Enhanced nucleic acid polymerization by the minor groove binding moiety

Abstract

The present invention relates to methods and compositions for improving nucleic acid polymerization, including DNA replication by in vitro primer extension to generate polymers for use, for example, in DNA template nanopore-based single-molecule sequencing. Nucleic acid polymerase reaction compositions containing polymerization enhancing moieties are provided, which allow for enhancement of DNA polymerase activity with nucleotide analogs, resulting in increased length of primer extension products for sequencing applications.

Description

[0001] Statement regarding sequence listing

[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 870225_422WO_SEQUENCE_LISTING.txt. The text file is 3.3KB, created on December 19, 2018, and submitted electronically via EFS-Web. Technical Field

[0003] The present invention generally relates to methods and compositions for polymerizing nucleic acids and / or affecting enzyme performance.

[0004] background

[0005] The measurement of biomolecules is the foundation of modern medicine and is widely used in medical research, more particularly in diagnostics and treatment and drug development. Nucleic acids encode information necessary for biological activity and reproduction, and are essentially the blueprint of life. Determining such blueprints can be used in pure research and applied science. In medicine, sequencing can be used to diagnose a variety of diseases and develop treatment methods for a variety of conditions (including cancer, heart disease, autoimmune disorders, multiple sclerosis and obesity). In industry, sequencing can be used to design improved enzymatic processes or synthetic organisms. In biology, this tool can be used, for example, to study the health of ecosystems, and therefore has a wide range of practicality. Similarly, the measurement of proteins and other biomolecules also provides markers and an understanding of disease and pathogen reproduction.

[0006] An individual's unique DNA sequence provides valuable information about their susceptibility to certain diseases. It also provides patients with the opportunity to be screened for early detection and / or receive preventive treatments. In addition, given a patient's personal blueprint, clinicians will be able to administer personalized therapies to maximize drug efficacy and / or minimize the risk of adverse drug reactions. Similarly, determining the blueprint of a disease-causing organism could yield new treatments for infectious diseases and more robust pathogen surveillance. Low-cost whole-genome DNA sequencing will lay the foundation for modern medicine. To achieve this goal, sequencing technology must continue to advance in terms of throughput, accuracy, and read length.

[0007] In the past decade, a large number of next-generation DNA sequencing technologies have become commercially available and have greatly reduced the cost of sequencing entire genomes. These include sequencing by synthesis ("SBS") platforms (Illumina, Inc., 454 Life Sciences, Ion Torrent, Pacific Biosciences) and similar ligand platforms (CompleteGenomics, Life Technologies Corporation). Many other technologies are being developed that utilize a variety of sample processing and detection methods. For example, GnuBio, Inc. (Cambridge, Mass.) uses picoliter reaction vessels to control millions of discrete probe sequencing reactions, while Halcyon Molecular (Redwood City, Calif.) attempts to develop technologies for direct DNA measurements using transmission electron microscopy.

[0008] Nanopore-based nucleic acid sequencing is a striking method that has been widely studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93: 13770-13773, 1996) characterized single-stranded polynucleotides because they were electrotransported by α-hemolysin nanopores embedded in lipid bilayers. It was demonstrated that during polynucleotide translocation, partial blockage of the nanopore aperture can be measured as a decrease in ionic current. However, the burden of polynucleotide sequencing in nanopores is that closely spaced bases (0.34 nm) must be resolved with small signal differences immersed in significant background noise. Due to the observation of rapid translocation rates of polynucleotides (usually about 1 base per microsecond), the measurement challenge of single-base resolution in nanopores has become more demanding. The translocation speed can be reduced by adjusting operating parameters (such as voltage, salt composition, pH, temperature and viscosity). However, such adjustments cannot reduce the translocation speed to a level that allows single-base resolution.

[0009] Stratos Genomics has developed a method called sequencing by amplification ("SBX") that uses a biochemical process to translate the sequence of DNA onto a measurable polymer called an "Xpandomer" (Kokoris et al., U.S. Patent No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion"). The transcribed sequence is encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are approximately 10 nm apart and designed for high signal-to-noise ratio, highly differentiated responses. These differences provide significant performance enhancements in the sequence read efficiency and accuracy of the Xpandomer relative to native DNA. Xpandomers can enable several next-generation DNA sequencing detection technologies and are well suited for nanopore sequencing.

[0010] Xpandomers are generated from non-natural nucleotide analogs (called XNTPs) (see Kokoris et al., U.S. Published Patent Application No. US20160145292A1, incorporated herein by reference in its entirety), characterized by lengthy substituents that enable the Xpandomer backbone to be amplified after synthesis. Due to their atypical structures, XNTPs and other nucleotide analogs (e.g., nucleotide analogs modified with detectable labeling moieties) introduce new challenges as substrates for currently available DNA polymerases. Kokoris et al. published PCT application WO2017 / 087281 (incorporated herein by reference in its entirety) describes engineered DP04 polymerase variants with enhanced primer extension activity using non-natural bulky nucleotide analogs as substrates.

[0011] In the DNA template itself, certain nucleotide sequence motifs are known to bring additional replication challenges to DNA polymerase. In particular, the result is several rounds of homopolymers or short repetitive DNA sequences, which can trigger slipped strand mismatches or "replication slips". Replication slips are believed to cover the following steps: (i) copying the first repetitive sequence by the replication mechanism, (ii) replication stagnation and polymerase dissociation from the newly synthesized end, (iii) making the newly synthesized chain unpaired and pairing it with the second repetitive sequence, and (iv) resuming DNA synthesis. The replication mechanism stagnates in the repeat region, thus causing mismatches of primers and templates. In vivo, mismatches of the two DNA chains during replication can lead to DNA rearrangements, such as deletions or duplications of various lengths. In vitro, replication slips lead to replication errors at the site of the slip event. Such reductions in polymerase persistence or accuracy significantly impair specific applications or desired genetic manipulations.

[0012] Therefore, new methods and compositions for enhancing polymerase reactions under conditions involving one or more reagents with atypical structures, such as in sequencing by amplification (SBX) and other applications within biotechnology and biomedicine (DNA amplification, conventional sequencing, labeling, detection, cloning, etc.) are necessary and would find value in the prior art. The present invention satisfies these needs and provides further related advantages.

[0013] All subject matter discussed in the background section is not necessarily prior art and should not be considered prior art simply because it is discussed in the background section. Along these lines, any problem discussed in the background section or in the prior art related to such subject matter should not be identified as prior art unless it is explicitly stated to be prior art. Rather, the discussion of any subject matter in the background section should be considered as part of the inventor's approach to solving a particular problem, which may itself be inventive.

[0014] Overview

[0015] In short, the present disclosure provides methods and compositions for enhancing nucleic acid polymerase activity. In certain embodiments, polymerase activity is enhanced in a polymerization reaction under conditions that introduce one or more challenges to the polymerase (e.g., conditions that include non-natural nucleotide analog substrates or template motifs that impair polymerase persistence). Such enhancement is achieved by supplementing the polymerization reaction with one or more additives from a class of compounds known in the art as "minor groove binders" (MGBs). Surprisingly and advantageously, the inventors of the present application have found that certain MGBs significantly enhance polymerase activity, particularly with non-natural, highly substituted nucleotide analog substrates.

