Methods, Kits, and Systems for Dual Labeling of Nucleic Acids

Abstract

A method is provided for introducing a modification at the end of a nucleic acid, thereby labeling the nucleic acid at a desired moiety including the 3'-end, 5'-end, or both the 3'-end and 5'-end. A kit for introducing such a modification at the end of a nucleic acid is also provided.

Description

Technical Field

[0001] Reference to Electronic Sequence Listing This application includes a sequence listing that was electronically submitted in XML format, which is hereby incorporated by reference in its entirety. The XML copy was created on May 26, 2023, named "YDBL-0005PCTUS-SequenceListing-20230526.xml", and is 7 KB in size. The sequence listing contained in this XML file is part of the specification, and the entirety of it is hereby incorporated by reference.

[0002] The present disclosure relates to modifications at both ends of a nucleic acid, and more specifically, to modifications at both the 3' and 5' ends of a nucleic acid.

Background Art

[0003] Labeling of nucleic acids is commonly performed in biomedical and biological applications, including identification and purification of unknown or target gene fragments, localization of target gene sequences, identification of nucleic acid-protein interactions, and visualization of cell and tissue dynamics. Generally, methods for labeling nucleic acids can be classified into chemical methods or enzymatic methods. Chemical labeling methods involve modifying the structure of a nucleic acid by modifying the 5'-phosphate group, 3'-hydroxyl group, or nucleobase or sugar moiety of the target nucleic acid with a chemically reactive compound such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), imidazole, hydrazine, sodium periodate, and sodium cyanoborohydride, and then attaching a chemical or functional moiety to the desired nucleic acid.

[0004] On the other hand, in enzymatic nucleic acid labeling methods, an enzyme such as alkaline phosphatase, nucleic acid kinase, or DNA / RNA polymerase is used to substitute, add, or incorporate a chemical or functional moiety such as a radioisotope, biotin group, or fluorescently labeled nucleotide into the target nucleic acid, thereby binding the desired label to the nucleic acid.

[0005] Using a number of nucleic acid labeling techniques, nucleic acid probes labeled with fluorophores, enzymes, and nucleotides modified with radioactive phosphates, or digoxigenin or biotin, etc. can be prepared. However, these methods usually involve redundant and complex procedures, use many types of enzymes, reactive chemicals, or radioactive isotopes, which require special enzymes or chemicals, or often require personal training for handling toxic or radioactive substances and waste. Therefore, nucleic acid labeling becomes a tedious and inefficient process.

[0006] Furthermore, the labeling efficiency of existing nucleic acid labeling methods varies depending on the length and type of nucleic acid, the position of the target site to be labeled (e.g., inside the nucleic acid sequence or terminal nucleotide(s)), and the chemical or functional moiety to be labeled. Therefore, there is a need for a simple, efficient, and environmentally friendly nucleic acid labeling method.

Summary of the Invention

[0007] The present disclosure provides a method for efficiently modifying or labeling the 3'-end of a nucleic acid or polynucleotide. This method introduces a specific modification or label to the 3'-end of a nucleic acid or polynucleotide by arranging a pair of functional moieties or chemical molecules that can stably or covalently bind to each other. For example, one component of the paired functional moiety or chemical molecule is labeled to the nucleobase or 3'-hydroxyl (3'-OH) group of a nucleotide, and it is incorporated by polymerase to the 3'-end of the target nucleic acid or polynucleotide. The thus obtained target nucleic acid or polynucleotide has a first functional moiety or chemical molecule at its 3'-end, which readily reacts with the other component of the paired moiety or molecule having the desired modification or label. As a result, a stable or covalently bonded connection is formed by the reaction between the paired functional moieties, whereby the target nucleic acid or polynucleotide can be modified or labeled with the desired molecule, particularly at its 3'-end.

[0008] In at least one embodiment, the method provided by the present disclosure includes modifying natural or synthetic deoxyribonucleic acid (DNA) at the 3' end. FIG. 1 is a schematic diagram showing an exemplary method of introducing a 3'-modification into a target nucleic acid. In some embodiments, the method provided by the present disclosure includes preparing a polynucleotide comprising a 3'-terminal nucleotide (e.g., shown as "nucleotide" in FIG. 1) having a reactive moiety (e.g., shown as "M1" in FIG. 1); and exposing the polynucleotide to a desired molecule (e.g., shown as "label" in FIG. 1) having a corresponding functional moiety (e.g., shown as "M2" in FIG. 1) that can react with the reactive moiety to form a bond, thereby binding the desired molecule to the 3' end of the polynucleotide. In some embodiments, the desired molecule has a labeling moiety (e.g., shown as "label" in FIG. 1) that is introduced into the polynucleotide to form a labeled polynucleotide.

[0009] In at least one embodiment, the method provided by the present disclosure further includes preparing a polynucleotide by template-independent enzymatic nucleic acid synthesis. In some embodiments, template-independent enzymatic nucleic acid synthesis includes using a DNA polymerase, an RNA polymerase, or an enzyme functionally equivalent thereto. In some embodiments, the DNA polymerase for template-independent enzymatic nucleic acid synthesis is an A-family DNA polymerase, a B-family DNA polymerase, or an X-family DNA polymerase. In at least one embodiment, the B-family DNA polymerase is a Thermococcus DNA polymerase. In at least one embodiment, the B-family DNA polymerase is a Thermococcus or Pyrococcus DNA polymerase. In at least one embodiment, the B-family DNA polymerase is selected from the group consisting of the B-family DNA polymerase of Thermococcus kodakarensis (Kod1), the B-family DNA polymerase of Pyrococcus furiosus (Pfu), the B-family DNA polymerase of Thermococcus litoralis (Vent), the B-family DNA polymerase of Thermococcus sp. 9°N (9°N), and the B-family DNA polymerase of Thermococcus gorgonarius (Tgo).

[0010] In at least one embodiment of the present disclosure, the template-independent enzymatic nucleic acid synthesis is carried out at a reaction temperature of 10°C to 100°C, such as 10°C to 90°C, 20°C to 90°C, 30°C to 90°C, 20°C to 80°C, 30°C to 80°C, 40°C to 80°C, 30°C to 70°C, 40°C to 70°C, or 50°C to 70°C.

[0011] In some embodiments, the method provided by the present disclosure further includes preparing the polynucleotide in solution phase. In other embodiments, the method provided by the present disclosure includes preparing the polynucleotide in solid phase, for example, preparing an initiator attached to a solid support. In some embodiments, the solid support is selected from the group consisting of particles, polymers, beads, resins, slides, chips, surface arrays, membranes, flow cells, wells, matrices, chambers, microfluidic chambers, channels, microfluidic channels, and gels.

[0012] In at least one embodiment, the method provided by the present disclosure further includes preparing an endonuclease to enzymatically release the polynucleotide from the initiator. In some embodiments, the endonuclease recognizes the second-to-last 3'-nucleotide of the initiator and cleaves the bond between the 3'-terminal nucleotide of the initiator and the polynucleotide, between the second-to-last 3'-nucleotide and the third-to-last 3'-nucleotide of the initiator, between the third-to-last 3'-nucleotide and the fourth-to-last 3'-nucleotide of the initiator, or between the fourth-to-last 3'-nucleotide and the fifth-to-last 3'-nucleotide of the initiator.

[0013] In at least one embodiment of the present disclosure, the endonuclease is derived from Thermococcus barophilus (Tba), Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac), Pyrococcus abyssi (Pab), Thermococcus kodakarensis (Tko), Thermococcus gammatolerans (Tga), or Bacillus subtilis (Bsu).

[0014] In at least one embodiment of the present disclosure, the 3'-terminal nucleotide is a natural nucleotide, a nucleotide analog, or an abasic (apurinic / apyrimidinic) nucleotide. In some embodiments, the 3'-terminal nucleotide is a ribonucleotide, a deoxyribonucleotide, or a heterologous nucleotide. In some embodiments, the reactive moiety is attached to the 2'-carbon or 3'-carbon of the ribose or to the nucleobase of the 3'-terminal nucleotide.

[0015] In at least one embodiment of the present disclosure, the corresponding functional moiety reacts with the reactive moiety by a bioorthogonal reaction. In some embodiments, the bioorthogonal reaction is a click conjugation, an oxime / hydrazine formation, a Staudinger ligation, a tetrazine ligation, or a quadricyclane ligation. In some embodiments, the click conjugation is selected from the group consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), an isocyanide-based click reaction, and inverse electron-demand Diels-Alder reaction (IEDDA).

[0016] In at least one embodiment of the present disclosure, the reactive moiety is selected from the group consisting of an azide group, an alkynyl group, a triallylphosphinyl group, a cyclooctynyl group, a thiol group, an alkenyl group, a nitrone group, an aldehydyl group, a ketonyl group, a dienyl group, and a dienophilyl group.

[0017] In at least one embodiment of the present disclosure, the corresponding functional moiety is a functional group selected from the group consisting of an azide group, an alkynyl group, a triallylphosphinyl group, a cyclooctynyl group, a thiol group, an alkenyl group, a nitrone group, an aldehydyl group, a ketonyl group, a dienyl group, and a dienophilyl group.

[0018] In some embodiments, the bioorthogonal reaction is carried out at a reaction temperature of 10°C to 100°C, such as, for example, 10°C to 90°C, 10°C to 80°C, 10°C to 70°C, 10°C to 60°C, 20°C to 80°C, 20°C to 70°C, 20°C to 60°C, 20°C to 50°C, 30°C to 70°C, 30°C to 60°C, 30°C to 50°C, or 30°C to 40°C. In some embodiments, the bioorthogonal reaction is carried out for a time of, for example, 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 hours, or more.

[0019] In at least one embodiment, the desired molecule is molecularly recognizable by detection of visible light, fluorescence, photoluminescence, electrochemiluminescence, laser, irradiation, fluorescence resonance energy transfer, fluorescence structural change, or fluorescence quenching. In some embodiments, the desired molecule is a chemical compound, fluorescent tag, dye, marker, reporter, quencher, amine, antigen, ligand, protein, antibody, antibody fragment, peptide, peptide analog, or quantum dot.

[0020] In at least one embodiment, the method provided by the present disclosure further includes a purification or concentration step for removing unlabeled nucleic acids or polynucleotides. For example, it includes preparing a protein having 3'→5' exonuclease activity and digesting nucleic acids or polynucleotides that have failed in 3'-terminal nucleotide synthesis by polymerase or have an incomplete binding reaction with a second reactive moiety.

