BHA, DMSO, urea, an SSB and D2O.

[0125] In some instances, the reagents for performing the Xpandomer synthesis reaction are provided in a single tube. In some embodiments, the single tube is suitable for storage at around -20 degrees Celsius. In other embodiments, the single tube is suitable for storage at around -80 degrees Celsius.

[0126] In other instances, the reagents for performing an Xpandomer synthesis reaction are provided in more than one tube. In some embodiments the kits may include a first tube and a second tube that each comprise one or more reagents selected from the group consisting of a DPO4 polymerase variant, as disclosed herein, XNTPs, a buffering agent, a salt, a sugar, a PEM, a polyphosphate, imidazole, a pyrazole, a triazole, an alkanediol, acetamide, glycerol, betaine, a PEG, NMP, BHA, DMSO, urea, an SSB and D2O, in which the reagents included in the first tube are different than the reagents included in the second tube. In certain embodiments, the first and second tubes may be stored at the same or different temperatures. In certain embodiments, the reagents in the first and second tubes may be in liquid form, frozen form, or lyophilized form.

[0127] In some instances, the kit may include a first tube comprising XNTPs and a solvent and a second tube comprising additional reagents necessary for performing the Xpandomer synthesis reaction. In one embodiment, the first tube may be stored as a frozen solid at -20 degrees Celsius.

[0128] In other instances, the kit may include a first tube comprising XNTPs, a solvent, a DPO4 polymerase variant, and a SSB and a second tubes comprising additional reagents necessary for performing the Xpandomer synthesis reaction.

[0129] In some instances, the kit may further include instmctions. The instructions may specify how to perform the Xpandomer synthesis step.

[0130] The Examples and polymerase variants provided below further illustrate and exemplify the compositions of the present invention and methods of preparing and using such compositions. It is to be understood that the scope of the present invention is not limited in any way by the scope of the following Examples.EXAMPLESEXAMPLE 1

[0131] IDENTIFICATION OF DPO4 AS A CANDIDATE TRANSLESION DNA POLYMERASE FOR INCORPORATION OF BULKY NUCLEOTIDE ANALOGS DURING TEMPLATE-MEDIATED DNA SYNTHESIS

[0132] To identify a DNA polymerase with the ability to synthesize daughter strands using “bulky” substrates (i.e., able to bind and incorporate heavily substituted nucleotide analogs into a growing nucleic acid strand), a screen was conducted of several commercially available polymerases. Candidate polymerases were assessed for the ability to extend an oligonucleotide -bound primer using a pool of dNTP analogs substituted with alkyne linkers on both the backbone a-phosphate and the nucleobase moieties (i.e., model bulky substrates, referred to herein generally as, “dNTP -2c”). Polymerases screened for activity included the following: VentR (Exo-), Deep VentR® (Exo-), Therminator, Therminator II, Therminator III, Therminator Y, 9°Nm, PWO, PWO SuperYield, PyroPhage 3173 (Exo-), Bst, Large Fragment, Exo- Pfu, Platinum Genotype TSP, Hemo Klen Taq, Taq, MasterAMP Taq, Phi29, Bsu, Large Fragment, Exo-Minus Klenow (D355A, E357A), Sequenase Version 2.0, Transcriptor, Maxima, Thermoscript, M-MuLV (RNase H-), AMV, M-MuLV, Monsterscript, and DPO4. Of the polymerases tested, DPO4 (naturally expressed by the archaea, Sulfolobus solfataricus) was most able to effectively extend a template-bound primer with dNTP-2c nucleotide analogs. Without being bound by theory, it was speculated that DPO4, and possibly other members of the translesion DNA polymerase family (i.e., class Y DNA polymerases), may be able to effectively utilize bulky nucleotide analogs as substrates owing to their relatively large substrate binding sites, which have evolved to accommodate naturally occurring, bulky DNA lesions.EXAMPLE 2