[0016] In one aspect, the present invention provides a method for enhancing a nucleic acid polymerase reaction, the method comprising the following steps: forming a nucleic acid polymerase reaction composition comprising a template nucleic acid, a nucleic acid polymerase, a mixture of nucleotides or nucleotide analogs, and at least one minor groove binding portion; and incubating the nucleic acid polymerase reaction composition under conditions that allow nucleic acid polymerization, wherein at least one minor groove binding portion increases the persistence, rate or fidelity of the nucleic acid polymerase reaction. In one embodiment, compared to a nucleic acid polymerase reaction lacking a minor groove binding portion, at least one minor groove binding portion increases the length of the resulting nucleic acid product. In another embodiment, at least one minor groove binding portion is selected from the group consisting of: distamycin A and its synthetic analogs, sphenothrin, (+)-CC-1065, duocarmycin, pyrrolobenzodiazepines, trabectin and its analogs, Hoechst dyes and their derivatives, lexitropsin, trabectin and its analogs, Hoechst dyes and their derivatives, lexitropsin, thiazotropsin A, bisamidines and polyamides. In certain embodiments, at least one minor groove binding portion is a Hoechst dye. In other embodiments, at least one minor groove binding portion includes multiple minor groove binding portions. In some embodiments, multiple minor groove binding portions include minor groove binding portions of different structural categories. In other embodiments, nucleic acid polymerase is DNA polymerase. In certain embodiments, DNA polymerase is DPO4 or its variant. In other embodiments, the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside triphosphoramidates (nucleosidetriphosphoramidates) polymerized tether moieties, wherein each of the nucleoside triphosphoramidates nucleoside triphosphoramidates comprises a core base and a polymerized tether moiety, and the core base is selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein the first end of the polymerized tether moiety is connected to the core base and the second end of the polymerized tether moiety is connected to the alpha phosphate of the nucleoside triphosphoramidate to provide the amplification of the nucleotide analog by the cutting of the phosphoramidate bond. In some embodiments, nucleic acid polymerization produces an amplifiable polymer of nucleotide analogs, wherein the amplifiable polymer encodes the core base sequence information of the template nucleic acid. In other embodiments, the conditions for allowing nucleic acid polymerization reaction include suitable polymerization buffer and oligonucleotide primers. In other embodiments, suitable buffer comprises Tris OAc, NH 4 OAc, PEG, DMF, polyphosphate 60, and MnCl 2In other embodiments, the reaction mixture further comprises a nucleic acid intercalator. In other embodiments, the reaction mixture further comprises a polyanion recognition moiety. In other embodiments, the mixture of nucleotides or nucleotide analogs comprises a nucleotide analog comprising a detectable label. In other embodiments, the detectable label is an optically detectable label selected from the group consisting of a luminescent label, a chemiluminescent label, a fluorescent label, a fluorescent label, a chromophore label, or a chromogenic label.

[0017] On the other hand, the present invention provides a composition comprising at least one minor groove binding portion and a mixture of nucleotide analogs, wherein the at least one minor groove binding portion increases the number and accuracy of nucleotide analogs incorporated into daughter chains during template-dependent polymerization reactions relative to the same polymerization reaction lacking at least one minor groove binding portion. In one embodiment, at least one minor groove binding portion is selected from the group consisting of: distamycin A and its synthetic analogs, sphenococcin, (+)-CC-1065, duocarmycin, pyrrolobenzodiazepines, trabectin and its analogs, Hoechst dyes and their derivatives, lexitropsin, thiazotropsin A, bisamidines and polyamides. In certain embodiments, at least one minor groove binding portion is a Hoechst dye. In other embodiments, at least one minor groove binding portion includes multiple minor groove binding portions. In some embodiments, multiple minor groove binding portions include minor groove binding portions of different structural categories. In some embodiments, the mixture of nucleotide analogs comprises nucleoside amino triphosphates, wherein each of the nucleoside amino triphosphates comprises a nucleobase and a polymeric tethering portion selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a first end of the polymeric tethering portion is attached to the nucleobase and a second end of the polymeric ether portion is attached to the alpha phosphate of the nucleoside amino triphosphate to provide amplification of the nucleotide analogs by cleaving the phosphoramidite bond. In other embodiments, the composition further comprises a buffer comprising Tris OAc, NH 4 OAc, PEG, DMF, polyphosphate 60, and MnCl 2 In other embodiments, the composition further comprises a DNA intercalator. In other embodiments, the composition further comprises a polyanion recognition moiety. In certain embodiments, the mixture of nucleotide analogs comprises a nucleotide analog comprising a detectable label. In some embodiments, the detectable label is an optically detectable label selected from the group consisting of a luminescent label, a chemiluminescent label, a fluorescent label, a fluorescent label, a chromophore label, or a chromogenic label.

[0018] In another aspect, the present invention provides a method for sequencing a DNA template, the method comprising the steps of: forming a DNA polymerase reaction composition comprising a DNA template, a replication primer complexed with the template, a DNA polymerase, a mixture of nucleotides or nucleotide analogs, and at least one minor groove binding moiety, incubating the DNA polymerase reaction composition under conditions that allow DNA polymerization, wherein the at least one minor groove binding moiety increases the rate, fidelity, or processivity of the DNA polymerase reaction; and determining the sequence of the nucleotides or nucleotide analogs in the resulting polymer of nucleotides or nucleotide analogs. In some embodiments, the at least one minor groove binding moiety is selected from the group consisting of: distamycin A and its synthetic analogs, sphenopsin, (+)-CC-1065, duocarmycin, pyrrolobenzodiazepines, trabectin and its analogs, Hoechst dyes and their derivatives, lexitropsin, thiazotropsin A, bisamidines, and polyamides. In other embodiments, the mixture of nucleotide analogs comprises nucleoside amino triphosphates, wherein each of the nucleoside amino triphosphates comprises a core base and a polymeric tethering portion, the core base being selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein the first end of the polymeric tethering portion is connected to the core base and the second end of the polymeric ether portion is connected to the α phosphate of the nucleoside amino triphosphate to provide amplification of the nucleotide analog by cutting of the amino phosphoric acid ester bond. In other embodiments, the DNA polymerase is DPO4 or a variant thereof. In other embodiments, the polymer of the obtained nucleotide analog is an amplifiable polymer. In other embodiments, the method further comprises the steps of: contacting an amplifiable polymer with an amino phosphoric acid ester cutting agent to produce a polymer of the amplified nucleotide analog. In certain embodiments, the polymeric tethering portion of each of the nucleotide analogs comprises a reporter portion specific to the core base of the analog. In other embodiments, the reporter portion produces a characteristic electronic signal. In other embodiments, the step of determining the sequence of the nucleotide analog comprises the step of translocating the polymer of the amplified nucleotide analog through a nanopore. Sequence Listing <110> Stratos Genomics, Inc. <120> Enhanced nucleic acid polymerization by the minor groove binding moiety <130> 870225.422WO <140> PCT <141> 2018-12-20 <150> US 62 / 614,114 <151> 2018-01-05 <160> 1 <170> PatentIn Version 3.5 <210> 1 <211> 352 <212> PRT <213> Sulfolobus solfataricus <400> 1 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The exemplary features of the present disclosure, its nature and various advantages will be apparent from the accompanying drawings and the detailed description of various embodiments below. Non-limiting and non-exhaustive embodiments are described with reference to the accompanying drawings, wherein like labels or reference numerals refer to similar parts throughout the multiple views unless otherwise indicated. The sizes and relative positions of the elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements are selected, enlarged and positioned to improve the readability of the drawings. The specific shapes of the drawn elements have been selected to facilitate identification of the drawings.

[0021] Figure 1A , 1B , 1C, and 1D are abbreviated schematic diagrams illustrating the main features of universal XNTPs and their use in sequencing by amplification (SBX).