[0021] In at least one embodiment, the present disclosure also provides a kit for modifying a polynucleotide at its 3'-end. The kit includes a nucleotide having a reactive moiety, a polymerase for incorporating the nucleotide having a reactive moiety at the 3'-end of the polynucleotide, and a desired molecule having a corresponding functional moiety that can react with the reactive moiety. In at least one embodiment, the kit for modifying a polynucleotide at its 3'-end includes a nucleotide having a reactive moiety, a polymerase, a desired labeling molecule, and a 3'→5' exonuclease. Here, the polynucleotide binds to the desired labeling molecule at the 3'-end to form a labeled polynucleotide that can be further enriched by the purified exonuclease described herein.

[0022] The present disclosure also provides a method for 5'-end labeling of a nucleic acid. The method includes preparing a target nucleic acid to be labeled; preparing a 5'-end glycosylase that reacts with the target nucleic acid to create an intermediate nucleic acid having an abasic site at the 5'-end of the target nucleic acid; and preparing an aldehyde-reactive compound having a detectable label to bind to the intermediate nucleic acid at the abasic site to form a labeled nucleic acid with the detectable label bound to the 5'-end.

[0023] In at least one embodiment, the nucleic acid is single-stranded or includes at least a double-stranded region formed by two complementary strands of the nucleic acid. In one embodiment, the nucleic acid is a DNA fragment or an RNA fragment. In another embodiment, the nucleic acid is newly synthesized or derived from a living organism. In some embodiments, the nucleic acid is immobilized on a solid surface or a polymer surface.

[0024] In at least one embodiment of the present disclosure, the aldehyde-reactive compound is a compound having at least one primary amine, a hydrazide, an acyl hydrazide, an aminooxy (ONH 2)It is a compound having a base, a compound having a naphthalene-containing aminooxy group, and / or a compound having a guanidine-containing aminooxy group. In some embodiments, the aldehyde-reactive compound is hydroxylamine biotin, aminooxy-poly(ethylene glycol)-azide, propargyl, aminooxy-poly(ethylene glycol)-DBCO, aminooxy-poly(ethylene glycol)-bicyclononine (BCN), a fluorescent dye-hydroxylamine such as Alexa Fluor488 hydroxylamine, an aldehyde-reactive probe (ARP), aminooxy-5(6)-FAM, aminooxy-5(6)-ROX, and aminooxy-5(6)-TAMRA, aminooxy-cyanine 555, aminooxy-cyanine 647, aminooxy-biotin, naphthalene-containing aminooxy-fluorescent dye, guanidine-containing aminooxy-fluorescent dye, naphthalene and / or guanidine-containing aminooxy-FAM, Cy5-PEG-aminooxy, or a fluorescent dye hydrazide such as a cyanine dye hydrazide or a CF dye hydrazide.

[0025] In at least one embodiment of the present disclosure, the nucleic acid contains a 5'-terminal nucleobase selected from the group consisting of hypoxanthine, cytosine, 3-alkyladenine, 8-oxoguanine (8-oxoG), uracil, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 5-fluorouracil, dihydroxyuracil, 5-formylcytosine, 5-carboxylcytosine, 3-methyladenine (3-meA), 3-methylguanine, 7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxylcytosine, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, a formamidopyrimidine derivative of adenine, and a formamidopyrimidine derivative of guanine.

[0026] In some embodiments, the 5'-end glycosylase of the present disclosure may be a monofunctional DNA glycosylase. In at least one embodiment of the present disclosure, the monofunctional DNA glycosylase is uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG; also called methylpurine DNA glycosylase (MPG)), single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD), 8-oxoguanine glycosylase 1 (OGG1) without abasic site lyase activity, endonuclease III-like glycosylase 1 (NTHL1) without abasic site lyase activity, endonuclease VIII-like glycosylase 1 (NEIL1) without abasic site lyase activity, endonuclease VIII-like glycosylase 2 (NEIL2) without abasic site lyase activity, endonuclease VIII-like glycosylase 3 (NEIL3) without abasic site lyase activity, enzymatic active fragments thereof, and any combination thereof.

[0027] In at least one embodiment of the present disclosure, the uracil-DNA glycosylase is derived from the family Micrococcaceae, Staphylococcaceae, or Caryophanaceae, including the genus Micrococcus, Stomatococcus, Staphylococcus, or Planococcus. In some embodiments, the uracil-DNA glycosylase is derived from the bacterium Micrococcus luteus.

[0028] In at least one embodiment of the present disclosure, the detectable label is selected from the group consisting of azide, alkyne, bicyclononyne (BCN), dibenzocyclooctyne (DBCO), maleimide, peptide, protein, antibody, dendrimer, biotin, radioisotope, chromogenic dye, fluorescent dye, luminescent dye, and any combination thereof.

[0029] In some embodiments, the method of the present disclosure further comprises preparing a 5'→3' exonuclease to remove unlabeled nucleic acids.In at least one embodiment, the 5'→3' exonuclease is selected from the group consisting of T5 exonuclease (T5exo), T7 exonuclease (T7exo), viral alkaline exonuclease, bacterial alkaline exonuclease, phage lambda exonuclease, for example, the 5'-exonuclease (ExoVI) of DNA polymerase I from Streptococcus pneumoniae or Helicobacter pylori, Escherichia coli exonuclease VIII (Exo VIII), for example, RecJ from Escherichia coli or Deinococcus radiodurans, RecJf derived from RecJ fused to maltose binding protein, Thermus thermophilus (Tth) RecJ, Mycoplasma pneumoniae (Mpn) NrnA, human exonuclease 5 (hEXO5), human exonuclease 1 (hEXO1), Saccharomyces cerevisiae-derived SNM1, human or bovine SNM1A, human SNM1B / Apollo, bovine SNM1B, for example, SXT-EXo from Vibrio cholerae, phospholipase D3 (PLD3), phospholipase D4 (PLD4), for example, Sso1391-Csa1 from Sulfolobus solfataricus, for example, Sto0027-Csa1 from Sulfolobus tokodaii, for example, Ttx1248-Csa1 from Thermoproteus tenax, for example, Sso1451-Csa1 from Sulfolobus solfataricus, for example, Sto2633-Csa1 from Sulfolobus tokodaii, for example, Pfu1793-Cas4 from Pyrococcus furiosus, for example, Sto2501 from Sulfolobus tokodaii, for example, Sso0001 from Sulfolobus solfataricus, for example, Sto2331-Cas4 from Sulfolobus tokodaii, for example, Ttx1245-Cas4 from Thermoproteus tenax, for example, Sso1449-Cas4 from Sulfolobus solfataricus, for example, Sto2635-Cas4 from Sulfolobus tokodaii, for example, Sso1392-Cas4 from Sulfolobus solfataricus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2) gp19, bacterial AddB, and any combination thereof.

[0030] In some embodiments, the method of the present disclosure further includes a nucleic acid synthesis step to obtain a unique 5'-terminal nucleobase. In some embodiments, the nucleic acid having a unique 5'-terminal nucleobase is synthesized by processes well known in the art, such as phosphoramidite-based nucleic acid synthesis processes and template-dependent and template-independent enzymatic nucleic acid synthesis processes.

[0031] In some embodiments, the method of the present disclosure further includes isolating nucleic acid fragments from a sample. For example, the nucleic acid may be isolated from a sample of untreated or disrupted virus or cells, such as eukaryotic cells including bacterial cells, archaeal cells, and human cells. Suitable samples include isolated cells and tissue samples such as biopsies including solid tissue or tumor biopsies. In some embodiments, the sample may be obtained from a formalin-fixed paraffin-embedded (FFPE) tissue sample or other storage sample of cellular material. The present disclosure also provides a kit for 5'-terminal labeling of nucleic acids, the kit comprising a 5'-terminal glycosylase and an aldehyde-reactive compound.

[0032] In some embodiments, the 5'-end glycosylase of the kit of the present disclosure is selected from the group consisting of uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG; also called methylpurine DNA glycosylase (MPG)), single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD), 8-oxoguanine glycosylase 1 (OGG1) without abasic site lyase activity, endonuclease III-like glycosylase 1 (NTHL1) without abasic site lyase activity, endonuclease VIII-like glycosylase 1 (NEIL1) without abasic site lyase activity, endonuclease VIII-like glycosylase 2 (NEIL2) without abasic site lyase activity, endonuclease VIII-like glycosylase 3 (NEIL3) without abasic site lyase activity, enzymatic active fragments thereof, and any combination thereof.

[0033] In at least one embodiment, the uracil DNA glycosylase of the kit of the present disclosure is derived from the family Micrococcaceae, Staphylococcaceae, or Caryophanaceae, including the genus Micrococcus, Stomatococcus, Staphylococcus, or Planococcus. In some embodiments, the uracil-DNA glycosylase is derived from Bacillus luteus.

[0034] In at least one embodiment, the aldehyde-reactive compound of the kit of the present disclosure is a compound having at least one primary amine, hydrazide, acylhydrazide, aminooxy (-ONH 2)It is a compound having a base and a compound having a naphthalene and / or guanidine-containing aminooxy group. In some embodiments, the aldehyde-reactive compound is hydroxylamine biotin, a fluorescent dye-hydroxylamine such as Alexa Fluor488, an aldehyde-reactive probe (ARP), an aminooxy-5(6)-FAM, an aminooxy-5(6)-ROX, and an aminooxy-5(6)-TAMRA, etc., an aminooxy-fluorescent dye, cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy-biotin, a naphthalene-containing aminooxy-fluorescent dye, a guanidine-containing aminooxy-fluorescent dye, a naphthalene and / or guanidine-containing aminooxy-FAM, Cy5-PEG-aminooxy, or a fluorescent dye hydrazide such as a cyanine dye hydrazide or a fluorescent CF dye hydrazide.

[0035] In at least one embodiment, the kit of the present disclosure further comprises a 5'→3' exonuclease for removing unlabeled nucleic acids. In some embodiments, the 5'→3' exonuclease is selected from the group consisting of T5 exonuclease, T7 exonuclease, phage lambda exonuclease, the 5'-exonuclease of DNA polymerase I (ExoVI), exonuclease VIII (Exo VIII), RecJ, RecJf, Tth RecJ, Mpn NrnA, human EXO5 (hEXO5), human exonuclease 1 (hEXO1), SNM1, SNM1A, human SNM1B / Apollo, bovine SNM1B, SXT-Exo, phospholipase D3 (PLD3), phospholipase D4 (PLD4), Sso1391-Csa1, Sto0027-Csa1, Ttx1248-Csa1, Sso1451-Csa1, Sto2633-Csa1, Pfu1793-Cas4, Sto2501, Sso0001, Sto2331-Cas4, Ttx1245-Cas4, Sso1449-Cas4, Sto2635-Cas4, Sso1392-Cas4, SIRV2 gp19, bacterial AddB, and any combination thereof.