[0133] IDENTIFICATION OF “HOT SPOTS” FOR DIRECTED MUTAGENESIS IN THE DPO4 PROTEIN AND SCREEN OF DPO4 MUTANT LIBRARIES TO IDENTIFY OPTIMIZED SEQUENCE MOTIFS

[0134] As an initial step in generating DPO4 variants with improved polymerase activity in the presence of non-natural substrates, the “HotSpot Wizard” web tool was used to identify amino acids in the DPO4 protein to target for mutagenesis. This tool implements a protein engineering protocol that targets evolutionarily variable amino acid positions located in, e.g., the enzyme active site. “Hot spots” for mutation are selected through the integration of structural, functional, and evolutionary information (see, e.g., Pavelka et al., ‘HotSpot Wizard: a Web Server for Identification of Hot Spots in Protein Engineering” (2009) Nuc Acids Res 37 doi: 10.1093 / nar / gkp410). Applying this tool to the DPO4 protein, it was observed that hot spot residues identified tended to cluster into certain zones, or regions, spread throughout the full amino acid sequence. Arbitrary boundaries were set to distinguish 15 such regions, designated “Mutl” - “Mutl5”, in which mutagenesis hot spots are concentrated. These 15 “Muf ’ regions are illustrated in FIG. 1 with hot spot residues identified by underscoring.

[0135] To screen for DPO4 variants with improved polymerase activity based on hot spot mapping, saturation mutagenesis libraries were created for the Mut regions, in which hot spot amino acids were changed, while conserved amino acids were left unaltered. Screening was conducted using a 96-well plate platform, and polymerase activity was assessed with a primer extension assay using “dNTP-OAc” nucleotide analogs as substrates. These model bulky substrates are substituted with triazole acetate moieties conjugated to alkyne substituents on both the a-phosphate and the nucleobase moieties. Screening results identified two Mut regions in particular that consistently produced DPO4 mutants with enhanced activity. These regions, “Mut4” and “Mutll”, correspond to amino acids 76-86 and amino acids 289-304, respectively, of the DPO4 protein.EXAMPLE 3

[0136] DEVELOPMENT OF A HIGH THROUGHPUT MULTIPLEX SCREENING ASSAY (“MUX”) FOR NEW SBX® POLYMERASES

[0137] Errors in SBX® sequence data can result from errors in the biochemical reactions producing the Xpandomer product and / or from errors in measurement as the Xpandomer translocates through the nanopore sensor. Notably, development of nextgeneration SBX® chemistry, including the improved XNTP translocation control elements, discussed with reference to FIG. 2, has led to significant improvements in measurement accuracy. Advantageously, this has enabled the inventors to use Xpandomer sequencing data as a metric to assess the accuracy, and other properties, of the SBX® polymerase. These advances have led to the implementation of high throughput, sequencing-based, screening assays to identify SBX® polymerases with improved properties in a highly efficient and effective manner.

[0138] One example of a high throughput multiplex screening assay is referred to herein as the “MUX” assay. The MUX assay utilizes a 96 well format to multiplex Xpandomer synthesis. In this format, individual SBX® reactions each extend a uniquely barcoded template present in the well. Following Xpandomer synthesis, individual reactions are pooled for Xpandomer processing and sequence determination. After nanopore-based sequence determination, the data can be “de-MUXed” based on barcode information to evaluate the performance of individual polymerase clones. Advantageously, the performance of 96 unique polymerase clones can be assessed in a single sequencing run, thereby reducing resource consumption and sequencing costs. In addition, the MUX assay offers numerous other advantages, including, but not limited to, reduction in workflow complexity (i.e., fewer steps in the workflow). For example, the MUX workflow introduces direct lysis of recombinant protein-expressing bacterial cells in culture without the need to, e.g., first pellet and resuspend the cells, and direct addition of Ni-NTA resin to bacterial lysates for recombinant protein purification without the need to, e.g., first perform nucleic acid removal or other clean-up steps.