[0022] Figure 2 is a schematic diagram illustrating further details of one embodiment of XNTP.

[0023] Figure 3 is a schematic diagram illustrating one embodiment of an Xpandomer passing through a biological nanopore.

[0024] Figure 4 is a gel showing primer extension products.

[0025] Figure 5 is a gel showing primer extension products.

[0026] Figure 6 is a gel showing primer extension products.

[0027] Fig. 7A and 7B is a histogram display of the aligned read population of nanopore-derived sequences.

[0028] Details

[0029] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, those skilled in the art will appreciate that the present invention can be practiced without these details. In other cases, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, in all subsequent specifications and claims, the word "comprise" and its synonyms and variants (such as, "having", "including", "comprises" and "comprising") should be interpreted as open, inclusive meanings, i.e., "including but not limited to". The term "essentially consisting of..." limits the scope of the claim to specified materials or steps, or those that do not substantially affect the basic and novel features of the claimed invention. In addition, the titles provided herein are for convenience only and do not explain the scope or meaning of the claimed invention.

[0030] Throughout this specification, references to "one embodiment" or "an embodiment" and variations thereof mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the various appearances of the phrases "in one embodiment" or "in an embodiment" throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Likewise, 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.

[0031] It should also be noted that, unless the disclosure and context clearly indicate inclusivity or exclusivity (as the case may be), the conjunction terms "and" and "or" are generally used in the broadest sense to include "and / or". Therefore, the use of alternatives (such as "or") should be understood to mean any one, two, or any combination of the alternatives. In addition, the combination of "and" and "or", when referred to herein as "and / or", is intended to cover embodiments that include all related items or concepts, as well as one or more other alternative embodiments that include less than all related items or concepts.

[0032] Where a range of values ​​is provided herein, it is understood that each intervening value between the upper and lower limits of the range (to one tenth of the lower limit unit, unless otherwise clearly specified herein) and any other described or intervening values ​​within the described range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present invention, subject to any explicitly excluded limits in the specified range. When the described range includes one or both of the upper and lower limits, the range excluding one or both of those included limits is also included in the present invention.

[0033] For example, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range provided herein should be understood to include any integer value within the listed range, and when appropriate, include fractions thereof (such as tenths and hundredths of integers). In addition, unless otherwise indicated, any numerical ranges cited herein relating to any physical feature, such as polymer subunits, size, or thickness should be understood to include any integer within the listed range. As used herein, unless otherwise indicated, the term "about" means ±20% of a specified range, value, or structure.

[0034] It should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It should also be understood that unless specifically defined herein, the terms used herein should be given their conventional meanings known in the relevant art.

[0035] Any title used in this article is only used to speed up the reader's review and should not be interpreted in any way as a limitation of the invention or the claims. Therefore, the titles and abstracts of the disclosures provided herein are only for convenience and do not interpret the scope or meaning of the embodiments.

[0036] definition

[0037] As used herein, "nucleic acid", also referred to as polynucleotide, is a series of covalently linked nucleotides, in which the 3' position of the pentose of one nucleotide is linked to the 5' position of the next by a phosphodiester group. Nucleic acid molecules can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination of the two. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are biologically present polynucleotides in which the nucleotide residues are linked in a specific sequence by phosphodiester bonds. As used herein, the terms "nucleic acid", "polynucleotide" or "oligonucleotide" encompass any polymer compound with a linear backbone of nucleotides. Oligonucleotides, also referred to as oligomers, are typically polynucleotides of shorter chains. If nucleic acid is targeted for sequencing, the nucleic acid is typically referred to as a "target nucleic acid" or "target sequence".

[0038] As used herein, "polymerization" refers to an in vitro method for preparing a new nucleic acid chain or extending an existing nucleic acid (i.e., DNA or RNA) in a template-dependent manner. Polymerization according to the present invention includes primer extension, which uses a polymerase to replicate the sequence of a polynucleotide template sequence. Nucleic acid polymerization (e.g., primer extension) results in the incorporation of nucleotides or nucleotide analogs into polynucleotides (i.e., primers) to form new nucleic acid molecules complementary to the polynucleotide template. The nucleic acid molecules formed can be used for single molecule sequencing or as a template for synthesizing another nucleic acid molecule.

[0039] As used herein, the term "template-dependent manner" is intended to refer to a process (e.g., DNA synthesis by a DNA polymerase) that involves template-dependent extension of a primer molecule. The term "template-dependent manner" refers to polynucleotide synthesis of RNA or DNA, wherein the sequence of the newly synthesized strand of the polynucleotide is determined by the well-known rules of complementary base pairing (see, e.g., Watson, JD et al., In: Molecular Biology of the Gene, 4th Edition, WA Benjamin, Inc., Menlo Park, Calif. (1987)).

[0040] As used herein, "nucleic acid polymerase" is an enzyme that is generally used to connect 3'-OH 5'-triphosphate nucleotides, oligomers and their analogs. Polymerases include, but are not limited to, DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, RNA-dependent RNA polymerases, T7 DNA polymerases, T3 DNA polymerases, T4 DNA polymerases, T7 RNA polymerases, T3 RNA polymerases, SP6 RNA polymerases, DNA polymerase 1, Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, DNA polymerase (NewEngland Biolabs), Deep DNA polymerase (New England Biolabs), Bst DNA polymerase large fragment, Stoeffel fragment, 9°N DNA polymerase, 9°N DNA polymerase, Pfu DNA polymerase, Tfl DNA polymerase, TthDNA polymerase, RepliPHI Phi29 polymerase, Tli DNA polymerase, eukaryotic DNA polymerase β, telomerase, Therminator TM Polymerase (New England Biolabs), KOD HiFi TMDNA polymerase (Novagen), KOD1 DNA polymerase, Q-β replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase. The polymerase according to the present invention may be a variant, mutant or chimeric polymerase.

[0041] As used herein, "DPO4-type DNA polymerase" is a DNA polymerase naturally expressed by the archaeon, Sulfolobus solfataricus or a related Y-family DNA polymerase, which is generally used in the replication of damaged DNA by a process called translesion synthesis (TLS). Y-family DNA polymerases are homologous to DPO4 polymerases (e.g., as listed in SEQ ID NO: 1); examples include prokaryotic enzymes, PolII, PolIV, PolV, archaeal enzymes, Dbh, and eukaryotic enzymes, Rev3p, Rev1p, Polη, REV3, REV1, and Polκ DNA polymerases, and chimeras thereof. The modified recombinant DPO4-type DNA polymerase comprises one or more mutations relative to a naturally occurring wild-type DPO4-type DNA polymerase, such as one or more mutations that increase the ability to utilize large nucleotide analogs as substrates or another polymerase property, and may include additional changes or modifications relative to a wild-type DPO4-type DNA polymerase, such as 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 labeling the polymerase enzyme). An example of a variant polymerase according to the invention is a variant of Sulfolobus solfataricus DPO4 disclosed in published PCT patent application WO2017 / 087281 A1, which is incorporated herein by reference in its entirety.