[0036] The present disclosure further provides a system for 5'-end labeling of nucleic acids. The system includes a reaction reservoir, chamber or vessel, a liquid handling / transfer device, a temperature control unit, and a time control unit, wherein the liquid handling / transfer device is configured to transfer a 5'-end glycosylase and an aldehyde-reactive compound to the nucleic acids in the reaction reservoir, chamber or vessel at a predetermined temperature controlled by the temperature control unit for a certain period of time.

[0037] The present disclosure further provides a kit for 5'-end labeling of nucleic acids, the kit comprising a 5'-end glycosylase and an aldehyde-reactive compound. In some embodiments, the kit further comprises a 5'→3' exonuclease for removing unlabeled nucleic acids, such as T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, phage lambda exonuclease, 5'-exonuclease of DNA polymerase I (ExoVI), exonuclease VIII (ExoVIII), RecJ, RecJf, Tth RecJ, Mpn NrnA, human exonuclease 5 (hEXO5), human exonuclease 1 (hEXO1), SNM1, SNM1A, human SNM1B / Apollo, bovine SNM1B, SXT-Exo, phospholipase D3 (PLD3), phospholipase D4 (PLD4), Sso1391-Csa1, Sto0027-Csa1, Ttx1248-Csa1, Sso1451-Csa1, Sto2633-Csa1, Pfu1793-Cas4, Sto2501, Sso0001, Sto2331-Cas4, Ttx1245-Cas4, Sso1449-Cas4, Sto2635-Cas4, Sso1392-Cas4, SIRV2 gp19, bacterial AddB, and any combination thereof.

[0038] In at least one embodiment, the 5'-terminal glycosylase is selected from the group consisting of uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG), single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD), 8-oxoguanine glycosylase 1 (OGG1) without apurinic / apyrimidinic lyase activity, endonuclease III-like glycosylase 1 (NTHL1) without apurinic / apyrimidinic lyase activity, endonuclease VIII-like glycosylase 1 (NEIL1) without apurinic / apyrimidinic lyase activity, endonuclease VIII-like glycosylase 2 (NEIL2) without apurinic / apyrimidinic lyase activity, endonuclease VIII-like glycosylase 3 (NEIL3) without apurinic / apyrimidinic lyase activity, enzymatic active fragments thereof, and any combination thereof. In at least one embodiment, the uracil-DNA glycosylase is derived from the family Micrococcaceae, Staphylococcaceae, or Caryophanaceae.

[0039] In at least one embodiment, the aldehyde-reactive compound is hydroxylamine biotin, fluorescent dye-hydroxylamine, aldehyde-reactive probe (ARP), aminooxy-fluorescent dye, aminooxy-poly(ethylene glycol)-azide, propargyl, aminooxy-poly(ethylene glycol)-DBCO, aminooxy-poly(ethylene glycol)-bicyclononyne (BCN), cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy-biotin, naphthalene-containing aminooxy-fluorescent dye, guanidine-containing aminooxy-fluorescent dye, Cy5-PEG-aminooxy, or fluorescent dye hydrazide.

[0040] The present disclosure also provides a method for 5' and 3' end labeling of nucleic acids. The method includes preparing a target nucleic acid to be labeled, adding a 5'-terminal glycosylase and a nucleotide having a reactive moiety to the target nucleic acid; creating an intermediate nucleic acid having an abasic site at the 5'-end of the target nucleic acid and incorporating a nucleotide having a reactive moiety at the 3'-end of the intermediate nucleic acid; preparing an aldehyde-reactive compound having a detectable label for binding to the intermediate nucleic acid at the abasic site at the 5'-end, and a desired molecule having a corresponding functional moiety capable of reacting with the reactive moiety; and exposing the intermediate nucleic acid to the aldehyde-reactive compound having a detectable label and the desired molecule having a corresponding functional moiety, thereby forming a labeled nucleic acid having a detectable label bound to the intermediate nucleic acid at the abasic site, and forming a labeled nucleic acid having a detectable label bound at the 5'-end and a bond formed between the reactive moiety and the corresponding functional moiety at the 3'-end.

[0041] The present disclosure also provides a kit for 5' and 3' end labeling of nucleic acids. The kit includes a 5'-terminal glycosylase, an aldehyde-reactive compound, a nucleotide having a reactive moiety, a polymerase for incorporating the nucleotide having a reactive moiety at the 3'-end of the nucleic acid, and a desired molecule having a corresponding functional moiety capable of reacting with the reactive moiety. The present disclosure will be more readily recognized and better understood by reference to the following description in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0042]

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Embodiments for Carrying Out the Invention

[0043] All terms, including descriptive or technical terms used herein, should be construed as having meanings that are obvious to those skilled in the art. However, the terms may have different meanings depending on the intention of those skilled in the art, precedents, or the emergence of new technologies. Also, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected terms will be explained in detail in the description of the present disclosure. Therefore, the terms used herein are defined based on the description throughout the specification together with the meaning of the terms.

[0044] In the practice of the present disclosure, unless otherwise indicated, conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the level of skill of the ordinary artisan, are utilized. Such techniques are well explained in the following references. For example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989), Cold Spring Harbor Press; "Oligonucleotide Synthesis" (M.J. Gait, 1984); "Methods in Molecular Biology", Humana Press; "Cell Biology: A Laboratory Notebook" (J.E. Cellis, ed., 1998) Academic Press; "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Handbook of Experimental Immunology" (Weir, 1996); "Introduction to Cell and Tissue Culture" (J.P. Mather and P.E. Roberts, 1998); "Cell and Tissue Culture: Laboratory Procedures" (A. Doyle, J.B. Griffiths and D.G. Newell, eds., 1993-8); "Methods in Enzymology" (Academic Press); "Handbook of Experimental Immunology" (D.M. Weir and C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells” (J.M. Miller and M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J.E.Edited by Coligan et al., 1991); "Short Protocols in Molecular Biology" (Wiley and Sons, 1999); "Immunobiology" (C.A. Janeway and P. Travers, 1997); "Antibodies" (P. Finch, 1997); "Antibodies: a practical approach" (Edited by D. Catty, IRL Press, 1988-1989); "Monoclonal antibodies: a practical approach" (Edited by P. Shepherd and C. Dean, Oxford University Press, 2000); "Using antibodies: a laboratory manual" (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); and "The Antibodies" (Edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995). Particularly useful techniques for specific embodiments are described in the items that follow. Those skilled in the art are considered to be able to make the most of the present disclosure based on the above description without further detailed explanation. Therefore, the following embodiments are for illustrative purposes only and should be construed not to limit the remainder of the present disclosure in any way. All publications cited herein are hereby incorporated by reference into this specification for the purposes or subject matter referred to herein.

[0045] The singular forms "a", "an", and "the" as used herein are intended to include the plural unless the context clearly dictates otherwise. The terms "includes", "comprises", "including", and "comprising" as used in the embodiments for carrying out the invention and / or in the claims are intended to be inclusive rather than exclusive, meaning they do not exclude other elements, materials, steps, etc. "sec", "min", and "hr" are used herein as abbreviations for "second", "minute", and "hour", respectively.

[0046] When a range is recited, endpoints are included in that range. Further, unless clearly stated otherwise, or not apparent from the context and the understanding of one of ordinary skill in the art, and unless the context clearly dictates otherwise, a value expressed as a range may assume any specific value or sub-range within the recited range in different embodiments of the present disclosure.

[0047] As used herein, the terms "about", "approximately", and "around" generally mean within 10%, 5%, 1%, or 0.5% of the recited value or range. Alternatively, the terms "about", "approximately", and "around" mean within the acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless otherwise clearly defined, all numerical ranges, amounts, values, and percentages of amounts of materials, periods, temperatures, operating conditions, ratios of ingredients, etc. disclosed herein are to be understood in all cases as being modified by the terms "about", "approximately", or "around".

[0048] As used herein, the term "derived from" when referring to a biological sample refers to a sample obtained from the source described at a certain point in time. For example, a biological sample derived from an organism can refer to a primary biological sample obtained directly from the organism (i.e., unmodified), or a sample that can be modified, for example, by introduction of a recombinant vector, by culturing under specific conditions, or by immortalization.

[0049] With respect to a list of one or more elements, the phrase "at least one" as used herein should be understood to mean at least one element selected from any one or more of the elements within the list of elements, and does not necessarily have to include at least one of each and every element listed in the list of elements, nor does it exclude any combination of the elements in the list. This definition also allows for the possibility that elements may optionally be present in addition to the elements specified within the list of elements to which the phrase "at least one" refers, whether or not related to the specified elements. Thus, by way of non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B", or, equivalently, "at least one of A and / or B") may, in one embodiment, refer to: at least one (optionally two or more) A (where B is not present) (and optionally including elements other than B); in another embodiment, at least one (optionally two or more) B (where A is not present) (and optionally including elements other than A); and in yet another embodiment, at least one (optionally two or more) A, and at least one (optionally two or more) B (as well as optionally including other elements).

[0050] As used herein, abasic sites, also known as depurinated / depyrimidinated (AP) sites, include any chemical structure after removal of the base moiety (including the entire base) by treatment of nucleotides (present in a polynucleotide chain) with an agent capable of cleaving the base moiety of the nucleotide, such as an agent (e.g., an enzyme, acidic conditions, or a chemical reagent) capable of cleaving the nucleotide base moiety. In one embodiment, an AP site is within the backbone of a nucleic acid such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) lacking a nucleobase, i.e., within the deoxyribose of the DNA backbone or the ribose of the RNA backbone that is not covalently bonded to any purine base such as adenine (A) or guanine (G), or any pyrimidine base such as cytosine (C), uracil (U), or thymine (T). An AP site may be within the nucleotide sequence of a nucleic acid at both the 5' and 3' ends of the nucleic acid, or at one end, either the 5' end or the 3' end, of the nucleic acid.

