Compositions for replicating a nucleic acid template

Abstract

The present invention relates to aqueous compositions, and more specifically aqueous compositions for replicating a nucleic acid template, wherein the composition comprises D2O, as well as to methods and uses relating to such compositions. The composition can be used, for example, in a method of storing the composition, or a method for replicating a nucleic acid template, for instance for the synthesis of Xpandomers for sequencing by expansion.

Description

COMPOSITIONS FOR REPLICATING A NUCLEIC ACID TEMPLATECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present disclosure claims the benefit of the filing date of U.S. Provisional Application No. 63 / 688,911 filed on August 30, 2024; the present disclosure also claims the benefit of the filing date of International Application No. PCT / US2024 / 061051, filed on December 19, 2024, the disclosures of which are hereby incorporated by reference herein in their entireties.FIELD OF THE INVENTION

[0002] The present invention relates to aqueous compositions, and more specifically aqueous compositions for replicating a nucleic acid template, wherein the composition comprises D2O, as well as to methods and uses relating to such compositions.BACKGROUND

[0003] Over the last two decades, biological membranes have emerged as an important tool in a variety of biomedical applications. This includes the use of lipid bilayer membranes in nanopore based sequencing applications, where nanopores provide a constant and reproducible physical aperture, through which a target molecule can be directed and sequenced.

[0004] One approach for nanopore-based sequencing of, for example, nucleic acids involves a sequencing-by-expansion approach by transcribing the sequence of nucleic acids into a simple to measure polymer molecule called an Xpandomer. Much like with polymerase chain reaction (PCR), Xpandomer synthesis is based on the natural function of DNA replication where expandable nucleoside triphosphates (XNTPs) act as substrates for replication.

[0005] Xpandomer synthesis is based on four easily differentiated XNTPs that include High Signal-to-Noise Reporters, one for each DNA base. Engineered polymerases incorporate these modified nucleotides into Xpandomers, producing a copy of the target nucleic acid template from the library. As the Xpandomer molecule transits through the nanopore, the distinct electrical signal of each base reporter is easily identifiable to enable highly accurate and high throughput nanopore-based nucleic acid sequencing. See, e.g., U.S. Pat. No. 7,939,259, titled“High Throughput Nucleic Acid Sequencing by Expansion;” and PCT publication WO 2020 / 236526 Al, titled “Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing”, both of which are hereby incorporated herein in their entirety.SUMMARY OF THE INVENTION

[0006] Aqueous compositions for replicating a nucleic acid template are commonly prepared using regular water, i.e. without addition of heavy water (D2O). The present disclosure provides an aqueous composition comprising added D2O, and more specifically a composition for replicating a nucleic acid template comprising added D2O. The present inventors have surprisingly found that adding D2O allows stable storage at, for example, -20 °C, compared to the same compositions without added D2O that could only be suitably stored at much lower temperatures, such as -80 °C. Specifically, after snap freezing, it was observed that compositions with added D2O could be maintained in a frozen solid state at -20 °C whereas compositions without added D2O would at least partially thaw. The inventors surprisingly found that the compositions disclosed herein comprising D2O may be capable of being frozen at -20 °C. In contrast, the same compositions without D2O (i.e. wherein essentially all the water is H2O) will not be in a frozen state at -20 °C (see Fig. 1). Without wishing to be bound by any theory, it is hypothesized that the frozen solid state contributes to the stable storage at -20 °C.

[0007] The disclosure also relates to methods using the aqueous composition disclosed herein, for example, a method for storing the composition, or a method for replicating a nucleic acid template.

[0008] The composition comprising D2O and methods relating thereto thus enable simple storage in freezers available in virtually any laboratory, as well as efficient replication of a nucleic acid template, for example for Xpandomer synthesis for sequencing by expansion.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 : Photography of an aqueous composition for replicating a nucleic acid template with or without added D2O stored at -20 °C. While the composition without added D2O (“H2O”) is liquid, the composition comprising D2O is in a frozen state.

[0010] FIG. 2: Representative gel electrophoresis for an exemplary Xpandomer product synthesized using an aqueous composition as disclosed herein with added D2O, after storage at -20 °C during which the composition had remained frozen solid.DETAILED DESCRIPTION OF THE INVENTION

[0011] The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0012] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton (Singleton et al., Dictionary of microbiology and molecular biology, 2nd ed., 1994, John Wiley and Sons, New York), Hale (Hale and Marham, The Harper Collins dictionary of biology, 1991, Harper Perennial, NY) and Walker (Walker and Cox, The Language of Biotechnology: A Dictionary of Terms. 1988, American Chemical Society, Washington, D.C. ISBN-0-8412-1499-1) provide one of skill with a general dictionary of many of the terms used in this invention. Practitioners are particularly directed to Sambrook (Sambrook et al., Molecular cloning: A laboratory manual, 1989, Cold Spring Harbor Laboratory Press), and Ausubel (Ausubel et al., Current protocols in molecular biology, 1993, John Wiley & Sons, Inc.), for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0013] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0014] In chemical structures shown herein, when not all natural valencies of an atom are filled by named groups, it should be understood that the unfilled valencies are filled by hydrogen. When a wavy line in a structure intersects a bond, then the intersected bond is the location where the structure joins to the remainder of a molecule.

[0015] When a structure depicts a molecule with one or more negatively charged oxygens, the structure likewise encompasses the molecule with the oxygen(s) in conjunction with H+ and / or any organic or inorganic cations. When a structure depicts a molecule with one or morehydroxyl groups, the structure likewise encompasses the molecule with the oxygen(s) from the hydroxyl group(s) in conjunction with H+ and / or any organic or inorganic cations.

[0016] Reference throughout this specification to "one embodiment" or "an embodiment" and variations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0017] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0018] The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.I. Terms

[0019] As used herein, the terms "analog" or "derivative" are used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called "reference" compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound. For example, a “nucleotide analog” includes a nucleotide with a modified nucleobase.

[0020] As used herein, the term "aliphatic" means a straight or branched hydrocarbon chain, which may be saturated or mono- or polyunsaturated. An unsaturated, aliphatic group contains one or more double and / or triple bonds. The branches of the hydrocarbon chain may include linear chains as well as non-aromatic cyclic elements. The hydrocarbon chainmay, unless otherwise stated, be of any length, and contain any number of branches. Both the main chain as well as the branches may furthermore contain heteroatoms as for instance B, N, O, P, S, Se or Si.

[0021] As used herein, the term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C1-C30 for branched chain). Moreover, the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkyl sulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. An "alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also includes the side chains of natural and unnatural amino acids.

[0022] As used herein, the term "alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. The term alkenyl further includes alkenyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkenyl group has 30 or fewer carbon atoms in its backbone (e.g., C2-C30 for straight chain, C3-C30 for branched chain). Moreover, the term alkenyl includes both "unsubstituted alkenyls" and "substituted alkenyls," the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkenyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkyl aryl ami no), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Other examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2- propenyl, 1-methyl-ethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 -methyl- 1-propenyl, 2-methyl-l- propenyl, l-methyl-2-propenyl, 2-methyl-2-propenyl; 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1-methyl-l-butenyl, 2-methyl-l-butenyl, 3-methyl-l-butenyl, l-methyl-2-butenyl, 2- m ethyl -2-butenyl, 3 -methyl -2-butenyl, 1 -methyl -3-butenyl, 2-m ethyl -3-butenyl, 3 -methyl -3- butenyl, l,l-dimethyl-2-propenyl, 1,2-dimethyl- 1-propenyl, l,2-dimethyl-2-propenyl, 1 -ethyl- 1- propenyl, l-ethyl-2-propenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl, 5 -hexenyl, 1-methyl-1-pentenyl, 2-methyl-l-pentenyl, 3-methyl-l-pentenyl, 4-methyl-l-pentenyl, l-methyl-2-pentenyl,2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, l-methyl-3 -pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3 -pentenyl, l-methyl-4-pentenyl, 2-methyl-4- pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, l,l-dimethyl-2-butenyl, l,l-dimethyl-3- butenyl, 1,2-dimethyl- 1-butenyl, l,2-dimethyl-2-butenyl, l,2-dimethyl-3-butenyl, 1,3-dimethyl-l- butenyl, l,3-dimethyl-2-butenyl, l,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3 -dimethyl- 1- butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-l-butenyl, 3,3-dimethyl-2- butenyl, 1-ethyl-l-butenyl, l-ethyl-2-butenyl, l-ethyl-3-butenyl, 2-ethyl- 1-butenyl, 2-ethyl-2- butenyl, 2-ethyl-3-butenyl, l,l,2-trimethyl-2-propenyl, 1 -ethyl- l-methyl-2-propenyl, l-ethyl-2-methyl- 1 -propenyl and l-ethyl-2-methyl-2-propenyl groups. Groups containing multiple double bonds may include but are not limited to buta-l,3-dienyl, penta-1, 3-dienyl or penta- 1,4-dienyl groups.