[0042] The polymerase activity of any of the above enzymes can be determined by methods known in the art. For example, the nucleotide incorporation assay of Hogrefe et al. (Methods in Enzymol. Vol. 334, pp. 91-116 (2001)) can be used to measure the polymerization rate. In short, polymerase activity can be measured as the incorporation of 32 The rate of incorporation of P-dCTP into activated salmon sperm DNA (purchased from Pharmacia; for activation protocols, see CC Richardson, Procedures in Nucl. Acid Res. (Cantoni and Davies, eds.), pp. 263-276 (1966), at p. 264). The reaction buffer can be, for example, 50 mM Tris-HCl (pH 8.0), 5 mM MgCl 2, 1mM dithiothreitol (DTT), 50μg / ml bovine serum albumin (BSA) and 4% (v / v) glycerol. The nucleotide substrate and DNA used are significantly excessive, usually at least 10 times the Km of the polymerase being measured, such as 200μgM of each dATP, dTTP and dGTP, 195μgM dCTP plus 5μgM labeled dCTP and 250μg / ml activated DNA. The reactants are quenched on ice, and aliquots of the reaction mixture are spotted onto ion exchange filters (e.g., Whatman DE81). Unincorporated nucleotides are washed, and then scintillation counting is performed to measure the radioactivity incorporated. As used herein, "enhanced rate" refers to an increase of 5-10%, 10-50% or 50-100% or more compared to the polymerization reaction lacking the additive that increases the rate as defined herein.

[0043] As used herein, "processivity" refers to the degree of polymerization of a nucleic acid polymerase during a single contact of the polymerase with its template, i.e., its property of continuing to act on a substrate rather than dissociating from the substrate. The degree of polymerization refers to the amount of nucleotides or nucleotide analogs added by the polymerase during a single contact between the polymerase and its template. Processivity may depend on the nature of the polymerase, the sequence of the template, the structure of the nucleotide or nucleotide analog substrate, and the reaction conditions, such as salt concentration, temperature, or the presence of specific additives.

[0044] As used herein, "enhanced processivity" refers to an increase of 5-10%, 10-50%, or 50-100% or greater compared to a polymerization reaction lacking an additive that increases processivity as defined herein. Methods for measuring processivity of nucleic acid polymerases are generally known in the art, for example, as described in Sambrook et al. 1989, In ​​Molecular Cloning, 2nd Edition, CSH Press, 7.79-7.83 and 13.8, as described in U.S. Published Patent Application No. 2002 / 0119467, Published PCT Application No. WO01 / 92501, and U.S. Pat. No. 5,972,603, the entireties of which are incorporated herein by reference in their entirety.

[0045] As used herein, the term "fidelity" refers to the accuracy of nucleic acid polymerization by a template-dependent nucleic acid polymerase. The fidelity of a DNA polymerase is measured by the error rate (the frequency of incorporating incorrect nucleotides (i.e., nucleotides that are not incorporated in a template-dependent manner)). The fidelity or error rate of a DNA polymerase can be measured using assays known in the art (see, e.g., Lundburg et al., 1991 Gene, 108: 1-6). As used herein, "enhanced fidelity" refers to an increase of 5-10%, 10-50%, or 50-100% or more compared to a polymerization reaction lacking an additive that increases fidelity as defined herein.

[0046] "Primer extension reaction" means a reaction between a target-primer hybrid and a nucleotide or nucleotide analog that results in the addition of a nucleotide or nucleotide analog to the 3'-end of the primer such that the incorporated nucleotide or nucleotide analog is complementary to the corresponding nucleotide of the target polynucleotide. Primer extension reagents typically include (i) a polymerase enzyme; (ii) a buffer; and (iii) one or more extendable nucleotides or nucleotide analogs. Primer extension reactions can be used to measure the length of the resulting nucleic acid product under specific experimental conditions, and to determine the effects of various polymerase reaction additives on polymerase activity by, for example, comparing the length of the extended primer product via gel electrophoresis.

[0047] As used herein, "minor groove binding moiety" (also referred to as "MGB") is an art-recognized term referring to a variety of small molecule DNA binders whose putative mechanism of action is to use the minor groove of double-stranded DNA. Typically, the structure of the MGB adopts a bent conformation that is complementary to the curve of the minor groove of B-form DNA. However, it should be understood that according to the present invention, the manner in which the MGB interacts with the template nucleic acid during the polymerase reaction has not yet been determined, and it is not clear whether the mechanism of action based on minor groove binding is crucial to the present invention. The inventors use this term solely due to its long-standing use in the art, rather than to describe any limiting mechanism of action. The MGB of the present invention may be derived from a natural product or may be synthetic. The MGB may be any analog or derivative of any known MGB. Exemplary MGBs include, but are not limited to, Hoechst dyes and derivatives (e.g., Hoechst 33258, Hoechst 34580, Hoechst 33342), trichomoniasis, distamycin A and its synthetic analogs (e.g., lexitropsin and thiazotropsin A), (+)-CC-1065, duocarmycin, pyrrolobenzodiazepines, trabectin and analogs, bisamidines (e.g., DAPI, Bernier, pentamidine, DB293 and 3,6-diaminoacridine hydrochloride), and polyamides.

[0048] As used herein, the term "plurality" means "at least two".

[0049] "XNTP" is an amplifiable 5' triphosphate-modified nucleotide substrate compatible with template-dependent enzymatic polymerization. XNTP has two different functional components; namely, a nucleobase 5'-amino triphosphate and a tether, which is attached to each nucleoside amino triphosphate at a position that allows for controllable amplification by intranucleotide cleavage of the phosphoramidate bond. XNTP is an exemplary "non-natural, highly substituted nucleotide analog substrate", as used herein. Exemplary XNTPs and methods for their preparation are described in PCT Application No. WO2016 / 081871, as disclosed by the applicant, which is incorporated herein by reference in its entirety.

[0050] "Xpandomer intermediates" are intermediate products (also referred to herein as "daughter strands") assembled from XNTPs and formed by polymerase-mediated template-directed assembly of XNTPs using a target nucleic acid template. The newly synthesized Xpandomer intermediate is a constrained Xpandomer. In the process step of cleaving the phosphoramidate bond provided by the XNTP, the constrained Xpandomer is no longer constrained and is an extended Xpandomer product as the tether is extended.

[0051] "Xpandomer" or "Xpandomer product" is a synthetic molecular construct produced by the extension of a constrained Xpandomer, which itself is synthesized by the assembly of a template-directed XNTP substrate. Xpandomer is extended relative to the target template from which it is produced. It consists of a series of subunits, tethers, and optionally part or all of the substrates, each subunit being a motif, each motif being a library member, which contains sequence information, all of which are derived from formative substrate constructs. Xpandomer is designed to be amplified to be longer than the target template, thereby reducing the linear density of sequence information of the target template along its length. In addition, Xpandomer optionally provides a platform to increase the size and abundance of reporters, which in turn improves the signal-to-noise ratio for detection. Lower linear information density and stronger signals improve resolution and reduce the sensitivity requirements for detecting and decoding template strand sequences.

[0052] "Tether" or "tether member" refers to a polymer or molecular construct having a substantially linear size and having an end portion at each of two opposite ends. The tether is connected to a nucleoside amino triphosphate using a linkage at the end portion to form an XNTP. The linkage is used to constrain the tether to a "constrained configuration". The tether has a "constrained configuration" and an "extended configuration". The constrained configuration is present in the XNTP and the daughter chain or Xpandomer intermediate. The constrained configuration of the tether is a precursor to the extended configuration found in the Xpandomer product. The transition from the constrained configuration to the extended configuration results in the cutting of the amino phosphorothioate bond that can be selectively cut. The tether contains one or more reporters or reporter constructs of the sequence information of the encodable substrate along its length. The tether provides a method to extend the length of the Xpandomer, and thereby reduce the linear density of the sequence information.