[0051] As used herein, the terms "nucleic acid", "nucleic acid sequence", and "nucleic acid fragment" refer to nucleotide sequences in single-stranded or double-stranded form, the source of which is not limited herein but generally includes nucleotides of natural origin or artificial chemical mimics. As used herein, the term "nucleotide" refers to a glycoside, with or without a nucleobase, and a monomeric unit of a nucleic acid or polynucleotide as described below, having one or more internucleotide linkages such as phosphodiester bonds. In some embodiments, the nucleobases include natural origin bases such as adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), non-natural origin bases such as xanthine, hypoxanthine, isoguanine, and isocytosine, and any analogs or derivatives thereof. In some embodiments, nucleotides having abasic sites (deletion of nucleobases) are also included within the scope of the present disclosure. In some embodiments, the sugar of the glycoside includes sugars of natural origin such as pentose sugars (e.g., deoxyribose and ribose), sugars of non-natural origin, and analogs thereof. In some embodiments, the nucleotides are linked via internucleotide linkages. Internucleotide linkages include, but are not limited to, phosphate, boranophosphate, phosphorothioate, phosphodiester, phosphotriester, H-phosphonate, aminophosphonic acid, methylphosphonic acid, phosphonoacetic acid, sulfurophosphonoacetic acid, or other variants of the phosphate backbone of natural nucleic acids. As used herein, the term "nucleotide" also encompasses structural analogs in place of natural or non-natural nucleotides such as modified nucleotides. For example, the term "xenonucleotide" refers to a nucleotide modified to have a sugar moiety different from that contained in natural DNA or RNA. Examples of nucleic acids having xenonucleotides, i.e., xeno nucleic acids (XNAs), include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), 1,5-anhydrohexitol nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), cyclohexene nucleic acid (CeNA), and FANA (fluoroarabino nucleic acid).

[0052] As used herein, the term "polynucleotide" generally refers to a polymer of nucleotides and encompasses any kind of nucleic acid such as natural or non-natural DNA or RNA, and modified nucleic acids such as the xeno nucleic acids (XNA) described herein. A polynucleotide can also include any combination of glycosides with or without nucleobases and internucleotide linkages. Unless otherwise specified, polynucleotides referred to herein have an inherent directionality from the 5' end of one nucleotide to the 3' end of the adjacent nucleotide, and in this case, template-independent synthesis of the polynucleotides described herein proceeds in the 5'→3' direction.

[0053] As used herein, when the term "polynucleotide" refers only to the polymer molecular structure, it is not intended to be characterized by the length of the nucleotide units. That is, polynucleotides as used herein are interchangeable with the term "oligonucleotide" and can range in size from a few monomer nucleotide units to thousands of monomer nucleotide units, for example, lengths of 2 - 5 nucleotides, 5 - 20 nucleotides, 20 - 100 nucleotides, 100 - 1,000 nucleotides, or more. A polynucleotide may consist entirely of modified or unmodified deoxyribonucleotides of natural or non-natural origin, entirely of modified or unmodified ribonucleotides of natural or non-natural origin, or a chimeric mixture thereof. The nucleobases (also known as nitrogenous bases) contained in a polynucleotide may be, for example, adenine, thymine, cytosine, guanine, uracil, xanthine, hypoxanthine, isocytosine, or isoguanine. Furthermore, a polynucleotide may contain one or more abasic sites (apurinic / apyrimidinic sites), also known as AP sites.

[0054] The terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and polymers thereof, and may be used interchangeably herein. These terms encompass known nucleotide analogs, or nucleic acids containing modified backbone residues or linkages, which are of synthetic, natural, and non-natural origin and / or have chemical properties similar to the reference nucleic acid and / or are metabolized in a manner similar to the reference nucleotide. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), complementary sequences, and sequences explicitly shown. In some embodiments, nucleotides are linked via nucleotide linkages. Nucleotide linkages include, but are not limited to, phosphate, boranophosphate, phosphorothioate, phosphodiester, phosphotriester, H-phosphonate, aminophosphonic acid, methylphosphonic acid, phosphonacetic acid, sulfur phosphonacetic acid, or other variants of the phosphate backbone of natural nucleic acids. The term "nucleotide" as used herein also encompasses structural analogs in place of natural or non-natural nucleotides such as modified nucleotides. For example, the term "xenonucleotide" refers to a nucleotide modified to have a sugar moiety different from that found in natural DNA or RNA. Examples of nucleic acids having xenonucleotides, i.e., xeno nucleic acids (XNA), include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), 1,5-anhydrohexitol nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), cyclohexene nucleic acid (CeNA), and fluoroarabino nucleic acid (FANA).

[0055] A nucleic acid, as used herein, may be from 5 bases to 10,000 bases in length, e.g., from 10 to 3,000 bases in length, from 10 to 1,000 bases in length, or from 10 to 100 bases in length. Nucleic acids isolated from biological sources may be longer than 1,000 bases and may be fragmented, e.g., by sonication, for use as described herein.

[0056] In some embodiments, the nucleic acid to be labeled by the methods of the present disclosure may be about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 65, about 75, about 85, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, or more nucleotides in length. In some embodiments, the nucleic acid may be at least about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 65, about 75, about 85, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, or more nucleotides in length. In other embodiments, the nucleic acid may be less than about 20, about 25, about 30, about 35, about 40, about 50, about 65, about 75, about 85, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, or about 650 nucleotides in length. It is understood that the length of the nucleic acid may represent the average size in a population.

[0057] As used herein, a glycosylase is an enzyme that can excise the base moiety of a nucleotide to create an AP site in a nucleic acid. Among them are included N-glycosylases, also referred to as "DNA glycosylases" or "glycosidases", examples of which include, but are not limited to, uracil N-glycosylase (UNG) that specifically cleaves dUTP and is interchangeably referred to as "uracil DNA glycosylase" (UDG); hypoxanthine-N-glycosylase; hydroxymethylcytosine-N-glycosylase; 3-methyladenine DNA glycosylase; 3- or 7-methylguanine DNA glycosylase; hydroxymethyluracil DNA glycosylase; and T4 endonuclease V. Glycosylases cleave the base moiety of a nucleotide in the middle of the nucleic acid, or at one or both ends. As used herein, a 5'-end glycosylase excises the base moiety of a nucleotide at the 5'-end of the nucleic acid.

[0058] As used herein, the term "initiator" refers to a nucleoside monomer, nucleotide monomer, oligonucleotide, polynucleotide, or a modified analog thereof, from which a nucleic acid is newly synthesized by a nucleic acid polymerase. The term "initiator" may also refer to an XNA having a 3'-hydroxyl group such as 3'-hydroxyl-PNA.

[0059] According to the present disclosure, the initiator may be linked or immobilized to a solid support, and the linking nucleotide binds to the 3'-terminal nucleotide of the initiator and the 5'-terminal nucleotide of the synthesized nucleic acid. The initiator may be directly attached to the solid support, attached to the support via a linker, or immobilized via physical interactions such as adsorption, electrostatic interaction, and hydrogen bonding. Examples of solid supports include, but are not limited to, microarrays, (coated or uncoated) beads, columns, optical fibers, wash cotton, nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicone, polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, magnetic particles, plastics (such as polyethylene, polypropylene, and polystyrene), gel-forming materials (such as proteins (e.g., gelatin), lipopolysaccharides, silicates, agarose, polyacrylamide, or methyl methacrylate polymers), sol-gels, porous polymers, hydrogels, nanostructured surfaces nanotubes (such as carbon nanotubes), and nanoparticles (such as gold nanoparticles or quantum dots).

[0060] As used herein, the term "polymerase" refers to an enzyme / protein capable of synthesizing nucleic acids, generally a DNA polymerase, an RNA polymerase, or a functionally equivalent enzyme, including enzymes of natural origin, modified enzymes, enzyme subunits, and derivatives thereof. For example, amino acid sequence modifications (such as mutations and functional group substitutions) can be applied to these enzymes for desired purposes such as removing 5'→3' exonuclease activity to improve polymerase activity, and modified enzymes with improved properties such as thermal stability / heat resistance and catalytic efficiency can be obtained.

[0061] According to the present disclosure, the term "polymerase" may be a template-dependent polymerase or a template-independent polymerase. Examples of polymerases include A family DNA polymerases (such as T7 DNA polymerase, Pol I, Pol γ, θ, and ν), B family DNA polymerases (such as Pol II, Pol B, Pol ζ, Pol α, δ and ε), C family DNA polymerases (such as Pol III), D family DNA polymerases (such as PolD), X family DNA polymerases (such as Pol β, Pol σ, Pol λ, Pol μ, and terminal deoxynucleotidyl transferase), Y family DNA polymerases (such as Pol ι, Pol κ, Pol η, DinB, Pol IV, and Pol V), reverse transcriptases (such as telomerase and hepatitis B virus), and enzymatically active fragments thereof.

[0062] Non-limiting examples of widely used template-dependent polymerases include T7 DNA polymerase of bacteriophage T7, which is a DNA-dependent DNA polymerase, and T3 DNA polymerase of bacteriophage T3; T7 RNA polymerase of bacteriophage T7, and T3 RNA polymerase of bacteriophage T3, which are DNA-dependent RNA polymerases; DNA polymerase I known as the Klenow fragment of Escherichia coli or a fragment thereof, which is a DNA-dependent DNA polymerase; Thermus aquaticus DNA polymerase, Tth DNA polymerase, and Vent DNA polymerase, which are heat-resistant DNA-dependent DNA polymerases; eukaryotic DNA polymerase β, which is a DNA-dependent DNA polymerase; telomerase, which is an RNA-dependent DNA polymerase; and non-protein catalytic molecules such as modified RNAs (ribozymes; Unrau & Bartel, 1998), and DNAs having template-dependent polymerase activity.

[0063] Non-limiting examples of template-independent polymerases include reverse transcriptase, polyA polymerase, DNA polymerase theta (θ), terminal deoxynucleotidyl transferase (TdT), and DNA polymerase mu (μ). Suitable polymerases for performing nucleic acid synthesis, nucleotide addition / incorporation, and nucleic acid synthesis steps are within the purview of one of ordinary skill in the art, and for the sake of brevity, further details thereof are omitted herein. Additionally, the B-family DNA polymerases previously provided by the inventors are also preferably used under template-independent conditions, and U.S. Patent No. 11591629B2 is hereby incorporated by reference in its entirety.