[0023] As used herein, the term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, the term "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. The term alkynyl further includes alkynyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkynyl group has 30 or fewer carbon atoms in its backbone (e.g., C2-C30 for straight chain, C3-C30 for branched chain). Moreover, the term alkynyl includes both "unsubstituted alkynyls" and "substituted alkynyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkenyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkyl aryl ami no), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Groups containing multiple triple bonds may include but are not limited to buta- 1,3 -diynyl, penta- 1,3 -diynyl or penta- 1,4-diynyl groups.

[0024] As used herein, the term "aromatic" means, unless otherwise stated, a planar cyclic hydrocarbon moiety of conjugated double bonds, which may be a single ring or include multiple fused or covalently linked rings. The main chain of the cyclic hydrocarbon moiety may, unless otherwise stated, be of any length and contain any number of heteroatoms, as for instance N, O and S. The aromatic group may be substituted by alkyl groups or heteroatoms like O, S, N, P or Si.

[0025] As used herein, the term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen , phosphorus, and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternate. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. A heteroalkyl is not cyclized. Examples include, but are not limited to: — CH2 — CH2 — O — CH3, — CH2 — CH2— NH— CH3, — CH2— CH2— N(CH3)— CH3, — CH2— S— CH2— CH3, — CH2— O— CH3, — S(O) — CH3, — CH2— CH2— S(O)2— CH3, CH=CH — O — CH3, — Si(CH3)3, — CH2— CH=N— OCH3, — CH=CH— N(CH3)— CH3, — O— CH3, — O— CH2— CH3, and — CN. Up to two heteroatoms may be consecutive, such as, for example, — CH2— NH — OCH3.

[0026] As used herein, the terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Cycloalkyls and heterocycloalkyl can be further substituted, e.g., with any of the substituents described herein.

[0027] Each of the above terms (e.g., "alkyl," "aromatic," "heteroalkyl," "cycloalkyl," etc.) includes both substituted and unsubstituted forms of the indicated radical. In that regard, whenever a group or moiety is described as being "substituted" or "optionally substituted" (or "optionally having" or "optionally comprising") that group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as being "substituted or unsubstituted" if substituted, the substituent(s) may be selected from one or more of the indicated substituents. If no substituents are indicated, it is meant that the indicated "optionally substituted" or "substituted" group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N- amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy,trihalomethanesulfonyl, trihalomethanesulfonamido, an ether, amino (e g. a mono-substituted amino group or a di-substituted amino group), and protected derivatives thereof. Any of the above groups may include one or more heteroatoms, including O, N, or S. For example, where a moiety is substituted with an alkyl group, that alkyl group may comprise a heteroatom selected from O, N, or S (e g. -(CH2-CH2-O-CH2-CH3)).

[0028] As used herein, the term "heteroatom" is meant to include atoms other than carbon, for example, boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). In some embodiments, a “heterocyclic ring” may comprise one or more heteroatoms. In other embodiments, an aliphatic group may comprise or be substituted by one or more heteroatoms.

[0029] As used herein, the term "nucleic acid template" refers to a (e.g. single stranded) nucleic acid molecule (such as a DNA molecule) or portion thereof that is capable of use as a guide for polymerase catalyzed replication.

[0030] As used herein, the term "nucleobase" refers to a heterocyclic moiety capable of non-covalently pairing with another nucleobase. The term "nucleobase" encompasses both "unmodified nucleobases" and "modified nucleobases." A "naturally occurring nucleobase" or an "unmodified nucleobase" (used interchangeably) refer to a nucleobase that is unmodified relative to its naturally occurring form. Likewise, a "modified nucleobase" means any substitution and / or change from a natural nucleobase. Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to oligonucleotides.

[0031] Modified nucleobases include, but are not limited to, 7-deazaguanine, N4Me cytosine, and 2,6 diaminopurine (DAP), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 7-methylguanine, 2-aminoadenine, 2-aminopurine, iso-C, iso-G, thioT, thioG, 5,6- dihydrouracil, 6-methyladenine, 2-propylguanine and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine such as 5- bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 5-propynyl ( — C=C — CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-aza uracil, cytosine and thymine, uracil-5-yl (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 8-azaguanine and 8-azaadenine, and 7-deazaadenine and 3- deazaguanine and 3 -deazaadenine, 8-aza-7-deazaguanine and 8-aza-7-deazaadenine. Additional nucleobases are disclosed in Greco et. al., Synthesis and site-specific incorporation of a simple fluorescent pyrimidine, Nature Protocols, vol.2, no.2, 2007; Dien et. al., Progress Toward a SemiSynthetic Organism with an Unrestricted Expanded Genetic Alphabet, J. Am. Chem. Soc. 2018, 140, 16115-16123; Zhang et. al., Evolution of Functional Six-Nucleotide DNA, J. Am. Chem. Soc. 2015, 137, 6734-6737; Biondi et. al. Artificially Expanded Genetic Information Systems for New Aptamer Technologies, Biomedicines 2018, 6, 53; Liu et. al., Helix-Forming Properties of Size-Expanded DNA, an Alternative Four-Base Genetic Form, J. Am. Chem. Soc. 9 Vol. 127, No. 5, 2005, 1396-1402; Tor et. al., Designing new isomorphic fluorescent nucleobase analogues: the thieno[3,2-d]pyrimidine core, Tetrahedron 63 (2007) 3608-3614; Laos et. al., Directed Evolution of Polymerases to Accept Nucleotides with Nonstandard Hydrogen Bond Patterns, Biochemistry 2013, 52, 5288-5294; Krueger et. al., Synthesis and Properties of Size-expanded DNAs: Toward Designed, Functional Genetic Systems, Acc Chem Res. 2007 February ; 40(2): 141-150; Srivatsan et. al., A highly fluorescent nucleoside analog based on thieno[3,4-d]pyrimidine senses mismatched pairing, Org. Biomol. Chem., 2008, 6, 1334-1338; Kim et. al., Synthesis and Properties of 5-Cyano-Substituted Nucleoside Analog with a Donor-Donor-Acceptor Hydrogen- Bonding Pattern, J. Org. Chem. 2012, 77, 3664-3669; and Noe et. al., Oligodeoxynucleotides Containing Multiple Thiophene-Modified Isomorphic Fluorescent Nucleosides, J. Org. Chem. 2013, 78, 8123-8128, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0032] As used herein, the term "nucleotide" refers to a nucleoside covalently attached to a phosphate or polyphosphate, such as adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP), adenosine 5'-tetraphosphate or its 2'- deoxy derivatives. A “nucleotide analog” includes, for example, a nucleotide with a modified nucleobase, such as 7-deaza dGTP, N4Me dCTP, 2,6 diaminopurine (DAP), or an XNTP.