[0139] The first stage of an exemplary MUX assay is a high throughput polymerase expression and purification workflow, which is largely automated and overcomes the need to produce polymerase protein samples at scale. This workflow may include the following steps: 1) providing a library of polymerase-encoding plasmids; 2) culturing bacteria transformed with the plasmid library in 96-well culture blocks (e.g., providing a 2ml deep well block in which each well is inoculated with an individual transformed bacterial colony, growing and inducing the cultures to express recombinant polymerase protein; 3) lysing the bacterial cells directly in the culture media (e.g., by adding an aliquot of lysis buffer including 36 mM tris-OAc, 90 mMNH40Ac, 3.6M Urea, 0.9% TWEEN-20, pH 8.3, 0.5mM PMSF, 2U / ml nuclease, and ReadyLys solution with a multichannel pipet, mixing and incubating the samples at 43 degrees C. for 20 minutes); and 4) purifying the recombinant polymerase protein using conventional IMAC -based purification (e.g., by adding an aliquot of Ni-NTA resin directly to each well of the block and mixing for 15 minutes, placing a 96 well PET filter plate on a flowthrough collection block and transferring lysate / resin slurry to the filter plate to selectively retain the polymerase-bound resin, placing the filter plate on a wash collection block and washing the resin with a wash buffer containing 20 mM tris-OAc, 50 mM imidazole, 0.01% triton X-100, 1000 mM NaCl, pH 7.5, placing the filter plate on an elution collection plate and adding elution buffer containing 20 mM tris base, 500 mM imidazole, 20% glycerol, 500 mM NaCl, pH 8.5 to collect eluted polymerase protein).

[0140] The second stage of the MUX assay is the Xpandomer synthesis reaction (i.e., SBX®) workflow, which may include the following steps: 1) providing a MUX synthesis reagent kit including XNTP substrates, PEMs, salts, solvent, MnCU and polyphosphate; 2) providing a 96 well plate including a unique dried lOOmer single stranded DNA template / extension-oligo complex in each well and filling each well with an aliquot of the MUX synthesis reagents; 3) adding a sample of a purified polymerase protein to each well and incubating the sample for 2 hr at 37 degrees C.; 4) terminating the Xpandomer synthesis reactions by quenching (e.g., by adding a sample of 50mM EDTA, 300mM Na2HPO4 pH 8, 1% Tween, and 0.5% SDS) and pooling the 96 individual reactions into a single pooled sample. The Xpandomer synthesis reaction is described in further detail in Applicant’s US provisional patent application no. 63 / 687,453, filed August 27, 2024, entitled, “Compositions for Replicating a Nucleic Acid Template,” which is herein incorporated by reference in its entirety for all purposes

[0141] The third stage of the MUX assay is the Xpandomer processing and precipitation workflow, which may include the following steps: 1) modification of the Xpandomers (with, e.g., a solution of 2M succinic anhydride); 2) cleavage of the Xpandomers (with, e.g., a solution of 35% DC1); 3) neutralization of the sample (with, e.g., a solution including lOmM imidazole and lOOmM HEPES pH 7.4); 4) ethanol precipitation of the Xpandomers (by adding, e.g., a solution of 5.91% Ficoll and 3.82M NaCl followed by 100% ethanol and a 90% ethanol wash); and 5) resuspending the purified Xpandomers in a buffer that is compatible with a nanopore sequencing system (e.g., a solution including 40% ACN,5% trehalose, and 0.1M imidazole that is compatible with the HTP nanopore sequencing system).

[0142] The fourth stage of the MUX assay is Xpandomer sequence determination and de-MUXing of the sequence data. Xpandomers may be sequencing using the Roche HTP High Throughput Nanopore Sequencing Platform, as described, e.g., in Applicant’s Published PCT Application No. PCT / EP2019 / 084581, which is herein incorporated by reference in its entirety. Secondary analysis of the sequence data is performed using suitable data analysis software.EXAMPLE 4

[0143] DESIGN OF NEXT-GENERATION SBX POLYMERASES

[0144] The inventors have utilized a variety of protein engineering approaches to transform the wildtype DPO4 polymerase into an Xpandomer synthetase. The development of high throughput screening assays, such as those described in Example 3, has greatly facilitated identification of DPO4 variants with improved properties for SBX®, e.g., one or more of improved accuracy, thermostability or solubility. This example describes a protein engineering approach based on specific mutagenic targeting of functional domains of interest in the DPO4 protein.