[0064] As used herein, the term "modification" refers to the alteration(s) of the chemical structure of a reactive molecule. When a nucleic acid is used as the reactive molecule, modifications include, but are not limited to, introducing additional chemical group(s) / moiety(ies) to the nucleic acid, removing or substituting native chemical group(s) / moiety(ies) from the nucleic acid, regardless of the nucleic acid source, or combinations thereof. Alternatively, the modification(s) may be introduced into a specific sequence of the nucleic acid during de novo nucleic acid synthesis, resulting in direct modification(s) to the nucleic acid. For example, a fluorophore-labeled nucleotide analog may be incorporated into a nucleic acid together with its native counterpart to obtain a "fluorescently labeled" nucleic acid. Similarly, site-specific modification(s) may be enzymatically inserted into a nucleic acid by incorporating nucleotide(s) with the desired modification(s). For example, a nucleoside triphosphate having a 3'-O-azidomethyl group can be enzymatically introduced to the 3'-end of a nucleic acid to directly add an azidomethyl modification to the 3'-end of the nucleic acid. Such modifications result in the addition of nucleotides to a target nucleic acid together with site-specific chemical groups.

[0065] As used herein, the term "detect" or "detection" refers to both quantitative and qualitative measurements, and thus, the terms "detect" or "detection" are used interchangeably herein with "analyze", "measure", etc. When a quantitative measurement is intended, phrases such as "measure the amount" are used. When either a qualitative or quantitative measurement is intended, the phrases "measure the level" or "detect the level" may be used.

[0066] As used herein, the term "exonuclease" refers to any wild-type or mutant enzyme that can cleave a phosphodiester bond (s) that joins the terminal nucleotide (s) of an oligonucleotide or polynucleotide, such as a 5'→3' exonuclease, a 3'→5' exonuclease, and a poly(A)-specific 3'→5' exonuclease. Non-limiting examples of exonucleases include exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VII, Xm1, and rat 1.

[0067] As used herein, the term "5'→3' exonuclease" refers to an exonuclease that cleaves a phosphodiester bond at the 5'-end of an oligonucleotide or polynucleotide. Non-limiting examples of 5'→3' exonucleases include T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, phage lambda exonuclease, the 5'-exonuclease of DNA polymerase I, exonuclease VIII, RecJ, RecJf, Tth RecJ, Mpn NrnA, human exonuclease 5, human exonuclease 1, SNM1, SNM1A, human SNM1B / Apollo, bovine SNM1B, SXT-Exo, phospholipase D3, phospholipase D4, Sso1391-Csa1, Sto0027-Csa1, Ttx1248-Csa1, Sso1451-Csa1, Sto2633-Csa1, Pfu1793-Cas4, Sto2501, Sso0001, Sto2331-Cas4, Ttx1245-Cas4, Sso1449-Cas4, Sto2635-Cas4, Sso1392-Cas4, SIRV2 gp19, and bacterial AddB.

[0068] As used herein, the term "3'-end" generally refers to the region or position of the same polynucleotide or oligonucleotide that is downstream of the 5'-region or position of the polynucleotide or oligonucleotide.

[0069] As used herein, the term "5'-end" generally refers to the region or position of the same polynucleotide or oligonucleotide that is upstream of the 3'-region or position of the polynucleotide or oligonucleotide.

[0070] As used herein, an aldehyde-reactive compound is a class of compounds that react with an aldehyde group or form a bond with an aldehyde group. In one embodiment, the aldehyde-reactive compound is structurally a compound having at least one primary amine, a hydrazide, an acyl hydrazide, an aminooxy (—ONH 2 ) group, a compound having a naphthalene-containing aminooxy group and / or a guanidine-containing aminooxy group.

[0071] As used herein, the term "label" (also referred to interchangeably as "detectable label") refers to a chemical group or functional moiety that binds or attaches to (also referred to interchangeably as "labels") a polynucleotide. A labeled polynucleotide can generally be detected directly or indirectly by a detectable signal. The detectable label may be attached (or bound) directly or via a non-interfering linking group to another moiety that can specifically bind to one or more sites to be labeled. The detectable label may be bound directly or indirectly, and may be bound covalently or non-covalently.

[0072] As used herein, the term "monofunctional DNA glycosylase" refers to a naturally occurring monofunctional glycosylase that essentially has only DNA glycosylase activity. The term "monofunctional DNA glycosylase" also refers to a monofunctional glycosylase derived from a bifunctional DNA glycosylase and naturally having both DNA glycosylase and abasic site lyase (AP lyase) activities, and having the AP lyase domain of the bifunctional DNA glycosylase removed or inactivated.

[0073] As used herein, the term "enzymatically active fragment" refers to a fragment of a catalytically or enzymatically active protein or polypeptide that has at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the activity of the protein or polypeptide from which the fragment is derived.

[0074] The step of signal detection is known in the art. Signal detection may be by visual inspection or using a suitable instrument appropriate for the label used, such as a spectrometer, fluorometer, luminometer, phosphoimager, Geiger counter, scintillation counter, or microscope. For example, if the label is a radioisotope, detection may be performed using, for example, a scintillation counter or photographic film such as autoradiography. When a fluorescent label is used, detection can be performed by exciting the fluorescent dye at an appropriate light wavelength and detecting the emitted fluorescence by a fluorescence microscope, visual inspection, photographic film, fluorometer, luminometer, charge-coupled device (CCD) camera, and scanner, etc. When an enzyme label is used, detection can be performed by preparing an appropriate substrate for the enzyme and detecting the resulting reaction product. For example, colored bodies can be obtained by many substrates of horseradish peroxidase such as o-phenylenediamine. Suitable instruments for high-sensitivity detection are known in the art. Alternatively, a signal amplification strategy can be optionally used to facilitate the detection of low-abundance molecular targets.

[0075] While the present disclosure is illustrated by specific embodiments and any features, it is understood that modifications and variations of the concepts disclosed herein can be made by those skilled in the art and that such modifications and variations are considered to be within the scope of the present disclosure.

Examples

[0076] In the following examples, exemplary embodiments according to the present disclosure are further described, but these should not be construed as limiting the scope of the present disclosure. The materials and methods used in the following examples are described in detail below. Materials used in the present disclosure but not annotated herein are commercially available.

[0077] Provided herein are examples of nucleic acid modifications using the methods of the present disclosure, showing several exemplary modifications of polynucleotides for generating nucleic acids whose 3'-ends are labeled with a fluorophore or a quencher.

[0078] As shown in Figure 1, the methods used in these modifications can actually be divided into three consecutive steps. 1) Using a polymerase, incorporate a modified nucleotide having a reactive moiety into the 3'-end of the target polynucleotide; 2) conjugate a desired molecule having a labeling moiety such as a fluorescent dye or a quencher and a corresponding functional moiety to the 3'-end of the target polynucleotide; 3) degrade the unlabeled or incompletely labeled polynucleotide with a 3'→5' exonuclease to enrich the desired labeled polynucleotide.

[0079] In some embodiments, an enzymatic synthesis technique is used to introduce a reactive moiety into the target polynucleotide. For example, nucleotide analog(s) are enzymatically added to the 3'-hydroxyl (3'-OH) end of a single-stranded nucleic acid initiator (target polynucleotide) by a template-independent synthesis technique to generate a polynucleotide having the desired reactive moiety. In at least one embodiment, the desired reactive moiety is an azide (N 3 ) or an azide group, and such a modification can be introduced at the 3'-end of the polynucleotide using a suitable reagent / compound such as a nucleotide analog containing an azide moiety such as a 3'-O-azidomethyl group. Based on this scenario, the following examples are further provided. Example 1: Preparation of a polynucleotide containing a 3'-O-azidomethyl group

[0080] As the target polynucleotide, a 45-mer DNA polynucleotide containing a fluorescein label at the 5'-end (5'-FAM-45-mer DNA) is used for 3'-modification or labeling. To introduce an azidomethyl group at the 3'-end of 5'-FAM-45-mer DNA, 3'-O-azidomethyl-deoxynucleoside triphosphate (3'-AZ-dNTP) is used as a template-independent DNA synthesis substrate for a selected Family B DNA polymerase or its variant. An exemplary polymerase used herein may be a self-polymerase variant derived from Vent DNA polymerase, which can efficiently incorporate 3'-AZ-dNTP at the 3'-end of the target polynucleotide. The nucleotide incorporation reaction is carried out with 100 nM of 5'-FAM-45-mer DNA target polynucleotide, 0.25 mM manganese chloride (MnC1 2) and in a reaction mixture (10 μL) containing 200 nM of polymerase. The reaction was initiated by adding 25 μM of 3’-O-azidomethyl-dTTP (3’-AZ-dTTP), and then incubated at 60 °C for a predetermined period. The period was from 2 minutes to 30 minutes. Thereafter, the reaction was terminated by adding 10 μL of quenching solution (95% deionized formamide and 25 mM EDTA) twice. The reaction mixture was further denatured at 95 °C for 10 minutes, and the reaction products were analyzed by 20% polyacrylamide gel electrophoresis containing 8 M urea (urea-PAGE). The reaction products were then visualized by imaging the gel with an Amersham Typhoon laser scanner- (Cytiva Life Sciences, Marlborough, Massachusetts, USA). At the end of the polymerase-dependent incorporation reaction of 3’-AZ-dTMP into 5’-FAM-45-mer DNA, 5’-FAM-46-mer DNA having an azide group at its 3’ end was formed (shown in lanes 2 of FIGS. 2A, 3, and 5, respectively) and could be used for subsequent labeling reactions. Alternatively, before the subsequent labeling reaction, 5’-FAM-46-mer DNA was further purified using a DNA QIAquick nucleotide removal kit (Qiagen, Germantown, Maryland). The preparation of 3’-azide-labeled target polynucleotides containing a 3’-O-azidomethyl group is shown in Scheme 1 below. JPEG2025517919000002.jpg45170 Example 2: 3’-Labeling of Polynucleotides Having an Azidomethyl Group at the 3’ End by Azide-Alkyne Click Reaction

[0081] As described in Example 1, after obtaining a polynucleotide containing an azidomethyl group at the 3’ end, the 3’-terminal azide (N 3 ) group can be directly used to label with any desired tag or functional molecule such as a fluorescent dye or quencher by an azide-alkyne click reaction.