[0033] Percent identity: The term “% identity” in the context of nucleic acid or amino acid sequences refers to the level of sequence identity between a nucleic acid sequence and a reference nucleic acid sequence or between an amino acid sequence and a reference amino acid sequence, when aligned using a sequence alignment program. For example, as used herein, 80%identity indicates that a sequence has greater than 80% sequence identity over a length of the reference sequence. Exemplary levels of sequence identity include, but are not limited to, 80% or more, 85% or more, 90% or more, 95% or more, and 98% or more sequence identity to a reference sequence, e.g., the wildtype sequence for any one of the polypeptides described herein. Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See also, Altschul et al., Basic local alignment search tool, 1990, J Mol Biol Vol. 215. Issue 3, pp. 403-10; and Altschul et al., Gapped BLAST and PSLBLAST: a new generation of protein database search programs, 1997, Nucleic Acids Res. Vol. 25, Issue 17, pp. 3389-402. Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program may be used for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. The BLASTP program may be used for searching amino acid sequence against amino acid sequences in the GenBank Protein Sequences and other public databases. All of BLASTN, BLASTX and BLASTP are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul et al., 1997, supra). In certain example embodiments, an alignment of selected sequences in order to determine “% identity” between two or more sequences, is performed using for example, the CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0034] As used herein, the term "polymerase" refers to any enzyme capable of catalyzing a polymerization reaction. Examples of polymerases include, without limitation, a nucleic acid polymerase, such as a DNA polymerase.

[0035] As used herein, the term "sequencing" refers to the determination of the identity and position of nucleobases in a nucleic acid.

[0036] Expandable nucleoside triphosphate: An “expandable nucleoside triphosphate”, “expandable NTP” or “XNTP” refers to a 5' phosphate modified non-natural nucleoside triphosphate (NTP) molecule (typically a non-natural 2’ -deoxynucleoside triphosphate (dNTP) molecule) compatible with template-dependent enzymatic polymerization. Each XNTPhas two distinct functional regions, i.e., a selectively cleavable bond (e g. a phosphoramidate bond) linking the 5’ a-phosphate to a sugar comprised in a nucleoside and a tether that is attached within the XNTP at positions that allow for controlled expansion by cleavage of the cleavable bond (e.g. a tether linking the 5’ a-phosphate and the nucleobase). A tether typically comprises a polymer. An XNTP can thus be present in a constrained configuration (when the cleavable bond is still intact) or in an expanded configuration (when the cleavable bond has been cleaved, e.g. via acid treatment).

[0037] Xpandomer: An “Xpandomer” or “Xp” refers to a molecule consisting of at least two monomers derived from XNTPs. An Xpandomer is obtainable, for example, by polymerase-mediated replication of a template nucleic acid using XNTPs as polymerase substrates to yield a Xp complementary strand. An expanded configuration of the Xpandomer can be obtained by cleavage of the phosphoramidate bond in the XNTPs, e.g. via acid treatment.II. Compositions

[0038] In some embodiments, the disclosure relates to an aqueous composition for replicating a nucleic acid template, the composition comprising a polymerase, a manganese or magnesium salt, nucleotides or nucleotide analogs, and D2O.

[0039] 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 less D2O, 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.

[0040] In some embodiments, the composition is capable of staying frozen at -20 °C for at least one day. In some embodiments, the composition is capable of staying frozen at -20 °C for at least one week. In some embodiments, the composition is capable of staying frozen at - 20 °C for at least two weeks. In some embodiments, the composition is capable of staying frozen when snap frozen and placed at -20 °C for at least one day. In some embodiments, the compositionis capable of staying frozen when snap frozen and placed at -20 °C for at least one week. In some embodiments, the composition is capable of staying frozen when snap frozen and placed at -20 °C for at least two weeks. Unless stated otherwise, frozen in the context of this application means frozen solid.

[0041] In some embodiments, the aqueous composition is suitable for replicating a nucleic acid template after storage at -20°C for at least one day, such as after storage at -20°C for one day, one week or two weeks.

[0042] 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.

[0043] In some embodiments, the polymerase is a DNA polymerase with strand displacement activity. The strand displacement activity describes the ability to displace downstream DNA hybridized to the nucleic acid template encountered during DNA synthesis. DNA polymerase with or without strand displacement activity are well-known in the art. Examples of DNA polymerases with strong strand displacement activity include Bst wildtype polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, Bsu polymerase or Klenow fragment. In some embodiments, the polymerase is a DNA polymerase with a similar or stronger strand displacement activity compared to Bst wildtype polymerase. For synthesis of Xpandomers, polymerases will typically be used comprising mutations that sterically allow the use of XNTPs as substrates. A suitable class of polymerases for incorporating XNTPs includes the translesion DNA polymerase family (i.e. Y-family polymerase) that includes e.g. the Dpo4 polymerase, polymerase K, or polymerase q, or variants thereof. Alternatives to Y-family polymerases include, for example, Therminator (Gardner et al. 2019, Front Mol Biosci. 6:28; available commercially under catalog # M0261 from New England Biolabs) or PrimPol (Rudd et al., 2014, Mol Cell Oncol. 1(2): e960754), or variants thereof.

[0044] Suitable variants include e.g. modified Dpo4 polymerases as described in WO 2017 / 087281, WO 2018 / 204707, WO 2019 / 118372, or WO 2025 / 082960, which are herein incorporated by reference in their entireties. For example the Dpo4 variant given as SEQ ID NO: 1, designated Dpo4_l herein, has the following amino acid substitutions with respect to wildtype Dpo4: F37T_D39L_K56Y_A57S_I59M_E63R_M76W_K78E_E79P_Q82W_Q83G_S86E_K152A_I153V_A155G D156S_M157K_D179N_P184Q_G187P_N188Y I189F E192QI248T_S272C_V 289W_T290R_E291 S D292R L293 W_D294N_I295 S_V296Q_S297Y_G299W_R300S_T301 W K321Q E324K E325K E327KA341-352. Other suitable examples include Dpo4 2-15, provided respectively as SEQ ID NOs: 2-15, respectively.

[0045] In some embodiments, the Dpo4 polymerase variant is selected from the group consisting of SEQ ID NOs: 1-15.

[0046] 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 SEQ ID NO: 1. Such a polymerase has DNA-dependent DNA polymerase activity, and more specifically, is capable of using XNTPs as polymerization substrate.

[0047] In some embodiments, the aqueous composition comprises a manganese salt. In some embodiments, the manganese salt is MnCU.

[0048] In some embodiments, the aqueous composition comprises a magnesium salt. In some embodiments, the manganese salt is MgCh.

[0049] In some embodiments, the nucleotides or nucleotide analogs comprise one or more nucleotide analogs selected from the group consisting of 7-deaza dGTP, N4Me dCTP and 2,6 diaminopurine (DAP).

[0050] In some embodiments, the nucleotides or nucleotide analogs comprise one or more XNTPs. In some embodiments, the composition comprises four different types of XNTPs, wherein the four different types base pair with guanine, adenine, thymine and cytosine, respectively. In some embodiments, the composition comprises four different types of XNTPs at isomolar amounts. In some embodiments, an XNTP comprises a phosphorami date bond, wherein the phosphoramidate bond links the alpha phosphate to the 5’ carbon, and a tether wherein the tether is a polymer j oined at a first end to the alpha phosphate and at a second end to the nucleobase.

[0051] In some embodiments, the polymerase is present at a concentration of 0.02- 0.2 pg / pl.

[0052] In some embodiments, the manganese salt is present at a concentration of 0.1-1 mM. In some embodiments, the magnesium salt is present at a concentration of 1-4.5 mM.

[0053] In some embodiments, the nucleotides or nucleotide analogs are present at a concentration of 20-150 pM.

[0054] 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 hydroxy anisole (BHA), a polyphosphate, urea, and a polymerase enhancing molecule (PEM).

[0055] In some embodiments, the buffering agent is selected from the group consisting of TrisCi, TrisOAc, NFEOAc, MES, and HEPES.

[0056] 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, NaSCU, sodium carbonate, sodium bicarbonate, sodium hexanoate, sodium glutamate, sodium perchlorate, CsCl, LiCl, LiOAc, LiF, lithium carbonate, LiPCE, KC1, KOAc, KF, KSO4, potassium phosphate monobasic, potassium phosphate dibasic, potassium carbonate, potassium bicarbonate, potassium glutamate, NH4CI, NH4F, NEEOAc, NH4SO4, NFUBr, 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.

[0057] In some embodiments, the sugar is maltose, trehalose, cellobiose or sucrose.

[0058] In some embodiments, the SSB is selected from the group consisting of KOD (SEQ ID NO: 16), Gp32 (SEQ ID NO: 17), TTH (SEQ ID NO: 18), SSB1 (SEQ ID NO: 19), RecA (SEQ ID NO: 20), RPA (SEQ ID NO: 21), and NCp7 (SEQ ID NO: 22).