[0053] A "tether element" or "tether segment" is a polymer having approximately linear dimensions with two ends, wherein the ends form terminal linkages for connecting tether elements. Tether elements are segments of a tether. Such polymers may include, but are not limited to: polyethylene glycol ( glycols), polyethylene glycols, polyglycols, polypyridine, polyisocyanate, polyisocyanate, poly(triarylmethyl)methacrylate, polyaldehyde, polypyrrolidone, polyurea, polyglycol phosphate diester, polyacrylate, polymethacrylate, polyacrylamide, polyvinyl ester, polystyrene, polyamide, polyurethane, polycarbonate, polybutyrate, polybutadiene, polybutyrolactone, polypyrrolidone, polyvinylphosphonate, polyacetamide, polysaccharide, polyhyaluronic acid, polyamide, polyimide, polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyterephthalate, polysilane, polyurethane, polyether, polyamino acid, polyglycine, polyproline, N-substituted polylysine, polypeptide, side chain N-substituted peptide, poly-N-substituted glycine Acids, peptoids, side chain carboxyl-substituted peptides, homotypic peptides, oligonucleotides, ribonucleic acid oligonucleotides, deoxynucleic acid oligonucleotides, oligonucleotides modified to prevent Watson-Crick base pairing, oligonucleotide analogs, polycytidylic acid, polyadenylic acid, polyuridylic acid, polythymidine, polyphosphates, polynucleotides, polyribonucleotides, polyethylene glycol-phosphodiester, peptide polynucleotide analogs, threonyl-polynucleotide analogs, diol-polynucleotide analogs, morpholino-polynucleotide analogs, locked nucleotide oligomer analogs, polypeptide analogs, branched polymers, comb polymers, star polymers, dendrimers, random, gradient and block copolymers, anionic polymers, cationic polymers, stem-loop forming polymers, rigid segments and flexible segments.

[0054] A "reporter" is composed of one or more reporter elements. The reporter is used to analyze the genetic information of the target nucleic acid.

[0055] A "reporter construct" comprises one or more reporters that produce one or more detectable signals, wherein the one or more detectable signals typically contain sequence information. The signal information is referred to as "reporter code" and is subsequently decoded into genetic sequence data. The reporter construct may also comprise a tether segment or other building blocks, including polymers, graft copolymers, block copolymers, affinity ligands, oligomers, haptens, aptamers, dendrimers, linker groups, or affinity binding groups (e.g., biotin).

[0056] The "reporter code" is the genetic information of the measured signal from the reporter construct. The reporter code is decoded to provide sequence-specific genetic information data.

[0057] Methods for nucleic acid polymerization enhanced by a minor groove binding moiety (MGB).

[0058] MGBs are a variety of small molecule DNA binders whose putative mechanism of action is to target the minor groove of double-stranded DNA (for a general review, see, e.g., JM Withers, G. Padroni, SM Pauff, AW Clark, SP Mackay and GA Burley, 5.07-DNA Minor Groove Binders as Therapeutic Agents, In Comprehensive Supramolecular Chemistry II, edited by Jerry L. Atwood, Elsevier, Oxford, 2017, pp. 149-178, ISBN 9780128031995, https: / / doi.org / 10.1016 / B978-0-12-409547-2.12561-2). Typically, the structure of MGB adopts a bent conformation that is complementary to the bend of the minor groove of B-form DNA. MGB can be any analog or derivative of any known MGB. Exemplary MGBs include, but are not limited to, Hoechst dyes and derivatives (e.g., Hoechst 33258, Hoechst 34580, Hoechst 33342), sphaerocyclin, distamycin A and its synthetic analogs (e.g., lexitropsin and thiazotropsin A), (+)-CC-1065, duocarmycin, pyrrolobenzodiazepines, trabectin and analogs, bisamidines (e.g., DAPI, Bernier, pentamidine, DB293 and 3,6-diaminoacridine hydrochloride), and polyamides. MGBs are available from a number of commercial sources, such as MilliporeSigma Corporation (St. Louis, MO).

[0059] According to certain embodiments of the present invention, MGB can enhance any nucleic acid polymerization reaction or improve the properties of the resulting nucleic acid, such as the length or accuracy of the reaction product. Polymerization reactions include, for example, primer extension reactions, PCR, mutagenesis, isothermal amplification, DNA sequencing, and probe labeling. Such methods are well known in the art. Enhancement can be provided by stimulating nucleotide incorporation through the following mechanisms: such as increasing the persistence of the polymerase (i.e., reducing the dissociation of the polymerase from the template), increasing the rate of substrate binding or enzymatic catalysis, and increasing the accuracy or fidelity of nucleotide incorporation. In addition, enhancement can be provided by reducing obstacles in the nucleic acid template, such as secondary structures and double-stranded DNA. Overcoming or improving such obstacles by adding MGB can make the polymerization reaction occur more accurately or more efficiently, or allow the use of lower denaturation / extension temperatures or isothermal temperatures.

[0060] In some embodiments, MGBs can be used in combination with other classes of PEM additives to enhance polymerase reactions.

[0061] Sequencing by Amplification (SBX)

[0062] An exemplary polymerase reaction that can be enhanced by MGB is the polymerization of unnatural nucleotide analogs called "XNTPs," which forms the basis of the "sequencing by amplification" (SBX) protocol developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion"). In general terms, SBX uses this biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer." The transcribed sequence is encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are approximately 10 nm apart and designed for high signal-to-noise ratio, highly differentiated responses. These differences provide significant performance enhancements in the sequence reading efficiency and accuracy of the Xpandomer relative to natural DNA. A general overview of the SBX process is depicted in Figure 1A –1D.

[0063] XNTP is a nucleotide substrate of amplifiable 5' triphosphate modification compatible with template-dependent enzymatic polymerization. Highly simplified XNTP is shown in 1A, which emphasizes the unique features of these nucleotide analogs: XNTP 100 has two different functional regions; that is, a selectively cleavable phosphoramidite bond 110 (connecting 5' α-phosphate 115 to nucleobase 105) and a tether 120 (which is connected to the position of controllable amplification in nucleoside amino triphosphates allowing cleavage within the nucleotide of the phosphoramidite bond). The tether of XNTP is composed of a joint arm portion 125A and 125B separated by a selectively cleavable phosphoramidite bond. Each joint is connected to an end of a reporter 130 via a connecting group (LG), as described in U.S. Patent No. 8,324,360 of Kokoris et al., which is incorporated herein by reference as a whole. XNTP100 is illustrated with a "constrained configuration", which is characterized by a subchain after XNTP substrate and polymerization. As found in Xpandomer products, the constrained configuration of polymerized XNTPs is a precursor to the extended configuration. The transition from the constrained configuration to the extended configuration occurs after the P--N bond of the phosphoramidate within the main backbone of the daughter chain is broken.

[0064] The synthesis of Xpandomers is outlined in Figure 1B and 1C During assembly, monomeric XNTPI substrates 145 (XATP, XCTP, XGTP, and XTTP) are polymerized onto the extendable end of the nascent daughter strand 150 through a template-directed polymerization process using a single-stranded template 140 as a guide. Typically, the process starts with a primer and proceeds in the 5' to 3' direction. Typically, a DNA polymerase or other polymerase is used to form the daughter strand, and conditions are selected so that a complementary copy of the template strand is obtained. After synthesis of the daughter strand, the coupled tether comprises a constrained Xpandomer, which further comprises the daughter strand. The tether in the daughter strand has a "constrained configuration" of the XNTP substrate. The constrained configuration of the tether is a precursor to the extended configuration, as found in the Xpandomer product. As Figure 1C As shown, the transition from the constrained configuration 160 to the extended configuration 165 is due to the cleavage of a selectively cleavable phosphoramidate bond (represented by an unshaded ellipse for simplicity) within the primary backbone of the daughter strand. In this embodiment, the tethers include one or more reporters or reporter constructs 130A, 130C, 130G, or 130T specific for the nucleobases to which they are attached, thereby encoding the sequence information of the template. In this way, the tethers provide a method to extend the length of the Xpandomer and reduce the linear density of the sequence information of the parent strand. Figure 1DXpandomer 165 is shown translocating from cis reservoir 175 to trans reservoir 185 through nanopore 180. After passing through the nanopore, each reporter of the linearized Xpandomer (labeled "G," "C," and "T" in this illustration) generates a unique and reproducible electronic signal (shown by superimposed trace 190) that is specific to the nucleobase to which it is attached.