[0082] For example, when it is desired to label the 3'-end of a target polynucleotide with a cyanine 5 (Cy5) fluorophore, a Cy5-alkyne molecule can be selected for an azide-alkyne coupling reaction with a polynucleotide having an azide (N 3 ) group at the 3'-end. In at least one embodiment, a polynucleotide having a 3'-azide group is reacted directly with Cy5-alkyne in the presence of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand, copper ions, and sodium ascorbate, to cause a Cu-catalyzed cycloaddition reaction between the 3'-azide group of the polynucleotide and the alkyne group of the Cy5-alkyne molecule. The azide-alkyne coupling reaction was carried out normally at 37 °C for 1 hour. Thereafter, unreacted Cy5-alkyne molecules were removed by using a DNA QIAquick nucleotide removal kit (Qiagen, Germantown, Maryland, USA). The purified reaction product was further subjected to 3'→5' exonuclease (e.g., 200 nM of 3'→5' exonuclease) treatment at 37 °C for 1 hour. The final reaction product was analyzed by 20% urea-PAGE and visualized by imaging the gel with an Amersham Typhoon laser scanner- (Cytiva Life Sciences, Marlborough, Massachusetts, USA). As a result of the azide-alkyne cycloaddition and enzyme-digestion reactions, a homogeneous target polynucleotide containing a Cy5 fluorophore at the 3'-end is produced (see lanes 3 in FIGS. 2A and 2B). The labeling of the Cy5 fluorophore to the 3'-end of the target polynucleotide is shown in Scheme 2 below. JPEG2025517919000003.jpg96170

[0083] Similarly, when it is desired to label the 3'-end of a target polynucleotide with a fluorescent quencher such as black hole quencher 1 (BHQ1), a BHQ1-alkyne molecule is used with an azide (N 3)It can be selected for the azide-alkyne coupling reaction with a target polynucleotide having a base. For example, a polynucleotide having a 3'-azide group was reacted with BHQ1-alkyne in the presence of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand, copper ions, and sodium ascorbate, causing a Cu-catalyzed cycloaddition reaction between the 3'-azide group and the alkyne group of the BHQ1-alkyne molecule. The azide-alkyne coupling reaction was carried out normally at 37 °C for 1 hour. Thereafter, unreacted BHQ1-alkyne molecules were removed by using a DNA QIAquick nucleotide removal kit (Qiagen, Germantown, Maryland, USA). The purified reaction product was further subjected to treatment with a 3'→5' exonuclease (e.g., 200 nM of 3'→5' exonuclease) at 37 °C for 1 hour. The final reaction product was analyzed by 20% urea-PAGE and visualized by imaging the gel with an Amersham Typhoon laser scanner (Cytiva Life Sciences, Marlborough, Massachusetts, USA). By performing the azide-alkyne cycloaddition and enzyme-digestion reactions successively, a homogeneous target polynucleotide containing a BHQ1 label at the 3'-end is generated (see lane 4 of Figure 3). The labeling of the fluorescent quencher to the 3'-end of the target polynucleotide is shown in Scheme 3 below. JPEG2025517919000004.jpg124170

[0084] The components of the BHQ1 labeling reaction and each experimental group are summarized in Table 1 below. The results of the labeling reactions corresponding to each experimental group are shown in lanes 1-4 of Figure 3, respectively. In Table 1, four experimental groups were planned, the reaction components are shown, the symbol "+" represents adding the specified reagent to the reaction in each experimental group, and the symbol "-" represents not adding the specified reagent to the reaction in the experimental group.

[0085]

Table 1

[0086] Alternatively, after obtaining a polynucleotide containing an azidomethyl group at the 3' end, the terminal azide group can be directly used to label with any desired tag or functional molecule such as a fluorescent dye or quencher by an azide-dibenzocyclooctyne (DBCO) ligation reaction.

[0087] For example, when a Cy5 fluorophore label is desired at the 3' end of a target polynucleotide, a Cy5-DBCO molecule can be selected for the azide-DBCO ligation reaction with a polynucleotide having an azide (N 3 ) group at the 3' end. In at least one embodiment, the polynucleotide having a 3'-azide group can be directly reacted with Cy5-DBCO in 1×TE buffer. The azide-DBCO ligation reaction was carried out normally at 37 °C for 1 hour. Thereafter, unreacted Cy5-DBCO molecules were removed by using a DNA QIAquick nucleotide removal kit (Qiagen, Germantown, Maryland, USA). The reaction product was analyzed by 20% urea-PAGE and visualized by imaging the gel with an Amersham Typhoon laser scanner- (Cytiva Life Sciences, Marlborough, Massachusetts, USA). The azide-DBCO ligation reaction generates a target polynucleotide having a Cy5 fluorophore at the 3' end (see lanes 2 in Figures 4A and 4B). The labeling of the Cy5 fluorophore at the 3' end of the target polynucleotide is shown in Scheme 4 below. JPEG2025517919000006.jpg108170

[0088] Similarly, when a fluorescent quencher label such as Black Hole Quencher 1 (BHQ1) is desired at the 3' end of a polynucleotide, a BHQ1-DBCO molecule is used with an azide (N 3)It can be used for the azide-DBCO ligation reaction with a target polynucleotide having a base. For example, a target polynucleotide having a 3'-azide group can be directly reacted with BHQ1-DBCO in 1×TE buffer. The azide-DBCO ligation reaction was normally carried out at 37°C for 1 hour. Thereafter, unreacted BHQ1-DBCO molecules were removed by using a DNA QIAquick nucleotide removal kit (Qiagen, Germantown, Maryland, USA). The purified reaction product was further subjected to treatment with a 3'→5' exonuclease (e.g., 200 nM 3'→5' exonuclease) at 37°C for 1 hour. The final reaction product was analyzed by 20% urea-PAGE and visualized by imaging the gel with an Amersham Typhoon laser (Marlborough, Massachusetts, USA). By continuously performing the azide-DBCO ligation and the enzyme-digestion reaction, a homogeneous target polynucleotide having a BHQ1 label at the 3' end is generated (see lane 4 of Figure 5). The labeling of the fluorescent quencher to the 3' end of the target polynucleotide is shown in the following scheme 5. JPEG2025517919000007.jpg122170

[0089] The components of the BHQl labeling reaction and each experimental group are summarized in Table 2 below. The results of the labeling reactions corresponding to each experimental group are shown in lanes 1 to 4 of Figure 5, respectively. In Table 2, four experimental groups were planned, the reaction components are shown, the symbol "+" represents adding the specified reagent to the reaction in each experimental group, and the symbol "-" represents not adding the specified reagent to the reaction in the experimental group.

[0090]

Table 2

[0091] A polynucleotide having a partial double-stranded region can also be used as a target polynucleotide for 3'-modification or labeling by addition of 3'-AZ-dNTP to the 3'-end of the target polynucleotide. For example, a partial double-stranded polynucleotide consisting of a 60-mer forward strand and a 20-mer reverse strand was used as the target polynucleotide. To introduce an azidomethyl group at the 3'-end of the partial double-stranded DNA, 3'-O-azidomethyl-deoxynucleoside triphosphate (3'-AZ-dNTP) was used as a template-independent DNA synthesis substrate for a selected B-family DNA polymerase or its variant. The nucleotide incorporation reaction was carried out in a reaction mixture (10 μL) containing 100 nM of the partial double-stranded target polynucleotide, 0.25 mM of manganese chloride (MnC1 2 ), and 200 nM of the polymerase. The reaction was initiated by adding 25 μM of 3'-O-azidomethyl-dTTP (3'-AZ-dTTP), and then incubated at 60 °C for 10 minutes. Thereafter, the reaction was terminated by adding 10 μL of quenching solution (95% deionized formamide and 25 mM EDTA) twice. The reaction mixture was further denatured at 95 °C for 10 minutes and then subjected to 20% polyacrylamide gel electrophoresis containing 8 M urea (urea-PAGE). The reaction product was visualized by imaging the gel with an Amersham Typhoon laser scanner- (Cytiva Life Sciences, Marlborough, Massachusetts, USA). After the polymerase-dependent 3'-AZ-dTMP incorporation reaction into the partial double-stranded polynucleotide, a 61-mer forward strand having an azide group at the 3'-end was formed (shown in lane 1 of Figure 6, where lane S indicates the electrophoretic position of the partial double-stranded polynucleotide before the 3'-end nucleotide incorporation reaction), and it can be used for subsequent labeling reactions.

[0092] In this example, the partial double-stranded polynucleotide containing an azidomethyl group at the 3'-end obtained above was subjected to subsequent labeling reactions via azide-dibenzocyclooctyne (DBCO) ligation as described above.

[0093] The results of the labeling reaction of the partial double-strand are shown in FIG. 6. Here, lane S shows the electrophoretic position of the partial double-stranded polynucleotide before the labeling reaction; lane 1 shows the result of the formation of the 61-mer forward strand having an azide group at the 3'-end; lane 2 shows the result after the labeling reaction; lane 3 shows the result after the purification reaction. The labeled product was further subjected to treatment with a 3'→5' exonuclease (e.g., 200 nM of 3'→5' exonuclease) at 37° C. for 1 hour. This example demonstrates the applicability of the 3'-labeling method of the present disclosure to polynucleotides having a partial double-stranded region or DNA consisting of chains of different lengths. Example 5: 3'-Labeling of Polynucleotides Having Different 3'-O-Azidomethyl Deoxynucleotides

[0094] A 38-mer DNA polynucleotide (5'-FAM-38-mer DNA) containing a fluorescein label at the 5'-end was used as a target polynucleotide for 3'-modification or labeling. To introduce an azidomethyl group at the 3'-end of 5'-FAM-38-mer DNA, different fluorescently labeled 3'-O-azidomethyl-deoxynucleoside triphosphates containing Cy5-labeled 3'-AZ-dATP, Cy5-labeled 3'-AZ-dGTP, and IF700-labeled 3'-AZ-dCTP were used as template-independent DNA synthesis substrates for the selected Family B DNA polymerase used. The exemplary polymerase used herein is Vent polymerase, which can efficiently incorporate various 3'-AZ-dNTPs at the 3'-end of the target polynucleotide. The nucleotide incorporation reaction was carried out with 100 nM of the 5'-FAM-38-mer DNA target polynucleotide, 0.25 mM manganese chloride (MnC1 2) and performed in a reaction mixture (10 μL) containing 200 nM polymerase. The reaction was initiated by adding 25 μM Cy5-labeled 3'-AZ-dATP, Cy5-labeled 3'-AZ-dGTP, or IF700-labeled 3'-AZ-dCTP, respectively, and then incubated at 60 °C for 20 minutes. The reaction was terminated by adding 10 μL of quenching solution (95% deionized formamide and 25 mM EDTA) twice to each reaction mixture, and further denatured at 95 °C for 10 minutes. The reaction products were then analyzed by 20% polyacrylamide gel electrophoresis containing 8 M urea (urea-PAGE), and the gel was visualized by imaging with an Amersham Typhoon laser scanner- (Cytiva Life Sciences, Marlborough, Massachusetts, USA). After incorporating Cy5-labeled 3'-AZ-dATP, Cy5-labeled 3'-AZ-dGTP, and IF700-labeled 3'-AZ-dCTP into the 5'-FAM-38-mer polynucleotide, 5'-FAM-39-mer polynucleotides having an azide group at the 3'-end were formed (shown in lanes 1, 2, and 3 of Figure 7, respectively).