[0059] In some embodiments, the imidazole includes (apart from imidazole as such) imidazole derivatives, such as imidazole chloride, imidazole acetate, 1 -methylimidazole, 2- methylimidazole, 1 -ethylimidazole, l-ethyl-3-methylimidazolium chloride, 2-methyl-2- imidazoline, l-butyl-3-methylimidazolium chloride, 1-methylimidazolium chloride, l-hexyl-3- methylimidazolium, 3 -octyl- 1-methylimidazolium, or l-decyl-3-methylimidazolium.

[0060] In some embodiments, the alkanediol is ethylenglycol, a propanediol, a butanediol, a pentanediol, or a hexanediol.

[0061] 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.

[0062] In some embodiments, PEG is PEG5k to PEG25k, such as PEG5k, PEG8k, PEGlOk, PEG15k or PEG20k.

[0063] In some embodiments, the polyphosphate is tripolyphosphate, tetrapolyphosphate, pentapolyphosphate, hexapolyphosphate, trimetaphosphate (TMP), hexametaphosphate (HMP), or polyphosphate 60.

[0064] In some embodiments, the PEM is a compound of the following formula that increases the processivity, rate, or fidelity of the nucleic acid polymerase reaction:

[0065] or a solvate, hydrate, tautomer, chelate or salt thereof, wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4; m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2;Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;L is a linking group;M is, at each occurrence, independently selected from hydrogen, halogen and Ci-C4alkyl;Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, Ci-C 6 alkyl, Ci-Ce haloalkyl , Ci-Cg heteroalkyl, Ci- C6cycloloalkyl, -OR0, -CONH2, -C(O)NR1R1', -C(O)(CH2)aNR1R1', -NR'R1', - NR1C(O)R3, -C(O)SR3, -COR3, -CO(CH2)aOC(O)R3, -OC(O)R3, -C(O)OR3, -C-O- R3, mercaptan, -R4-H, -SOR1, -S(O)2R1, -S(O)2NR1R1', -CH2-NR1S(O)2R3, - NR'S(O)2R3, and -C(CH3)=N-(phenyl)-O-CH2-C-CH;R° is, at each occurrence, independently selected from Ci-Ce alkyl, Ci-Ce haloalkyl, C2-Ce alkenyl, C2-Ce alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;R1and R1are, at each occurrence, independently selected from H, hydroxyl, Ci-Ce alkyl, C1-C6 haloalkyl, C1-C20 heteroalkyl, C1-C10 heteroalkyl-NH2, C2-Ce alkenyl, C2-Ce alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl (e.g., substituted with one or more R3groups), substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl, benzyl, -C(=NH)NH2, -CH2CO2R0, -C(H)(CHa(COOH))2, - CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R3, (CH2)aC(O)NH(CH2)aC(O)OR3, -(CH2)a-CN, spermine, -(CH2)a-NH2, -C(O)R3, - (CH2)aC(O)OR3, -(CH2)aOR3, -C(H)((CH2)aOH)a', -CHCH(OH)(CH2)aOH, - (CH2)aOH, CI-C6-C(O)OH, -(CH2)a-heterocycle which may be substituted (e.g., such as with an R3group) or unsubstituted,wherein R1and R1together form a substituted or unsubstituted heterocyclic ring, including, but not limited to, azetidine, pyrrolidine, piperidine, piperazine, morpholine,R2is, at each occurrence, independently selected from C2-C6 alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;R3is, at each occurrence, independently selected from H, Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, -Ci-Ce-OH, -Ci-Ce-C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;R4is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene, wherein the heteroatom is O, S, NH, or a combination thereof;Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, - (CH2)aAr2, -(CH2)3PO(OEt)2, or -CH2CO2Me;Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G1, G2, G3, G4and G5, wherein: when Ar2 is monosubstituted, G1is, at each occurrence, independently selected from oxo, Ci-Ce alkyl, Ci-Ce haloalkyl, -E-O-R3, -E-C(R1)(R1')(R3), -NH2, -N02, -SO3R3, -SO3O , -SO3N(H)(R1), -E-C(O)R3, -E-CO2H, -B(OH)2, -C(O)NR1R1', -E- PO(OR1)2, and aryl substituted with G2, G3, G4and G5;G2, G3, G4and G3are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO2, -SO3R3, Ci-Ce alkyl, Ci- C6haloalkyl, -E-O-R3, — E-(CH2)aC(O)R3, -E-CO2H, -E-CHO, -E-C(O)R3, -E- C(O)NH(OH), -E-C(O)NHR1, -E-C(O)N(H)C(H)(R1)(R1'), -E-C(O)N(R1)((CH2)aP(O)(O)OH)2, -E-C(O)N(R1)((CH2)aSO3H), -E-C(O)NR1R1, -E- NRJRr, -E-OR2, -C(O)-heterocycloalkyl (where the heterocycloalkyl is substituted or unsubstituted), -C(O)-N(H)-CH(COOH)((CH2)a-heteroaryl) (where heteroaryl is substituted or unsubstituted), wherein E is, at each occurrence, independently selected from a direct bond, and Ci-Ce alkylene.

[0066] In some embodiments, the PEM is a compound of the following formula that increases the processivity, rate, or fidelity of the nucleic acid polymerase reaction:

[0067] wherein independently at each occurrence: m is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2; Ari is optionally substituted aryl; Ar2 is selected from 5- and 6-membered monocyclic aromatic rings and 9- and 10-membered fused bicyclic rings comprising two 5- and / or 6- membered monocyclic rings fused together, where at least one of the two monocyclic rings is an aromatic ring, where Ar2 is optionally substituted with one or more substituents selected from halide, Ci-Ce alkyl, Ci-Ce haloalkyl, ECO2R0, ESO3R0, E- CONH2, E-CHO, E-C(O)NH(OH), E-N(R°)2, and E-OR°, where E is selected from a direct bond and Ci-Cealkylene; and R° is selected from H, Ci-Cealkyl and Ci-Ce haloalkyl, M is selected from hydrogen, halogen and C1-C4 alkyl; and L is a linking group; or a solvate, hydrate, tautomer, chelate or salt thereof.

[0068] In some embodiments, Ari is an aryl group, also referred to as an aromatic moiety. The aromatic moiety may be a carbocyclic or heterocyclic aromatic moiety, where eachof the aromatic ring atoms is carbon in a carbocyclic aromatic moiety, while at least one of the aromatic ring atoms is nitrogen, oxygen or sulfur in a heterocyclic aromatic moiety. In some embodiments, Ari may be a 5- or 6-membered monocyclic aromatic ring that is optionally substituted, where optionally at least one ring atom may be selected from oxygen, sulfur and nitrogen, with the remainder being carbon atoms. Ari may include, for example, pyridine, N-ethyl isonicotinamide, N,N-diethyl isonicotinamide, pyridine-4-ethylsulfonamide, 4-methyl pyridine, ethyl isonicotinate, 4-cyanopyridine, and furan.

[0069] In some embodiments, Ar2 is a 5- or 6-membered monocyclic aromatic rings, where Ar2 is optionally substituted with one or more substituents selected from halide, Ci- C6alkyl, Ci-C6haloalkyl, ECO2R°, ESO3R0, E-CONH2, E-CHO, E-C(O)NH(OH), E-N(R°)2, and E-OR°, where E is selected from a direct bond and Ci-Ce alkylene; and R° is selected from H, Ci- Ce alkyl and Ci-Ce haloalkyl, M is selected from hydrogen, halogen and Ci-C4alkyl; and L is a linking group; or a solvate, hydrate, tautomer, chelate or salt thereof. In some embodiments, Ar2 is a 5- or 6-membered monocyclic aromatic rings, where Ar2 is optionally substituted with one or more substituents selected from Ci-Ce alkyl, Ci-Ce haloalkyl, ECO2R0, ESO3R0, E-CONH2, E- CHO, E-C(O)NH(OH), E-N(R°)2, and E-OR°, where E is a direct bond; and R° is selected from H, Ci-C6alkyl and Ci-Cehaloalkyl, M is hydrogen; and L is a linking group; or a solvate, hydrate, tautomer, chelate or salt thereof. Ar2 includes, for example, substituted (e.g. hydroxyl or trifluoromethyl) or unsubstituted benzoic acid, substituted (e.g. hydroxyl or trifluoromethyl) or unsubstituted benzamide (optionally with one or two N-linked Cl -C4 alkanes) or substituted (e.g. hydroxyl or trifluoromethyl) or unsubstituted benzenesulfonic acid. In some embodiments, Ar2 is salicilic acid, 2-hydroxy benzoic acid, 2-trifluoromethyl benzoic acid, 2-hydroxy N-hydroxy benzamide, trifluoroacetophenone, and benzenesulfonic acid. For example, Ar2 may be linked to the rest of the molecule in meta or para position with respect to a carboxy (ester), amide, sulfonate or trifluoroacetyl group, when present.