[0145] As is known in the art, the crystal structure of DPO4 in ternary complex with DNA and an incoming nucleotide has been solved (see, e.g., Ling et al. “Crystal Structure of a Y-Family DNA Polymerase in Action: A Method for Error-Prone and Lesion-Bypass Replication”. 2001. Cell, Vol. 107, 91-112). These structures indicate that DPO4 adopts a similar overall shape to other polymerase proteins, which has been likened to a right hand composed of palm, finger, and thumb domains. The palm domain (amino acids 1-10 and 78-166) forms the catalytic center of the polymerase near the interface with the finger domain. The finger domain (amin acids 11-77) tightly envelopes the Watson-Crock base pair formed between a template base and the incoming nucleotide and plays a critical role in the induced-fit mechanism to discriminate against mismatches. The little finger domain, unique to DPO4, at the C terminus of the protein (amino acids 244-341) is physically located next to the finger domain and interacts with DNA in the major groove. The thumb domain (amino acids 167-233) and little finger domain grip the DNA template and primer across the minor groove from underneath and the major groove from above. The template base and incoming nucleotide are surrounded by the palm and finger domains.

[0146] Based on such observations, e.g., that the finger domain surrounds the incoming nucleotide, the palm domain forms the active site of the polymerase, and the thumb and little finger domain form the “clamp” that contacts the backbone of the DNA primer and template, the inventors speculated that mutations in these regions may further evolve DPO4 and known variants into an Xpandomer synthetase. Accordingly, saturation mutagenesis libraries were designed to target residues in these regions of the protein. Resulting variants were screened, e.g., using the high throughput MUX assay to assess the accuracy of Xpanodmer synthesis.

[0147] After screening tens of thousands of variants, several new amino acid positions and new mutations at previously identified positions were identified that further optimize DPO4 for Xpandomer synthesis. A non-limiting list of exemplary amino acid positions and mutations identified is set forth in Table 4. Exemplary combinations of specific amino acid substitutions is set forth in Table 3.Table 4Exemplary Amino Acid positions and MutationsEXAMPLE 5

[0148] Optimization of Next-Generation SBX Polymerases for Duplex Sequencing by Expansion

[0149] Recently, the SBX technology has advanced to enable sequencing of libraries of duplex template constructs. Duplex template constructs include a double stranded nucleic acid library fragment joined on one end to a Y adapter and on the other end to a hairpin adapter, which covalently joins the two strands of the library fragment. Advantageously, duplex sequencing provides sequence information from both the sense and antisense strands of the library fragment in a single sequencing read. Duplex sequencing and duplex template construct libraries are described further in Applicant’s published PCT application no. WO2025 / 132779, the contents of which are herein incorporated by reference in their entirety.

[0150] The use of duplex constructs as templates for the Xpandomer synthesis reaction has led to changes to prior reaction chemistries (e.g., those used for replicating a non-duplexed template). An exemplary Xpandomer synthesis reaction for replicating a duplexed template may include the following reagents: 50mM TrisCi, pH 8.0; 125mM NaCl; 250mM imidazole; 5% pentanediol; 12.5mM first PEM; 2.5mM second PEM; 0.35mM MnCh; 0.05pg / pL SSB; 0. ImM XNTP mix; 1% glycerol in 74% D2O. Typically, around 0.07pg / pL polymerase protein is added to the reaction. A 70pL reaction may be run at 37 degrees C. for 120 minutes and terminated by incubation at 60 degrees C. for five minutes. Such changes to the SBX template and reaction chemistry have required further optimization of the SBX polymerase.