[0065] Figure 2 The general structure of XNTP is described in more detail. XNTP 200 is composed of a nucleobase amidotriphosphate 210 having a linker arm portion 220A and 220B separated by a selectively cleavable phosphoramidite bond 230. The tether is attached to the nucleoside amidotriphosphate at linking groups 250A and 250B, wherein the first tether end is attached to a heterocycle 260 (represented herein by cytosine, although the heterocycle may be any of the four standard nucleobases A, C, G or T), and the second tether end is attached to the alpha phosphate 270 of the nucleobase backbone. It will be appreciated by those skilled in the art that many suitable coupling chemistries known in the art can be used to form the final XNTP substrate product, for example, the tether conjugation can be completed by triazole linkage.

[0066] In this embodiment, tether 275 is composed of several functional elements, including enhancers 280A and 280B, reporter codes 285A and 285B, and translation control elements (TCE) 290A and 290B. Each of these features has a unique function during the displacement of Xpandomer through the nanopore and the generation of unique and reproducible electronic signals. Tether 275 is designed for displacement control (TCH) by hybridization. As described, TCE provides a hybridization zone that can be paired with a complementary oligomer (CO) and is positioned adjacent to the reporter code. Determine the size of different reporter codes to prevent ions from flowing through the nanopore at different measurable levels. Specific reporter codes can be effectively synthesized using phosphoramidite chemistry commonly used for oligonucleotide synthesis. Reporters can be designed by selecting a series of specific phosphoramidites from commercially available libraries. Such libraries include, but are not limited to, polyethylene glycols of 1 to 12 or more ethylene glycol units in length, aliphatics of 1 to 12 or more carbon units in length, deoxyadenosine (A), deoxycytosine (C), deoxyguanosine (G), deoxythymine (T), abasic (Q). The double-stranded TCE associated with the reporter code also contributes to ion current blocking, so the combination of the reporter code and TCE can be referred to as a "reporter". Following the reporter code is an enhancer, which in one embodiment includes a spermine polymer.

[0067] Figure 3An embodiment of the process of translocation of a cleaved Xpandomer through an α-hemolysin nanopore is shown. The biological nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two electrolyte pools. A typical electrolyte is 1 molar KCl (buffered to pH 7.0). When a small voltage, typically 100 mV, is applied across the bilayer, the nanopore restricts the flow of ionic current and is the main resistor in the circuit. Xpandomer reporters are designed to provide specific levels of ionic current blockade, and when the sequence of the reporter translocates the nanopore, the sequence information can be read by measuring the sequence of ionic current levels.

[0068] The α-hemolysin nanopore is usually oriented so that translocation occurs by entering the vestibule side and exiting the stalk side. Figure 3 As shown, the nanopore is oriented to capture the Xpandomer from the stem side first. When using the TCH method, this orientation is advantageous because it causes less clogging artifacts to occur when first entering the vestibule. Unless otherwise stated, the stem side will first be the assumed displacement direction. When the Xpandomer is displaced, the reporter enters the stem until its double-stranded TCE stops at the stem entrance. The diameter of the duplex is ~2.4nm and the stem entrance is ~2.2nm, so that the reporter is kept in the stem until the complementary strand 395 of the duplex dissociates (releases), so the displacement proceeds to the next reporter. Since the Xpandomer is still displaced and diffuses out from the hole, it is very unfavorable for the free complementary strand to enter the nanopore.

[0069] In one embodiment, each member of the reporter code (after the duplex) is formed by an ordered selection of phosphoramidites, which can be selected from many commercial libraries. Each constitutive phosphoramidite contributes to the net ionic resistance according to its position in the nanopore (after the duplex termination), its displacement, its charge, its interaction with the nanopore, its chemical and thermal environment, and other factors. The charge on each phosphoramidite is partially due to the phosphate ion with a nominal charge of -1, but can be effectively reduced by counterion shielding. The force applied to the duplex is due to these effective charges along the reporter, which are subject to the action of the local electric field. Since each reporter can have a different charge distribution, it can apply different forces on the duplex for a given applied voltage. The force transmitted along the reporter backbone is also used to stretch the reporter out to provide a repeatable blocking response.

[0070] Compositions for enhancing nucleic acid polymerization using a minor groove binding moiety (MGB).

[0071] In one embodiment, the present disclosure provides a composition comprising at least one MGB as disclosed herein and a plurality of nucleotides and / or nucleotide analogs. In another embodiment, the present disclosure provides a composition comprising at least one MGB as disclosed herein and a buffer. In another embodiment, the present disclosure provides a composition comprising at least one MGB as disclosed herein and a polynucleotide. In another embodiment, the present disclosure provides a composition comprising at least one and a protein as disclosed herein, wherein optionally the protein is a polymerase comprising any one of the polymerases described above.

[0072] In one embodiment, the present disclosure provides an aqueous (water-containing) composition comprising at least one MGB and a buffer (particularly a buffer suitable for carrying out a DNA polymerization reaction), wherein Tris HCl is an exemplary buffer of this type. In one embodiment, the present disclosure provides a composition comprising at least one MGB and a DNA polymerase protein. In one embodiment, the present disclosure provides a composition comprising at least one MGB and a polynucleotide (such as a 20-90mer, 20-60mer, 30-90mer or 30-60mer) oligonucleotide. In one embodiment, the present disclosure provides a composition comprising each of these components, i.e., an aqueous composition comprising at least one MGB, a buffer, a DNA polymerase protein and a polynucleotide.

[0073] In some embodiments, MGBs can be used in combination with another class of additives to enhance polymerase reactions. One exemplary class of additives is polymerization enhancing moieties (PEMs). More information about using PEMs to enhance polymerase reactions can be found in Applicant's co-application entitled ENHANCEMENT OF NUCLEIC ACID POLYMERIZATION BY AROMATIC COMPOUNDS. Example

[0074] Example 1

[0075] Screening additives for enhancing XNTP polymerization

[0076] The sequencing by amplification (SBX) method developed by the inventors provides a significant performance enhancement in the efficiency and accuracy of sequence reading of Xpandomers relative to natural DNA. However, the initial transcription of a sequence of a natural DNA template onto a measurable Xpandomer relies on the ability of a DNA polymerase to utilize XNTPs as substrates (referenced herein). Figure 1A and Figure 2The general structure of XNTP is discussed. The inventors found that most DNA polymerases cannot effectively polymerize XNTP. In order to improve the efficiency and accuracy of XNTP polymerization into Xpandomer, a variety of additives were screened to enhance the ability of DNA polymerase primer extension reaction using XNTP as substrate.