[0070] In some embodiments, n is 0. In some embodiments, m is 2, M is H, and n is 0.

[0071] In embodiments with n = 0, the PEM is a compound of the following formula that increases the processivity, rate, or fidelity of the nucleic acid polymerase reaction:wherein Ari, M, Ar2 and m are as defined herein.

[0072] A halogen can be, for example, F, Cl, Br, or I.

[0073] In some embodiments, the PEM compounds may be any compound disclosed in Applicant’s published PCT applications no.s WO / 2019 / 135975, WO / 2020 / 038682, and WO / 2025 / 137293, the contents of which are herein incorporated by reference in their entireties.

[0074] 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

[0075] The aqueous composition for replicating a nucleic acid template may be used as a premix for part of or all components required for replicating the nucleic acid template. For example, the aqueous composition for replicating a nucleic acid template may comprise a polymerase, a manganese salt, nucleotides or nucleotide analogs, and D2O, and further components required for replication may be added later at a suitable time, e.g. shortly before or at the same with combining the aqueous composition with the nucleic acid template and / or primers. In another example, the aqueous composition for replicating a nucleic acid template may comprise all components required for replicating the nucleic acid template; thereby the composition may serve as master mix that can be simply combined with the nucleic acid template and / or primers.

[0076] 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 nucleic acid comprising two complementary strands that are joined on a first end to a hairpin adapter.

[0077] 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.

[0078] 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.

[0079] In some embodiments, the aqueous composition consists of the following components in water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mM manganese salt, 20-150 pM XNTPs, 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, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 2-8% (w / v) alkanediol, 2-10% (w / v) acetamide, 0.1-1% (w / v) glycerol, 200-400 mM betaine, 10-30% (w / v) PEG, 3-10 % (v / v) NMP, 0.5-1.5 mM BHA, and 1-5 % (v / v) DMSO, and further optionally 0.02-1 pg / pl SSB.

[0080] In some embodiments, the aqueous composition consists of the following components, plus water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mM manganese salt, 20-150 pM XNTPs, 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 HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 200-400 mM betaine, 10-30% (w / v) PEG, 3-10 % (v / v) NMP, 0.5-1.5 mM BHA, and 1-5 % (v / v) DMSO, and further optionally 0.02-1 pg / pl SSB.

[0081] In some embodiments, the aqueous composition consists of the following components, plus water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1mM MnCh, 20-150 pM XNTPs, 20-100 mM buffering agent selected from 20-100 mM Tris and 0-5 mM MES, 100-200 mM inorganic salt, 0.05-0.7 % (w / v) sugar, 0.1-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 200-400 mM betaine, 10-30% (w / v) PEG8k, 3-10 % (v / v) NMP, 0.5-1.5 mM BHA, and 1-5 % (v / v) DMSO, and further optionally 0.02-1 pg / pl SSB.

[0082] In some embodiments, the aqueous composition consists of the following components in water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mM manganese salt, 20-150 pM XNTPs, 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 HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w / v) PEG, and 2-8% (w / v) alkanediol, and further optionally 0.02-1 pg / pl SSB.

[0083] In some embodiments, the aqueous composition consists of the following components in water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mM MnCh, 20-150 pM XNTPs, 20-100 mM buffering agent selected from 20-100 mM Tris and 0-5 mM MES, 100-200 mM inorganic salt, 0.05-0.7 % (w / v) sugar, 0.1-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w / v) PEG8k, and 2-8% (w / v) alkanediol, and further optionally 0.02-1 pg / pl SSB.

[0084] In some embodiments, the aqueous composition consists of the following components in water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mM manganese salt, 20-150 pM XNTPs, 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 HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w / v) PEG, and 2-10% (w / v) acetamide, and further optionally 0.02-1 pg / pl SSB.

[0085] In some embodiments, the aqueous composition consists of the following components in water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mM MnCh, 20-150 pM XNTPs, 20-100 mM buffering agent selected from 20-100 mM Tris and 0-5 mM MES, 100-200 mM inorganic salt, 0.05-0.7 % (w / v) sugar, 0.1-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w / v) PEG8k, and 2-10% (w / v) acetamide, and further optionally 0.02-1 pg / pl SSB.

[0086] In some embodiments, the aqueous composition consists of the following components in water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mMmanganese salt, 20-150 pM XNTPs, 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 HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w / v) PEG, 0.1-1% (w / v) glycerol, 2-8% (w / v) alkanediol, and 2-10% (w / v) acetamide, and further optionally 0.02-1 pg / pl SSB.

[0087] In some embodiments, the aqueous composition consists of the following components in water (including added D2O): 0.02-0.2 pg / pl Dpo4 polymerase variant, 0.1-1 mM MnCh, 20-150 pM XNTPs, 20-100 mM buffering agent selected from 20-100 mM Tris and 0-5 mM MES, 100-200 mM inorganic salt, 0.05-0.7 % (w / v) sugar, 0.1-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w / v) PEG8k, 0.1-1% (w / v) glycerol, 2-8% (w / v) alkanediol, and 2-10% (w / v) acetamide, and further optionally 0.02-1 pg / pl SSB.

[0088] In some embodiments, any of the aqueous compositions disclosed herein may include more than one PEM, for example the aqueous compositions may include one or more, two or more, three or more, or four or more PEMs.

[0089] Specific examples of the aqueous composition are given in the following Table 2, wherein the composition consists of the components given in water (including added D2O).Table 2

[0090] In some embodiments, the aqueous composition is selected from compositions Al-Zl in Table 2.

[0091] In some embodiments, the aqueous composition is selected from compositions A2-Z2 in Table 2.

[0092] In some embodiments, the aqueous composition is selected from compositions A3-Z3 in Table 2.

[0093] In some embodiments, the aqueous composition is selected from compositions A4-Z4 in Table 2.

[0094] In some embodiments, the aqueous composition is selected from compositions A5-Z5 in Table 2.

[0095] In some embodiments, the aqueous composition is selected from compositions A6-Z6 in Table 2.

[0096] It has further been found that an aqueous composition as disclosed herein may allow efficient replication of a nucleic acid template (e.g. to generate an Xpandomer (complementary) replicate strand), regardless of the presence of added D2O. The disclosure thus also provides aqueous compositions for replicating a nucleic acid template, consisting of the components given in any one of

[0077] -

[0085] or Table 2 in water (without added D2O).III. Methods

[0097] The composition disclosed herein can be used, for example, for replicating a nucleic acid template. An application may be, for example, the synthesis of Xpandomer for sequencing by expansion.

[0098] The disclosure provides a method for storing the aqueous composition comprising D2O disclosed herein, the method comprising storing the aqueous composition at a temperature between -10°C and -30°C. In some embodiments, the method comprises storing the composition at -15°C to - 25°C, such as at -20 °C. In some embodiments, the aqueous composition is stored in a frozen state. In some embodiments, the aqueous composition is stored for at least one day, such as for at least one week or at least two weeks. In some embodiments, the composition is stored for a period of one day to three months.

[0099] The disclosure also provides a method for replicating a nucleic acid template, comprising contacting an aqueous composition as disclosed herein with the nucleic acid template. In some embodiments, the method further comprises (b) incubating the product of step (a) at a temperature suitable for replicating the nucleic acid template.