[0095] In this example, the 5'-FAM-39-mer polynucleotides containing different azidomethyl groups at the 3'-end obtained above were subjected to a subsequent labeling reaction via the direct incorporation of fluorescently labeled 3'-O-azidomethyl-deoxynucleoside triphosphate.

[0096] The results of the labeling reaction of the partially double-stranded polynucleotides are shown in Figure 7. Here, lanes S1, S2, and S3 indicate the electrophoretic positions of the 5'-FAM-38-mer and 5'-FAM-39-mer polynucleotides before the labeling reaction, respectively; lanes 1, 2, and 3 indicate the electrophoretic positions of the fluorescently labeled 5'-FAM-38-mer and 5'-FAM-39-mer polynucleotides after the labeling reaction, respectively; lanes 1-1, 2-1, and 3-1 show the results after the purification reaction. The purified reaction products were further subjected to treatment with 3'→5' exonuclease (e.g., 200 nM 3'→5' exonuclease) at 37 °C for 1 hour.

[0097] This example demonstrates the applicable use of different 3'-O-azidomethyl-deoxynucleoside triphosphates for introducing an azidomethyl group to the 3'-end of a target polynucleotide using the 3'-labeling method of the present disclosure. Example 6: 5'-End Labeling of Nucleic Acids Using Aldehyde-Reactive Compounds

[0098] A 45-mer single-stranded DNA (ssDNA; 5'- / deoxyU / CTCGGCCTGGCACAGGTCCGTCTCAGTGCTGCGGCGACCACCGA-3' (SEQ ID NO: 1)) containing a uracil residue at the 5'-end and a fluorescein (FAM) dye at the 3'-end was synthesized. To excise uracil and subsequently perform abasic site labeling, 100 nM of the 45-mer uracil-containing ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Luttrell's bacterium (MluUDG) and 5 mM of an aldehyde-reactive probe ((N-aminooxyacetyl)-N'-biotinylhydrazine). The reaction was initiated by adding MluUDG and the aldehyde-reactive probe at 37 °C for 15 minutes. The reaction was terminated by adding an equal volume of quenching solution twice (30 mM EDTA and 95% (v / v) deionized formamide), followed by denaturation at 95 °C for 10 minutes. The reaction products were analyzed by denaturing 20% polyacrylamide gel electrophoresis (urea-PAGE) containing 8 M urea. The results were visualized by scanning the gel with an Amersham Typhoon imager (Cytiva Life Sciences, Marlborough, Massachusetts, USA) and are shown in Figure 8. As shown in Figure 8, the addition of MluUDG and the aldehyde-reactive probe further generated a higher molecular weight band in the gel image, indicating the presence of the labeled ssDNA product.

[0099] Similarly, in another example, a partially double-stranded DNA molecule was labeled and analyzed with an aldehyde-reactive probe. The double-stranded DNA was prepared by annealing a 45-mer uracil-containing ssDNA (SEQ ID NO: 1) to a 15-mer complementary strand (5’-TGTGCCAGGCCGAGA-3’ (SEQ ID NO: 2)) at a molar ratio of 1:1.5 in a 1× Tris-EDTA (TE) buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98 °C for 3 minutes in a thermal cycler and then gradually cooling it to 4 °C (e.g., 30 seconds every 5 °C). The resulting double-stranded DNA was treated with MluUDG to excise uracil and then subjected to subsequent abasic site labeling under the above-described steps and conditions for labeling ssDNA. The reaction was terminated by adding an equal volume of quenching solution twice and then denatured at 95 °C for 10 minutes. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager and are shown in Figure 8. In Figure 8, the addition of MluUDG and the aldehyde-reactive probe to the double-stranded DNA further generated higher molecular weight bands in the gel, indicating the presence of labeled DNA products. Thus, by the provided method, both single-stranded and double-stranded DNA were labelable with an aldehyde-reactive probe. Example 7: 5’-End Labeling of Nucleic Acids Using Aminooxy-5(6)-FAM

[0100] A 45-mer single-stranded DNA (ssDNA; SEQ ID NO: 1) containing a uracil residue at the 5'-end and a cyanine 5 (Cy5) dye at the 3'-end was synthesized. To excise uracil and subsequently label the abasic site, 100 nM of ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Lutheus bacteria (MluUDG) and 2 mM of aminooxy-5(6)-FAM. The reaction was initiated by adding MluUDG and aminooxy-5(6)-FAM and incubated at 37 °C for 60 minutes. The reaction was terminated by adding an equal volume of quenching solution twice (30 mM EDTA and 95% (v / v) deionized formamide), followed by denaturation at 95 °C for 10 minutes. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As shown in Figure 9, the addition of MluUDG and aminooxy-5(6)-FAM further generated higher molecular weight bands in the gel, indicating the presence of labeled FAM-ssDNA products.

[0101] Similarly, in another example, partial double-stranded DNA molecules were labeled and analyzed with aminooxy-5(6)-FAM. Double-stranded DNA was prepared by annealing a 45-mer uracil-containing ssDNA (SEQ ID NO: 1) to a 15-mer complementary strand (5’-TGTGCCAGGCCGAGA-3’ (SEQ ID NO: 2)) at a molar ratio of 1:1.5 in a 1×TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98 °C for 3 minutes in a thermal cycler and then gradually cooling it to 4 °C (e.g., 30 seconds every 5 °C). The resulting double-stranded DNA was treated with MluUDG to excise uracil and then subjected to abasic site labeling under the above-described steps and conditions for labeling ssDNA. The reaction was terminated and the product was analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As also shown in Figure 9, the addition of MluUDG and aminooxy-5(6)-FAM to double-stranded DNA resulted in the further formation of higher molecular weight bands in the gel, indicating the presence of labeled FAM-DNA products. Thus, it was shown that both single-stranded and double-stranded DNA were labeled with aminooxy-5(6)-FAM by the provided method. Example 8: 5’-End Labeling of DNA with Naphthalene- and Guanidine-Containing Aminooxy-FAM

[0102] A 47-mer single-stranded DNA (ssDNA; 5’- / DeoxyU / CTCGGCCTGGCACAGGTCCGTCTCAGTGCTGCGGCGACCACCGAGG-3 (SEQ ID NO: 3)) containing a uracil residue at the 5’ end was synthesized. To excise uracil and subsequently label the abasic site, 100 nM of ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Lutius bacteria (MluUDG) and 2 mM of naphthalene and guanidine-containing aminooxy-FAM. The reaction was initiated by adding MluUDG and naphthalene and guanidine-containing aminooxy-FAM, and incubated at 37 °C for 60 minutes. The reaction was terminated by adding an equal volume of quenching solution twice (30 mM EDTA and 95% (v / v) deionized formamide), and then denatured at 95 °C for 10 minutes. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As shown in Figure 10, the addition of MluUDG and naphthalene and guanidine-containing aminooxy-FAM further generated higher molecular weight bands in the gel, indicating the presence of the labeled FAM-ssDNA product.

[0103] Similarly, in another example, partial double-stranded DNA molecules were also labeled and analyzed with naphthalene- and guanidine-containing aminooxy-FAM. Double-stranded DNA was prepared by annealing a 47-mer uracil-containing ssDNA (SEQ ID NO: 3) to a 15-mer complementary strand (SEQ ID NO: 2) at a 1:1.5 molar ratio in 1×TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98 °C for 3 minutes in a thermal cycler and then gradually cooling it to 4 °C (e.g., 30 seconds every 5 °C). The resulting double-stranded DNA was subjected to the above-described steps and conditions for excising uracil and labeling ssDNA, and then to abasic site labeling. The reaction was terminated and the product was analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As shown in Figure 10, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAM to the double-stranded DNA further generated higher molecular weight bands in the gel, indicating the presence of FAM-labeled double-stranded DNA products. Thus, by the provided method, both single-stranded and double-stranded DNA were labelable with naphthalene- and guanidine-containing aminooxy-FAM. Example 9: 5'-End Labeling of 5'-Phosphorylated DNA with Aldehyde-Reactive Compounds and Concentration of Labeled DNA Using Phage Lambda Exonuclease

[0104] A 45-mer single-stranded DNA (ssDNA; SEQ ID NO: 1) containing a uracil residue at the 5’ end and fluorescein (FAM) at the 3’ end was synthesized. First, in the presence of adenosine triphosphate (ATP), the DNA was 5’-end phosphorylated with T4 polynucleotide kinase at 37 °C for 10 minutes. To excise uracil and subsequently perform abasic site labeling, 100 nM of 5’-phosphorylated ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Luttrellspiraceae bacterium (MluUDG) and 5 mM of an aldehyde-reactive probe (N-(aminooxyacetyl)-N’-biotinylhydrazine). The reaction was initiated by adding MluUDG and the aldehyde-reactive probe simultaneously at 37 °C over 15 minutes. To remove unlabeled ssDNA, 2 units of phage lambda exonuclease were added, and then the mixture was incubated at 37 °C for an additional 3.5 hours. The reaction was stopped by adding an equal volume of quenching solution twice (30 mM EDTA and 95% (v / v) deionized formamide), and then denatured at 95 °C for 10 minutes. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As shown in Figure 11, the addition of MluUDG and the aldehyde-reactive probe resulted in the further formation of a higher molecular weight band in the gel, indicating the presence of labeled ssDNA product. When the reaction mixture was further treated with phage lambda exonuclease to degrade unlabeled DNA (i.e., the purification step), a portion of the labeled ssDNA product was further concentrated.

[0105] Thus, by the provided method, DNA can be labeled with an aldehyde-reactive probe, and the labeled DNA fraction can be further concentrated by treatment with phage lambda exonuclease.