[0077] A typical primer extension reaction included the following reagents: 2 pmol primer, 2.2 pmol 45mer oligonucleotide template, 50 pmol of each XNTP (XATP, XCTP, XGTP, and XTTP), 50 mM Tris HCl, pH 6.79, 200 mM NaCl, 20% PEG, 5% NMS, 0.5 nmol polyphosphate 60.19, 0.3 mM MnCl 2 and 0.6 μg purified recombinant DNA polymerase protein. The reaction was run at 23°C for 1 hour. The reaction product (i.e., constrained Xpandomer) was treated to cleave the phosphoramidate bond to generate a linear Xpandomer. The reaction products were analyzed on a 4-12% acrylamide gel using gel electrophoresis to resolve and visualize Xpandomer products of different lengths. For additive screening, reaction additives are typically tested in the micromolar to millimolar range.

[0078] Table 1 below lists an overview of the additive screening results. As expected based on previous experiments, sodium polyphosphate and PEG8000 enhanced primer extension using XNTPs. However, most previously untested additives either had no effect or actually inhibited the primer extension reaction. Surprisingly, several known MGBs, including Hoescht 33258, Hoescht 34580, Hoescht 33342, 3,6-diaminoacridine hydrochloride, and sphaerothrin dihydrochloride, significantly and reproducibly enhanced primer extension with XNTPs. Representative gels demonstrating this enhancement are shown in Table 1. Figure 4 . As can be seen in lane 1, in the absence of additives, the DNA polymerase extends the template-bound primer up to about 14 XNTPs under these conditions. The absence of 14-mer extension products in lanes 2 and 10 indicates that DAPI and furanobisamidine have an inhibitory effect on XNTP polymerization. The extension products in lanes 3 to 9 appear to exceed the length of those in the lane 1 control (e.g., the arrow in lane 3 points to the 18-mer product), demonstrating the enhancing effect of these MGBs on XNTP polymerization. Therefore, MGBs become the focus of further optimization of XNTP polymerization.

[0079] Table 1

[0080]

[0081]

[0082]

[0083] Example 2

[0084] Optimizing MGB-mediated enhancement of XNTP polymerization

[0085] Using the primer extension reaction described in Example 1, titration experiments were performed to determine the optimal amount of MGB for enhancing XNTP polymerization. Results from a representative experiment are presented in Figure 5 These results indicate that each of the MGBs tested (Hoescht 33258, Hoescht 33342, and sphingomyelin, ranging from 75 to 600 μM) had a dose-dependent enhancement, with the highest concentrations showing the most robust enhancement of primer extension. Further titration experiments showed that 0.6 mM Hoescht 33258 and 0.8 mM Hoescht 33342 showed robust enhancement of XNTP polymerization. Representative gels showing these effects are presented in Figure 6 middle.

[0086] Example 3

[0087] MGB Enhanced Sequencing by Amplification (SBX)

[0088] To investigate the precision of XNTP polymerization enhancement, primer extension products were sequenced using the SBX protocol. Briefly, the constrained Xpandomer product of XNTP polymerization was cleaved to generate a linear Xpandomer. This was first accomplished by quenching the extension reaction with a solution containing 100 mM EDTA, 2 mM THPTA, and 2% Tween-20. The sample was then washed with 1 M NaHCO in DMF. 3 Amine modification was performed with a solution of 1 M succinic anhydride. Cleavage of the phosphoramidate bond was performed with 37% HCl, and the linear Xpandomer was purified using a QIAquick column (QIAGEN, Inc.).

[0089] For sequencing, α-hemolysin was prepared by mixing it in buffer B1 (containing 2 M NH 4 Cl and 100 mM HEPES, pH 7.4) to insert the DPhPE / hexadecane bilayer members to prepare the protein nanopore. The cis well was washed with buffer B2 (containing 0.4 M NH 4Cl, 0.6M GuCl and 100mM HEPES, pH 7.4.) were perfused. The Xpandomer sample was heated to 70°C for 2 minutes, cooled completely, and then 2 μL of sample was added to the cis well. A voltage pulse of 90mV / 390mV / 10μs was then applied and data was collected via Labview acquisition software.

[0090] The sequence data were analyzed by presenting a histogram of the sequence read population from a single SBX reaction. The analysis software aligns each sequence read to the sequence of the template and trims the sequence range that does not align to the correct template sequence at the end of the read. A representative histogram of SBX sequencing of a 45mer template is presented in Fig. 7A (no additive control) and Figure 7B (SBX in the presence of Hoescht 33258). It can be seen that in the absence of the additive, sequence reads beyond template base 20 are imprecise. Notably, adding MGB to the SBX reaction improves the accuracy of sequence reads completely to the end of the 45mer template.

[0091] All references disclosed herein, including patent references and non-patent references, are hereby incorporated by reference in their entirety, as if each reference was individually incorporated.

[0092] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent disclosures mentioned in this specification and / or listed in the application data sheet, including but not limited to U.S. Provisional Patent Application 62 / 614,114, are incorporated herein by reference in their entirety. In order to describe and disclose materials and methods such as described in the disclosure, such documents may be incorporated by reference, which may be used in conjunction with the invention currently described. The disclosure discussed above and throughout the entire text is provided prior to the filing date of the present application only for its disclosure. Nothing herein shall be construed as an admission that the inventor has no right to predate any cited publication by virtue of prior invention.

[0093] All patents, publications, scientific articles, websites, and other documents and materials cited or mentioned herein are indicative of the level of skill of those skilled in the art to which the present invention relates, and each such cited document and material is hereby incorporated by reference to the same extent as if it were individually incorporated by reference in its entirety or set forth in its entirety herein. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, websites, electronically obtained information, and other reference materials or documents.

[0094] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Therefore, the claims should not be limited by the disclosure herein.

[0095] In addition, the written description portion of the patent includes all claims. In addition, all claims, including all original claims and all claims from any and all priority documents, are hereby incorporated by reference in their entirety into the written description portion of the specification, and applicant reserves the right to physically incorporate any and all such claims into the written description or any other part of this application. Thus, for example, in no case shall a patent be construed arbitrarily as not providing a written description for a claim, asserting that the exact wording of the claim is not set forth in the same words in the written description portion of the patent.

[0096] The claims will be construed as such. However, in no event shall any adjustment or modification of any claim or any portion thereof during the prosecution of one or more applications cause this patent to be construed as a waiver of any and all equivalents that do not form part of the prior art, even though it is alleged or readily understood or difficult to interpret.

[0097] Other non-limiting embodiments are within the following claims. This patent may not be construed as limited to the specific embodiments or non-limiting embodiments or methods specifically and / or expressly disclosed herein. In no event shall a patent be limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office unless such statement is specifically and expressly adopted by the applicant in a respondent written document without qualification or reservation.

Claims

1. A method for enhancing a nucleic acid polymerase reaction, the method comprising: a) forming a nucleic acid polymerase reaction composition comprising: (i) a template nucleic acid, (ii) nucleic acid polymerase, (iii) a mixture of nucleotides or non-natural nucleotide analogs, (iv) at least one minor groove binding moiety; and b) incubating the nucleic acid polymerase reaction composition under conditions permissive for nucleic acid polymerization, wherein the at least one minor groove binding moiety increases the processivity of the nucleic acid polymerase reaction; wherein the mixture of nucleotides or non-natural nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside amino triphosphates, wherein each of the nucleoside amino triphosphates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tethering moiety, wherein a first end of the polymeric tethering moiety is attached to the nucleobase and a second end of the polymeric tethering moiety is attached to the alpha phosphate of the nucleoside amino triphosphate to provide for amplification of the non-natural nucleotide analog by cleavage of the phosphoramidate bond, wherein the mixture of nucleotides or non-natural nucleotide analogs is an XNTP; wherein the nucleic acid polymerase is a DNA polymerase, and the DNA polymerase is a DPO4 DNA polymerase; and The at least one minor groove binding moiety is selected from the group consisting of Hoechst dye and its derivatives, sphingomyelin and 3,6-diaminoacridine hydrochloride, wherein the Hoechst dye and its derivatives are selected from the group consisting of Hoechst 33258, Hoechst 34580 and Hoechst 33342.