[0100] When the aqueous composition comprises a nucleic acid template, the disclosure also provides a method for replicating a nucleic acid template, the method comprising incubating the aqueous composition as disclosed herein at a temperature suitable for replicating the nucleic acid template.

[0101] The method provides at least one replicate strand of the nucleic acid template. A replicate strand can either be complementary to the nucleic acid template, or it can have the same sequence as the template nucleic acid. When using XNTPs in the composition, a replicate strand generated by such a method will be an Xpandomer (in constrained configuration).Typically, a single complementary replicate strand of the nucleic acid template is generated when using XNTPs.

[0102] The method of replicating a nucleic acid template may be preceded by a method for storing the aqueous composition comprising D2O as disclosed herein, wherein the composition is thawed before contacting the composition comprising D2O as disclosed herein with the nucleic acid template.

[0103] The temperature suitable for replicating the nucleic acid template is not particularly limited. In some embodiments, the temperature suitable for replicating the nucleic acid template is a temperature of 37°C or higher. In some embodiments, the temperature suitable for replicating the nucleic acid template is a temperature of 37-45 °C. In some embodiments, the temperature suitable for replicating the nucleic acid template is a temperature of 40-45 °C, such as 42 °C.

[0104] The type of nucleic acid template is not particularly limited, and includes a DNA or RNA template. In some embodiments, the nucleic acid template is a DNA template, such as a genomic DNA or a cDNA template. In some embodiments, the DNA template is a cell-free DNA (cfDNA) template.

[0105] The nucleic acid can be part of a library of nucleic acids. For example, the library can be a library of genomic DNA, cDNA or cfDNA.

[0106] In some embodiments, the nucleic acid template is a duplex nucleic acid template comprising two complementary strands that are joined on a first end to a hairpin adapter. In some embodiments, the duplex nucleic acid template further comprises a Y adapter. In such embodiments, the two complementary strands may be joined on a second end to the Y adapter.

[0107] The replication of a nucleic acid template is typically primed by a primer. Thus, in some embodiments, the method comprises hybridizing a primer to the nucleic acid template to initiate the replication. The design and generation of primers is known in the art. The primer to be used is not particularly limited and can be designed, for example, to hybridize with the nucleic acid at a position so as to allow the generation of the complementary strand to parts of the nucleic acid that are of interest, including full-length. When a library of nucleic acids is to be sequenced, it is possible, for example, to use a standard primer binding to all nucleic acids of interest in the library, or a random primer mixture.

[0108] In some embodiments, the method can comprise hybridizing a primer to the nucleic acid template, followed by contacting the nucleic acid with an aqueous composition as disclosed herein.

[0109] In some embodiments, the primer is attached to a solid support.

[0110] If necessary, the nucleic acid template can also be denatured, e.g. to facilitate primer hybridization. Means for denaturation are not particularly limited, and include e.g. applying heat (e.g. 90°C-100°C). Thus, in some embodiments, the method can comprise denaturing the nucleic acid template and then hybridizing a primer to the nucleic acid template, followed by contacting the nucleic acid template with an aqueous composition as disclosed herein.

[0111] Typically, the nucleic acid template is replicated by using a polymerase, such as a (DNA-dependent) DNA polymerase.

[0112] The method typically comprises the use of four different types of the nucleoside triphosphate, wherein the four different types of the nucleoside triphosphate base pair with guanine, adenine, thymine and cytosine, respectively.

[0113] The disclosure also provides a method for sequencing a nucleic acid using the aqueous composition comprising XNTPs as disclosed herein.

[0114] The disclosure thus provides a method for determining the sequence of a nucleic acid template, comprising the following steps in order:1) Generating a complementary replicate strand of the nucleic acid template by the method for replicating a nucleic acid template as disclosed herein by using XNTPs as polymerase substrates;2) Selectively cleaving the P-N bond within the XNTPs incorporated into the replicate strand to generate an expanded replicate strand;3) Sequencing the expanded replicate strand,4) Determining the sequence of the nucleic acid template based on the sequence of the expanded replicate strand.

[0115] In some embodiments, the complementary replicate strand is separated from the nucleic acid template after step 1), for example by denaturation. The complementary replicate strand can optionally be purified before proceeding with step 2).

[0116] The P-N bond can be selectively cleaved in step 2) under acidic conditions, for example. This can be achieved by addition of an acid, such as DC1. Cleavage in step 2) typicallyyields an Xpandomer in expanded configuration. The product of step 2) can optionally be purified before proceeding with step 3).

[0117] In preferred embodiments, the expanded complementary replicate strand is sequenced in step 3) by nanopore-based sequencing. Methods for nanopore-based sequencing are known in the art, see e.g. WO 2020 / 236526. For example, nanopore-based sequencing can comprise:(a) providing a chip for nanopore-based sequencing comprising:(i) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side;(ii) a nanopore inserted into the barrier, wherein the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier;(b) contacting the cis side of the barrier with the expanded complementary strand;(c) applying a voltage across the barrier of the chip to translocate the expanded complementary strand to the trans side;(d) determining one or more changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by the expanded complementary strand during the translocation; and(e) determining, based on the one or more changes in the electrical characteristic of the nanopore, a sequence for the expanded complementary strand.

[0118] The barrier is typically a lipid bilayer membrane, such as a DPhPE / hexadecane bilayer membrane. A nanopore, such as a a-hemolysine nanopore, can be inserted into the membrane by electroporation in a buffer, such as a buffer of 2 M NH4CI and 100 mM HEPES, pH 7.4. The cis well can be perfused with a buffer containing 0.4M NH4CI, 600mM GuanCl, lOOmM HEPES; pH 7.4, and 5% glycerol and the trans well can be perfused with buffer containing 0.4M NH4C1, 600mM GuanCl, 5% ethyl acetate, lOmM HEPES; pH 7.4, before introducing the Xpandomer to the cis side for sequencing.IV. Examples

[0119] Aqueous compositions comprising a Dpo4 polymerase variant, MnCE and XNTPs according to the disclosure were prepared with or without added D2O, snap frozen in liquid nitrogen and then placed at -20 °C for at least one day. The compositions to which D2O had beenadded remained in a frozen solid state, whereas the composition without added D2O at least partially thawed at -20 °C (see Fig. 1). The storage at -20 °C was tested with 50% D2O and close to 100% D2O, and in both cases the composition remained frozen solid after at least one day of storage.

[0120] Compositions were then used for Xpandomer synthesis. Xpandomers can be synthesized and sequenced according to the methods previously described in e.g. Example 35- 38 of WO 2019 / 135975, e.g. using to the compositions disclosed herein for Xpandomer synthesis. The compositions without added D2O that had at least partially thawed after storage at -20 °C were not reasonably suited for Xpandomer synthesis anymore. In contrast, the composition with added D2O allowed for efficient Xpandomer synthesis as exemplified in Fig. 2.SEQUENCES

[0121] SEQ ID NO: 1 (Dpo4_l)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSG MPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDY REAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGSKAKPNGIKVIDDEEVKRLIRELN IADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTR VRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWRSRWNSQYRW S WFPHGISKET A YSE S VKLLQQILKKDKRKIRRIGVRF SKF

[0122] SEQ ID NO: 2 (Dpo4_2)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAG IPIVEAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYR EAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIAADMAKPNGIKVIDDEEVKRLIRELDI ADVPGIGNITAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRV RKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFP HGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKF

[0123] SEQ ID NO: 3 (Dpo4_3)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSG MPIVRAKKILPNAVYLPWREVVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWRSRWNSQYR WSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0124] SEQ ID NO : 4 (Dpo4_4)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREVVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWKQRWNSQYR WSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0125] SEQ ID NO: 5 (Dpo4_5)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWKSYWNSQYR WSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0126] SEQ ID NO : 6 (Dpo4 6)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWKSRWNSQYR WSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0127] SEQ ID NO : 7 (Dpo4_7)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRT RVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWKSYWNSTYR WSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0128] SEQ ID NO: 8 (Dpo4_8)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWRSRWNSQYR WSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0129] SEQ ID NO : 9 (Dpo4_9)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWKQRWNSQYRWSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0130] SEQ ID NO: 10 (Dpo4_10)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREVVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGRMAKPNGIKVIDDEEVKRLIRELDIADVKGIPYFTAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWKSYWNSQYR WSWFPHGISKETAYSESVKLLQQILKKDKRKIRRIGVRFSKF