[0151] The ability to analyze SBX polymerase performance on the Roche HTP high throughput nanopore sequencing platform has enabled evaluation of the impact of changes in the polymerase amino acid sequence on individual sequencing metrics, such as accuracy, length, coverage depth and uniformity, Q score, GC content, and the like. This level of analysis offers considerable advantages over prior methods of analyzing polymerase performance, e.g., those based on gel analysis of the products of the Xpandomer synthesis reaction.

[0152] One approach towards designing DPO4 polymerase variants optimized for duplex library sequencing has involved a deeper interrogation of certain “mut regions”, which are illustrated in FIG. 1. These are regions that are predicted to tolerate multiple mutations andare notably not obvious from examination of the crystal structure of the polymerase. For example, sub-regions of mut 6 (amino acids 152-158) and mut 11 (amino acids 289-301) were further interrogated with the objective of identifying optimized combinations of 2-3 amino acid substitutions, in particular those at positions that may occupy the same 3-D space. This approach led to the identification of, e.g., positions 290 and 291 as a combination that has a positive impact on duplex sequencing when altered from the wildtype identities. Other optimized sequences of SBX polymerase are disclosed throughout the present disclosure, e.g., in SEQ ID NO:s 2-59.

[0153] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet, including but not limited to U.S. Patent No. 7,939,259 , PCT Publication No. WO 2016 / 081871, U.S. Provisional Patent Application No. 62 / 597,109 and U.S. Provisional Patent Application No. 62 / 656,696, are incorporated herein by reference, in their entirety. Such documents may be incorporated by reference for the purpose of describing and disclosing, for example, materials and methodologies described in the publications, which might be used in connection with the presently described invention.

Claims

CLAIMS1. An isolated recombinant DNA polymerase, which recombinant DNA polymerase comprises an amino acid sequence that is at least 80% identical to amino acids 1-340 of SEQ ID NO: 1, which recombinant DNA polymerase comprises a mutation at an amino acid position selected from the group consisting of 8, 32, 37, 39, 48, 49, 53, 59, 74, 179, and 272, wherein identification of positions is relative to wildtype DPO4 polymerase (SEQ ID NO:1), which recombinant DNA polymerase further includes a deletion to remove the terminal 12 amino acids (the PIP box region) of the SEQ ID NO: 1and which recombinant DNA polymerase exhibits polymerase activity.

2. The DNA polymerase of claim 1, wherein the DNA polymerase comprises an amino acid sequence that is at least 85% identical to amino acids 1-340 of SEQ ID NO: 1.

3. The DNA polymerase of claim 1 or 2, wherein the mutation at amino acid position 8 is F8Y, the mutation at amino acid position 32 is A32N, the mutation at amino acid position 37 is F37G, F37S, or F37T, the mutation at position 39 is D39L, D39N, or D39F, the mutation at amino acid position 48 is Y48H, the mutation at amino acid position 49 is E49Y, the mutation at amino acid position 53 is F53E, the mutation at position 59 is I59M, the mutation at amino acid position 74 is L74I, the mutation at position 179 is D179N, and the mutation at position 272 is S272C, or S272A.

4. The DNA polymerase of any one of claims 1-3, comprising mutations at amino acid positions 37, 39, 59, 179, and 272.

5. The DNA polymerase of claim 4, wherein the mutation at amino acid position 37 is F37T, the mutation at amino acid position 39 is D39L, the mutation at position 59 is I59M, the mutation at amino acid position 179 is D179N, and the mutation at amino acid position 272 is S272C.