2. The method of claim 1, wherein the at least one minor groove binding moiety increases the length of the resulting nucleic acid product compared to a nucleic acid polymerase reaction lacking the minor groove binding moiety.

3. The method of claim 1, wherein the at least one minor groove binding moiety is Hoechst 33258, Hoechst 34580 or Hoechst 33342.

4. The method of claim 1, wherein the at least one minor groove binding moiety comprises a plurality of minor groove binding moieties.

5. The method of claim 4, wherein the plurality of minor groove binding moieties comprises minor groove binding moieties of different structural classes.

6. The method of claim 1, wherein the nucleic acid polymerization reaction produces an amplifiable polymer of nucleotide analogs, wherein the amplifiable polymer encodes nucleobase sequence information of the template nucleic acid.

7. The method of claim 6, wherein the conditions for allowing nucleic acid polymerization reaction include a suitable polymerization buffer and oligonucleotide primers.

8. The method of claim 7, wherein the suitable buffer comprises Tris OAc, NH4OAc, PEG, DMF, polyphosphate 60, and MnCl2.

9. The method of claim 1, wherein the reaction composition further comprises a nucleic acid intercalator.

10. The method of claim 1, wherein the reactive composition further comprises a polyanion recognition moiety.

11. The method of claim 1, wherein the mixture of nucleotides or non-natural nucleotide analogs is an XNTP comprising a detectable label.

12. The method of claim 11, wherein the detectable label is an optically detectable label selected from the group consisting of a luminescent label, a fluorescent label, or a chromophore label.

13. The method of claim 11, wherein the detectable label is selected from a chemiluminescent label, a fluorescent label, or a chromogenic label.

14. A composition for enhancing the processivity of a DNA polymerase reaction, comprising at least one minor groove binding moiety, a mixture of non-natural nucleotide analogs and said DNA polymerase, said DNA polymerase being DPO4 DNA polymerase, wherein the mixture of non-natural nucleotide analogs comprises nucleoside amino triphosphates, wherein each of the nucleoside amino triphosphates comprises a nucleobase and a polymeric tethering moiety, the nucleobase being selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a first end of the polymeric tethering moiety is attached to the nucleobase and a second end of the polymeric tethering moiety is attached to the alpha phosphate of the nucleoside amino triphosphate to provide for amplification of the non-natural nucleotide analogs by cleavage of the phosphoramidate bond, wherein the mixture of non-natural nucleotide analogs is XNTP; and The at least one minor groove binding moiety is selected from the group consisting of Hoechst dye and its derivatives, sphingomyelin and 3,6-diaminoacridine hydrochloride, wherein the Hoechst dye and its derivatives are selected from the group consisting of Hoechst 33258, Hoechst 34580 and Hoechst 33342.

15. A composition comprising at least one minor groove binding moiety, a mixture of non-natural nucleotide analogs, and a DNA polymerase, wherein the DNA polymerase is DPO4 DNA polymerase, wherein the at least one minor groove binding moiety increases the number of nucleotide analogs incorporated into a daughter strand during a template-dependent polymerization reaction relative to the same polymerization reaction in the absence of the at least one minor groove binding moiety, wherein the mixture of non-natural nucleotide analogs comprises nucleoside amino triphosphates, wherein each of the nucleoside amino triphosphates comprises a nucleobase and a polymeric tethering moiety, the nucleobase being selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a first end of the polymeric tethering moiety is attached to the nucleobase and a second end of the polymeric tethering moiety is attached to the alpha phosphate of the nucleoside amino triphosphate to provide for amplification of the non-natural nucleotide analogs by cleavage of the phosphoramidate bond, wherein the mixture of non-natural nucleotide analogs is XNTP; and The at least one minor groove binding moiety is selected from the group consisting of Hoechst dye and its derivatives, sphingomyelin and 3,6-diaminoacridine hydrochloride, wherein the Hoechst dye and its derivatives are selected from the group consisting of Hoechst 33258, Hoechst 34580 and Hoechst 33342.

16. The composition of claim 14, wherein the at least one minor groove binding moiety is Hoechst 33258, Hoechst 34580 or Hoechst 33342.

17. The composition of claim 14, wherein the at least one minor groove binding moiety comprises a plurality of minor groove binding moieties.

18. The composition of claim 17, wherein the plurality of minor groove binding moieties comprises minor groove binding moieties of different structural classes.

19. The composition of claim 14, further comprising a buffer comprising Tris OAc, NH4OAc, PEG, DMF, polyphosphate 60, and MnCl2.

20. The composition of claim 14, further comprising a DNA intercalator.

21. The composition of claim 14, further comprising a polyanion recognition moiety.

22. The composition of claim 14, wherein the mixture of non-natural nucleotide analogs is a detectably labeled XNTP.

23. The composition of claim 22, wherein the detectable label is an optically detectable label selected from the group consisting of a luminescent label, a fluorescent label, or a chromophore label.

24. The composition of claim 22, wherein the detectable label is selected from a chemiluminescent label, a fluorescent label, or a chromogenic label.

25. A method for sequencing a DNA template, the method comprising the steps of: a) forming a DNA polymerase reaction composition comprising: (i) DNA template, (ii) a replication primer complexed with the template, (iii) DNA polymerase, (iv) a mixture of nucleotides or non-natural nucleotide analogs, (v) at least one minor groove binding moiety, b) incubating the DNA polymerase reaction composition under conditions permissive for DNA polymerization, wherein the at least one minor groove binding moiety increases the processivity of the DNA polymerase reaction; and c) determining the sequence of the nucleotides or non-natural nucleotide analogues in the resulting polymer of nucleotides or non-natural nucleotide analogues; wherein the DNA polymerase is DPO4 DNA polymerase; wherein the mixture of nucleotides or non-natural nucleotide analogs comprises nucleoside amino triphosphates, wherein each of the nucleoside amino triphosphates comprises a nucleobase and a polymeric tethering moiety, the nucleobase being selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a first end of the polymeric tethering moiety is attached to the nucleobase and a second end of the polymeric tethering moiety is attached to the alpha phosphate of the nucleoside amino triphosphate to provide for amplification of the non-natural nucleotide analog by cleavage of the phosphoramidate bond, wherein the mixture of nucleotides or non-natural nucleotide analogs is an XNTP; and The at least one minor groove binding moiety is selected from the group consisting of Hoechst dye and its derivatives, sphingomyelin and 3,6-diaminoacridine hydrochloride, wherein the Hoechst dye and its derivatives are selected from the group consisting of Hoechst 33258, Hoechst 34580 and Hoechst 33342.

26. The method of claim 25, wherein the resulting polymer of nucleotide analogs is an amplifiable polymer.

27. The method of claim 26, further comprising the step of contacting the amplifiable polymer with a phosphoramidate cleavage agent to produce an amplified nucleotide analog polymer.

28. The method of claim 27, wherein the polymeric tether portion of each of the nucleotide analogs comprises a reporter portion specific for the nucleobase of the analog.

29. The method of claim 28, wherein the reporter moiety produces a characteristic electronic signal.

30. The method of claim 29, wherein the step of determining the sequence of the nucleotide analogs comprises the step of translocating the amplified nucleotide analog polymers through a nanopore.

Images