[0131] SEQ ID NO: 11 (Dpo4_l l)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGSKAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWHTRHYSTYRYKWFPHGISKETAYSESVKLLQKILAKDTRKIRRIGVRFSKF

[0132] SEQ ID NO: 12 (Dpo4_12)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGSKAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAGMDRHYSTYRYKWFPHGISKETAYSESVKLLQKILAKDTRKIRRIGVRFSKF

[0133] SEQ ID NO: 13 (Dpo4 13)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGSKAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWDQRHYSTYRYKWFPHGISKETAYSESVKLLQKILAKDTRKIRRIGVRFSKF

[0134] SEQ ID NO: 14 (Dpo4_14)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGSRGKPGGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAWTGRHYSTYRYKWFPHGISKETAYSESVKLLQKILAKDTRKIRRIGVRFSKF

[0135] SEQ ID NO: 15 (Dpo4_15)MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRTELSGAVATANYEARKFGVYSGMPIVRAKKILPNAVYLPWREPVYWGVSERIMNLLREYSEKIEIASIDEAYLDISDKVRDY REAYNLGLEIKNKILEKEKITVTVGISKNKVFAAVAGSKAKPNGIKVIDDEEVKRLIRELNIADVQGIPYFTAQKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTR VRKSIGRTVTMKRNSRNLEEIKPYLFRAIEECYYKLDKRIPKAIHVVAYFDRHYSTYRYK WFPHGISKETAYSESVKLLQKILAKDTRKIRRIGVRFSKF

[0136] SEQ ID NO: 16 (KOD, Thermococcus kodakarensis)MEVLTKDEIINRIIRERGLSRSEIEEKIRELAKMHGVSENAAAVMLAEELGVSLGKEEEMLYIKDLVPGMTGVNIVARIKRKFPPREYTRRDGSTGRVADLIIYDSTGQARLVLWDAMVAKYYDDLNVGDVIKVIDPTVKEGMRGVELHANFRTRIIKNPEDPRVEEIPPLEEVRSYNYRRVQIKELQGGERFVEVRGTIAKLYRVLVYDACPECRRRVDYDPSTDTWICPEHGPVNP VKITVLDFGLDDSTGYIRTTLFGDSAAELIGEEPEVIDEKLKKLIDEGLTPKEAGKRLAED EYYPLIGKEIVVRGSVVEDKFLGTLFKARSWDEVNEKAEIERVRRELYRELKEYGLE

[0137] SEQ ID NO: 17 (Gp32, Bacteriophage T4)MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSY WANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELN TKFGQVMGT AVMGGAAAT AAKKADKV ADDED AFNVDDFNTKTEDDFMS SSSGSSSSA DDTDLDDLLNDL

[0138] SEQ ID NO: 18 (TTH, Thermus thermophilus)MARGLNRVFLIGALATRPDMRYTPAGLAILDLTLAGQDLLLSDNGGEREVSWYHRVRLLGRQAEMWGDLLDQGQLVFVEGRLEYRQWEREGEKRSELQIRADFLDPLDDRGKERAEDSRGQPRLRAALNQVFLMGNLTRDPELRYTPQGTAVARLGLAVNERRQGAEERTHFV E VQ A WRDLAE W A AELRKGDGLF VIGRLVNDSWTS S SGERRFQTRVEALRLERPTRGPA QAGGSRSREVQTGGVDIDEGLEDFPPEEELPF

[0139] SEQ ID NO: 19 (SSB1, Escherichia coli)MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHR VVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLG GRQGGGAPAGGNIGGGQPQSGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMD FDDDIPF

[0140] SEQ ID NO: 20 (RecA, Escherichia coli)MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGSLSLDIALGAGGLPM GRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIYARKLGVDIDNLLCSQP DTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDSHMGLAARMMSQAMRK LAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASVRLDIRRIGAVKEGENV VGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVKEKLIEKAGAWYSYKGE KIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSVDDSEGVAETNEDF

[0141] SEQ ID NO : 21 (RPA, homo sapiens)MVGQLSEGAIAAIMQKGDTNIKPILQVINIRPITTGNSPPRYRLLMSDGLNTLSSFMLATQ LNPLVEEEQLSSNCVCQIHRFIVNTLKDGRRVVILMELEVLKSAEAVGVKIGNPVPYNEG LGQPQVAPPAPAASPAASSRPQPQNGSSGMGSTVSKAYGASKTFGKAAGPSLSHTSGGT QSKVVPIASLTPYQSKWTICARVTNKSQIRTWSNSRGEGKLFSLELVDESGEIRATAFNE QVDKFFPLIEVNKVYYFSKGTLKIANKQFTAVKNDYEMTFNNETSVMPCEDDHHLPTV QFDFTGIDDLENKSKDSLVDIIGICKSYEDATKITVRSNNREVAKRNIYLMDTSGKVVTATLWGEDADKFDGSRQPVLAIKGARVSDFGGRSLSVLSSSTIIANPDIPEAYKLRGWFDAE GQALDGVSISDLKSGGVGGSNTNWKTLYEVKSENLGQGDKPDYFSSVATVVYLRKENC MYQACPTQDCNKKVIDQQNGLYRCEKCDTEFPNFKYRMILSVNIADFQENQWVTCFQE SAEAILGQNAAYLGELKDKNEQAFEEVFQNANFRSFIFRVRVKVETYNDESRIKATVMD VKPVDYREYGRRLVMSIRRSALM

[0142] SEQ ID NO : 22 (NCp7, HIV- 1 )MQRGNFRNQRKMVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWP S YKGRPGNF

Claims

What is claimed is:

1. An aqueous composition for replicating a nucleic acid template, the composition comprising a polymerase, a manganese or magnesium salt, nucleotides or nucleotide analogs, and D2O.

2. The aqueous composition of claim 1, comprising at least 40% D2O.

3. The aqueous composition of claim 1 or 2, wherein the composition is capable of staying frozen at -20 °C for at least one day.

4. The aqueous composition of any one of claims 1-3 that is suitable for replicating a nucleic acid template after storage at -20°C.

5. The aqueous composition of any one of claims 1-4, wherein the polymerase is a strand displacing DNA polymerase.

6. The aqueous composition of any one of claims 1-4, wherein the polymerase is Bst wildtype polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, Bsu polymerase or Klenow fragment.

7. The aqueous composition of any one of claims 1-4, wherein the polymerase is a Y family DNA polymerase.

8. The aqueous composition of claim 7, wherein the Y family DNA polymerase is Dpo4 polymerase, or a variant thereof.

9. The aqueous composition of any one of claims 1-8, wherein the manganese or magnesium salt is MnCh or MgCh.

10. The aqueous composition of any one of claims 1-9, wherein the nucleotides or nucleotide analogs comprise one or more nucleotide analogs selected from the group consisting of 7-deaza dGTP, N4Me dCTP, and 2,6 diaminopurine (DAP).

11. The aqueous composition of any one of claims 1-10, wherein the nucleotides or nucleotide analogs comprise one or more expandable nucleoside triphosphates (XNTPs).

12. The aqueous composition of claim 11, wherein the one or more XNTPs comprise a phosphorami date bond, wherein the phosphoramidate bond links the alpha phosphate to the 5’carbon, and a tether wherein the tether is a polymer joined at a first end to the alpha phosphate and at a second end to the nucleobase.

13. The aqueous composition of any one of claims 1-12, further comprising one or more components selected from the group consisting 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, urea and a polymerase enhancing molecule (PEM).

14. The aqueous composition of claim 13, wherein the buffering agent is selected from TrisCi, TrisOAc, NH4OAc, MES, and HEPES.