6. The DNA polymerase of any one of claims 1 -5 , further comprising at least one mutation at an amino acid position selected from the group consisting of 57, 152, 153, 155, 157, 158, 161, 192, 249, 250, 289, 290, 291, 292, 293, 294, 296, 300, 324, 325, and 327, wherein the mutation at position 57 is A57S, the mutation at position 152 is K152H, K152N, or K152W, the mutation at position 153 is I153N or I153L, the mutation at position 155 is A155S, the mutation at position 157 is M157W, M157H, or M157K, the mutation at position 158 is A158C, A158G, A158V, A158Q, or A158M, the mutation at position 161 is N161A, N161S, N161G, N161E, N161M, or N161Q, the mutation at position 192 is E192Q, the mutation atposition 249 is V249R, the mutation at position 250 is T250M, the mutation at position 290 is T290H, T290F, T290I, T290S, T290D, T290M, T290H, T290W, T290A, T290Q, T290V, or T290Y, the mutation at position 291 is E291G, E291S, E291T, E291N, E292H, E291D, or E291Q, the mutation at position 292 is D292R, the mutation at position 293 is L293H or L293W, the mutation at position 294 is D294Y or D294N, the mutation at position 296 is V296T or V296Q, the mutation at position 300 is R300K or R300K, and the mutation at position 327 is E327T, E327K, E327P or E327I.

7. The DNA polymerase of any one of claims 1-6, comprising mutations T290H and E291T or T290D and E291Q.

8. The DNA polymerase of any one of claims 1 -7, further comprising at least one mutation at an amino acid position selected from the group consisting of 56, 63, 76, 78, 79 82, 83, 86, 152, 153, 155, 156, 184, 187, 188, 189, 248, 289, 295, 297, 299, 301, 321, 324, and 325.

9. The DNA polymerase of claim 8, wherein the mutations at amino acid positions 56, 63, 76, 78, 79 82, 83, 86, 152, 153, 155, 156, 184, 187, 188, 189, 248, 289, 295, 297, 299, 301, 321, 324, and 325 are K56Y, E63R, M76W, K78E, E79P, Q82W, Q83G, S86E, K152A, I153V, A155G, D156S, P184Q, G187P, N188Y, I189F, I248T, V289W, I295S, S297Y, G299W, T301W, E324K, and E325K.

10. The DNA polymerase of any one of claims 1-9, comprising a deletion of at least one amino acid.

11. The DNA polymerase of claim 10, wherein the amino acid is selected from the group consisting of amino acid 291, amino acid 292, amino acid 293, and amino acid 294.

12. The DNA polymerase of claim 10 or 11, wherein the amino acid comprises two amino acids.

13. An isolated recombinant DNA polymerase comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 2 - 59.

14. A composition comprising a recombinant DNA polymerase as set forth in any one of claims 1-13.

15. The composition of claim 14, further comprising at least one non-natural nucleotide analog substrate.

16. The composition of claim 15, wherein the non-natural nucleotide analog substrate is an XNTP.

17. The composition of any of claims 14-16, further comprising a polymerase enhancing molecule (PEM) or a manganese salt.

18. The composition of any one of claims 14-17, further comprising one or more of a buffering agent, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, polyethylenglycol (PEG), dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and urea.

19. The composition of any one of claims 14-18, further comprising D2O.

20. The composition of claim 19, wherein the composition comprises between 70% w / v and 75% w / v D2O.

21. A kit for performing an Xpandomer synthesis reaction, wherein the kit comprises a DPO4 polymerase variant as set forth in any one of claims 1-13 and one or more reagents selected from a buffering agent, a salt, a sugar, a polymerase enhancing molecule (PEM), a polyphosphate, imidazole, a pyrazole, a triazole, an alkanediol, acetamide, glycerol, betaine, a polyethylenglycol (PEG), N-methyl-2 -pyrrolidone (NMP), butylated hydroxyanisole (BHA), dimethyl sulfoxide (DMSO), urea, a single stranded binding protein (SSB), XNTPs, and D2O.

22. The kit of claim 21 , wherein the DPO4 polymerase variant and the one or more reagents are provided in a single tube.

23. The kit of claim 21 , wherein the DPO4 polymerase variant and the one or more reagents are provided in more than one tube.

24. The kit of any one of claims 21-23, wherein the kit is suitable for storage at -20 degrees Celsius.

25. The kit of any one of claims 21-23, wherein the kit is suitable for storage at -80 degrees Celsius.

26. A modified nucleic acid encoding a modified DPO4-type DNA polymerase as set forth in any one of claims 1-13.

27. A host cell comprising the modified nucleic acid of claim 25.

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