15. The aqueous composition of claim 13 or 14, wherein the salt is selected from the group consisting of NaCl, NaBr, NaOAc, NaF, sodium formate, sodium phosphate monobasic, sodium phosphate dibasic, NaSCE, sodium carbonate, sodium bicarbonate, sodium hexanoate, sodium glutamate, sodium perchlorate, CsCl, LiCl, LiOAc, LiF, lithium carbonate, LiPCE, KC1, KOAc, KF, KSCE, potassium phosphate monobasic, potassium phosphate dibasic, potassium carbonate, potassium bicarbonate, potassium glutamate, NEUC1, NEUF, NEEOAc, NEUSCE, NEUBr, ammonium citrate, ammonium carbonate, ammonium bicarbonate, ammonium sulfite, ammonium glutamate, ammonium phosphate monobasic, tetramethylammonium chloride (TMAC1), trimethylamine N-oxide (TMAO), tetraethyl am monium chloride (TEACI), guanidinium chloride, guanidinium thiocyanate, guadinium carbonate.

16. The aqueous composition of any one of claims 13-15, wherein the sugar is maltose, trehalose, cellobiose or sucrose.

17. The aqueous composition of any one of claims 13-16, wherein the SSB is selected from the group consisting of KOD (SEQ ID NO: 16), Gp32 (SEQ ID NO: 17), TTH (SEQ ID NO: 18), SSB1 (SEQ ID NO: 19), RecA (SEQ ID NO: 20), RPA (SEQ ID NO: 21), and NCp7 (SEQ ID NO: 22).

18. The aqueous composition of any one of claims 13-17, wherein the alkanediol is ethylene glycol, a propanediol, a butanediol, a pentanediol, or a hexanediol.

19. The aqueous composition of claim 18, wherein the pentanediol is 1,2-pentanediol, 1 ,3- pentanediol, 1,5-pentanediol, or 2,4-pentanediol.

20. The aqueous composition of claim 18, wherein the hexanediol is 1,6-hexanediol or 2,5- hexanediol.

21. The aqueous composition of any one of claims 13-20, wherein the PEM is a compound of the following formula that increases the processivity, rate, or fidelity of the nucleic acid polymerase reaction:or a solvate, hydrate, tautomer, chelate or salt thereof, wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4; m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2;Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;L is a linking group;M is, at each occurrence, independently selected from hydrogen, halogen and Ci-C4alkyl;Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole,wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, Ci-C 6 alkyl, Ci-Ce haloalkyl , Ci-Ce heteroalkyl, Ci- C6cycloloalkyl, -OR0, -CONH2, -C(O)NR1R1', -C(O)(CH2)aNR1R1', -NRJRr, - NR1C(O)R3, -C(O)SR3, -COR3, -CO(CH2)aOC(O)R3, -OC(O)R3, -C(O)OR3, -C-O- R3, mercaptan, -R4-H, -SOR1, -S(O)2R1, -S(O)2NR1R1', -CH2-NR1S(O)2R3, - NR1S(O)2R3, and -C(CH3)=N-(phenyl)-O-CH2-C-CH;R° is, at each occurrence, independently selected from Ci-Ce alkyl, Ci-Ce haloalkyl, C2-Cg alkenyl, C2-Cs alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;R1and R1' are, at each occurrence, independently selected from H, hydroxyl, Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-C2o heteroalkyl, C1-C10 heteroalkyl-NH2, C2-C > alkenyl, C2-Ce alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl (e.g., substituted with one or more R3groups), substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl, benzyl, -C(=NH)NH2, -CH2CO2R°, -C(H)(CHa(COOH))2, - CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R3, (CH2)aC(O)NH(CH2)aC(O)OR3, -(CH2)a-CN, spermine, -(CH2)a-NH2, -C(O)R3, - (CH2)aC(O)OR3, -(CH2)aOR3, -C(H)((CH2)aOH)a', -CHCH(OH)(CH2)aOH, - (CH2)aOH, CI-C6-C(O)OH, -(CH2)a-heterocycle which may be substituted (e.g., such as with an R3group) or unsubstituted,wherein R1and R1together form a substituted or unsubstituted heterocyclic ring, including, but not limited to, azetidine, pyrrolidine, piperidine, piperazine, morpholine,R2is, at each occurrence, independently selected from C2-Ce alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;R3is, at each occurrence, independently selected from H, Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-Ce heteroalkyl, -Ci-Ce-OH, -Ci-Ce-C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;R4is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene, wherein the heteroatom is O, S, NH, or a combination thereof;Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, - (CH2)aAr2, -(CH2)3PO(OEt)2, or -CH2CO2Me;Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G1, G2, G3, G4and G5, wherein: when Ar2 is monosubstituted, G1is, at each occurrence, independently selected from oxo, Ci-Ce alkyl, Ci-Ce haloalkyl, -E-O-R3, -E-C(R1)(R1)(R3), -NH2, -NO2, - SO3R3, -SO3O , -SO3N(H (R' ), -E-C(O)R3, -E-CO2H, -B(OH)2, -C(O)NR1R1', -E- PO(OR1)2, and aryl substituted with G2, G3, G4and G5;G2, G3, G4and G are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO2, -SO3R3, Ci-Ce alkyl, Ci- Ce haloalkyl, -E-O-R3, — E-(CH2)aC(O)R3, -E-CO2H, -E-CHO, -E-C(O)R3, -E- C(O)NH(OH), -E-C(O)NHR1, -E-C(O)N(H)C(H)(R1)(R1'), -E-C(O)N(R1)((CH2)aP(O)(O)OH)2, -E-C(O)N(R1)((CH2)aSO3H), -E-C(O)NR1R1, -E- NRJRr, -E-OR2, -C(O)-heterocycloalkyl (where the heterocycloalkyl is substituted or unsubstituted), -C(O)-N(H)-CH(COOH)((CH2)a-heteroaryl) (where heteroaryl is substituted or unsubstituted), wherein E is, at each occurrence, independently selected from a direct bond, and Ci-Ce alkylene.

22. The aqueous composition of claim 21, where the PEM compound is selected from the group consisting of:

23. The aqueous composition of any one of claims 1-22, further comprising a nucleic acid template.

24. The aqueous composition of claim 23, wherein the nucleic acid template is a duplex nucleic acid comprising two complementary strands that are joined on a first end to a hairpin adapter.

25. A method for storing the aqueous composition of any one of claims 1-24, comprising storing the composition at a temperature between -10°C and -30°C.

26. The method of claim 25, comprising storing the composition at -20 °C.

27. The method of claim 25 or 26, wherein the aqueous composition is stored in a frozen state.

28. A method for replicating a nucleic acid template, comprising the step:(a) contacting the aqueous composition of any one of claims 1-22 with a nucleic acid template.

29. The method of claim 28, further comprising:(b) incubating the product of step (a) at a temperature suitable for replicating the nucleic acid template.

30. A method for replicating a nucleic acid template, comprising incubating the composition of claim 23 or 24 at a temperature suitable for replicating the nucleic acid template.31 . The method of claims 29 or 30, wherein the temperature suitable for replicating the nucleic acid template is a temperature of 37°C or higher.

32. The method of any one of claims 29-31, wherein the temperature suitable for replicating the nucleic acid template is a temperature of 37-45 °C.

33. The method of any one of claims 29-32, wherein the temperature suitable for replicating the nucleic acid template is a temperature of 40-45 °C.

34. The method of any one of claims 28-33, wherein the nucleic acid template is comprised in a library of nucleic acids.

35. The method of any one of claims 28-34, wherein the nucleic acid template is a DNA template.

36. The method of any one of claims 28-35, wherein the nucleic acid template is a duplex nucleic acid template comprising two complementary strands that are joined on a first end to a hairpin adapter.

37. The method of claim 36, wherein the two complementary strands are joined on a second end to the Y adapter.

38. The method of any one of claims 28-37, comprising hybridizing a primer to the nucleic acid template to initiate the replication.

39. The method of any one of claims 28-38, wherein the aqueous composition comprises XNTPs.

40. A method for determining the sequence of a nucleic acid template, comprising the following steps in order:1) generating a complementary replicate strand of the nucleic acid template by the method of claim 39;2) selectively cleaving the P-N bond within the XNTPs incorporated into the replicate strand to generate an expanded replicate strand;3) sequencing the expanded replicate strand; and4) determining the sequence of the nucleic acid template based on the sequence of the expanded replicate strand.

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