[0106] Example 10: 5’-End Labeling of 5’-Phosphorylated DNA with Naphthalene- and Guanidine-Containing Aminooxy-FAM, and Purification and Concentration of FAM-Labeled DNA Using Phage Lambda Exonuclease

[0107] A 47-mer single-stranded DNA (ssDNA; SEQ ID NO: 3) containing a uracil residue at the 5'-end was synthesized. First, in the presence of adenosine triphosphate (ATP), the DNA was 5'-end phosphorylated with T4 polynucleotide kinase at 37°C for 30 minutes. To excise uracil and subsequently perform abasic site labeling, 100 nM of ssDNA was mixed with 115 ng of uracil-DNA glycosylase from Lutius bacteria (MluUDG), and 1 mM of naphthalene and guanidine-containing aminooxy-FAM. The reaction was initiated by adding MluUDG and naphthalene and guanidine-containing aminooxy-FAM, and incubated at 37°C for 30 minutes. To remove unlabeled ssDNA, 2 units of phage lambda exonuclease were added, and then incubated at 37°C for an additional 30 minutes. The reaction was stopped by adding an equal volume of quenching solution twice (30 mM EDTA and 95% (v / v) deionized formamide), and then denatured at 95°C for 10 minutes. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As shown in Figure 12, the addition of MluUDG and naphthalene and guanidine-containing aminooxy-FAM further generated higher molecular weight bands in the gel, indicating the presence of FAM-labeled ssDNA products. When the reaction mixture was further treated with phage lambda exonuclease to degrade unlabeled DNA, the FAM-labeled ssDNA products were concentrated.

[0108] Similarly, in another example, 5'-phosphorylated double-stranded DNA molecules were also labeled with naphthalene and guanidine-containing aminooxy-FAM, and then subjected to phage lambda exonuclease treatment to enrich the FAM-labeled double-stranded DNA. The double-stranded DNA was prepared by annealing a 47-mer uracil-containing ssDNA (SEQ ID NO: 3) to a 15-mer complementary strand (SEQ ID NO: 2) at a 1:1.5 molar ratio in a 1×TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out in a thermal cycler by heating the DNA mixture to 98 °C for 3 minutes and then gradually cooling it to 4 °C (e.g., 30 seconds every 5 °C). First, the resulting double-stranded DNA was 5'-end phosphorylated with T4 polynucleotide kinase in the presence of ATP, then subjected to uracil excision and subsequent abasic site labeling, and further phage lambda exonuclease treatment was performed under the above-described steps and conditions for labeling ssDNA. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As shown in Figure 12, the addition of MluUDG and naphthalene and guanidine-containing aminooxy-FAM to the double-stranded DNA resulted in the further generation of higher molecular weight bands in the gel, indicating the presence of the FAM-labeled DNA product. When the reaction mixture was further treated with phage lambda exonuclease to degrade the unlabeled DNA, the FAM-labeled DNA product was enriched.

[0109] Thus, by the provided method, both single-stranded and double-stranded DNA can be labeled with naphthalene and guanidine-containing aminooxy-FAM, and the labeled DNA fraction can be further enriched by treatment with phage lambda exonuclease. Example 11: Dual Labeling of Double-Stranded DNA with Guanidine-FAM at the 5' End and Direct Incorporation at the 3' End

[0110] 21-mer uracil-containing ssDNA was obtained. Its electrophoretic position is shown in lane 1 of Figure 13. Double-stranded DNA was prepared from this ssDNA by annealing 21-mer uracil-containing ssDNA and a 22-mer complementary strand at a 1:1 molar ratio in a 1×TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98 °C for 3 minutes in a thermal cycler and then gradually cooling it to 4 °C (e.g., 30 seconds every 5 °C).

[0111] The resulting double-stranded DNA was subjected to 5'-labeling by reacting 100 nM of the double-stranded DNA obtained above with 100 ng of MluUDG, 1 mM of guanidine-FAM, and 20 mM of p-phenylenediamine in a 10 μL reaction mixture at 37 °C for 60 minutes. To remove unlabeled ssDNA, 1 μM of phage lambda exonuclease was added and then incubated at 37 °C for an additional 60 minutes. The reaction was stopped by adding an equal volume of quenching solution twice (25 mM EDTA and 95% (v / v) deionized formamide), and then denatured at 95 °C for 10 minutes. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon imager. As shown in lane 2 of Figure 13, the addition of MluUDG and guanidine-FAM to the double-stranded DNA caused the band to migrate to a higher molecular weight position in the gel, indicating the presence of the labeled FAM-DNA product.

[0112] 3 In another reaction, the resulting double-stranded DNA was subjected to 3'-labeling by directly incorporating nucleotides with a fluorescent dye. The reaction was carried out in a 10 μL mixture containing 100 nM of double-stranded DNA, 200 nM of Vent polymerase, and 20 μM of N

[0113] -dTTP-Cy3 at 37 °C for 60 minutes. The results of the 3'-labeling are shown in lane 3 of Figure 13.100 nM double-stranded DNA, 100 ng of MluUDG, 1 mM guanidine-FAM, 20 mM p-phenylenediamine, 200 nM Vent polymerase, and 20 μM of N 3 A double-labeling reaction was performed by preparing a 10 μL mixture containing -dTTP-Cy3. The reaction was initiated by adding MluUDG and Vent polymerase and incubated at 37 °C for 60 minutes. The results of double-stranded DNA double-labeling at both the 5'-end and 3'-end are shown in lane 4 of Figure 13, indicating that the shift in electrophoretic position is a double-labeled product with a higher molecular weight than both the 5'-end labeled product and the 3'-end labeled product. Example 12: Double-labeling of double-stranded DNA by guanidine-FAM at the 5'-end and azide-DBCO ligation reaction at the 3'-end

[0114] 26-mer uracil-containing ssDNA was obtained. Its electrophoretic position is shown in lane 1 of Figure 14. Double-stranded DNA was prepared from this ssDNA by annealing 26-mer uracil-containing ssDNA and a 27-mer complementary strand (shown in lane 2 of Figure 14) at a 1:1 molar ratio in a 1×TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out in a thermal cycler by heating the DNA mixture to 98 °C for 3 minutes and gradually cooling it to 4 °C (e.g., 30 seconds per 5 °C).

[0115] The resulting double-stranded DNA was combined with the 100 nM double-stranded DNA obtained above, 200 nM Vent polymerase, 100 μM of N 3The 3'-labeling was performed by reacting 100 nM of double-stranded DNA, 100 ng of MluUDG, 1 mM of guanidine-FAM, 20 mM of p-phenylenediamine, 200 nM of Vent polymerase, 100 μM of N

[0116] -dTTP, and 100 μM of DBCO-Cy5 at 37 °C for 60 minutes. The result of the 3'-labeling is shown in lane 3 of Figure 14. The electrophoretic shift of the 3'-labeled product compared to the positions in lanes 1 and 2 suggests successful labeling with Cy5. The obtained labeled product was further concentrated by 3'-exonuclease digestion. To remove the unlabeled DNA strands, the reaction was initiated by adding 1 μM of hTREX1 to the labeling mixture at 37 °C. After 60 minutes, the digestion reaction was stopped by adding 10 μL of quenching solution (95% deionized formamide and 25 mM EDTA) twice. The result is shown in lane 4 of Figure 14. A darker band is shown compared to the band at the same position in lane 3. 3 A double-labeling reaction was carried out by preparing a 10 μL mixture containing 100 nM of double-stranded DNA, 100 ng of MluUDG, 1 mM of guanidine-FAM, 20 mM of p-phenylenediamine, 200 nM of Vent polymerase, 100 μM of N

[0117] The double-labeled product was further subjected to 5'-exonuclease and 3'-exonuclease digestion to remove unlabeled DNA strands. This is also referred to as the purification step. The reaction was initiated by adding 1 μM of lambda exonuclease and 1 μM of hTREX1 to the above-labeled mixture at 37°C. After 60 minutes, the reaction was stopped by adding 10 μL of quenching solution (95% deionized formamide and 25 mM EDTA) twice. The results are shown in lane 6 of Figure 14. A darker band is shown compared to the band at the same position in lane 5, indicating that the labeled product was efficiently purified. Example 13: Double-labeling of double-stranded DNA with guanidine-FAM at the 5' end and BHQ1 at the 3' end

[0118] A 25-mer uracil-containing ssDNA was obtained, and its electrophoretic position is shown in lane 1 of Figure 15. Double-stranded DNA was prepared from this ssDNA by annealing the 25-mer uracil-containing ssDNA and a 26-mer complementary strand (shown in lane 2 of Figure 15) at a 1:1 molar ratio in 1×TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98°C for 3 minutes in a thermal cycler and gradually cooling it to 4°C (e.g., 30 seconds at every 5°C).

[0119] First, the obtained double-stranded DNA was subjected to 5'-labeling by reacting 100 nM of the double-stranded DNA obtained above with 100 ng of MluUDG, 1 mM of guanidine-FAM, and 20 mM of p-phenylenediamine in a 10-μL reaction mixture at 37°C for 60 minutes. Then, 200 nM of Vent polymerase, 100 μM of N 3By adding -dCTP-BHQ1 at 37°C, the 5'-labeled mixture was subjected to 3'-labeling and further incubated for 60 minutes. Thereafter, to remove unlabeled DNA strands, 1 μM of lambda exonuclease and 1 μM of hTREX1 were added to the obtained labeled mixture at 37°C. After 60 minutes, the reaction was stopped by adding 10 μL of quenching solution (95% deionized formamide and 25 mM EDTA) twice. The results are shown in lane 3 of Figure 15. It is shown that the band position has shifted compared to lanes 1 and 2.

[0120] In another experiment, simultaneous labeling at the 5'-end and 3'-end was performed by mixing 100 nM of double-stranded DNA, 100 ng of MluUDG, 1 mM of guanidine-FAM, 20 mM of p-phenylenediamine, 200 nM of Vent polymerase, and 100 μM of N 3 -dCTP-BHQ1 in a 10 μL reaction volume. The reaction was initiated by adding MluUDG and Vent polymerase at 37°C. The reaction was carried out for 60 minutes before subjecting it to exonuclease digestion. To remove unlabeled DNA strands, 1 μM of lambda exonuclease and 1 μM of hTREX1 were added to the labeled mixture at 37°C and incubated for 60 minutes. The reaction was stopped by adding 10 μL of quenching solution (95% deionized formamide and 25 mM EDTA) twice. The results of simultaneous double-labeling and exonuclease digestion are shown in lane 4 of Figure 15. Lanes 3 and 4 show similar band intensities, indicating that the simultaneous reaction does not affect the labeling efficiency of double-stranded DNA. The band intensity represents the amount of labeled product.

[0121] 100 μM of N in the above 3'-end labeling reaction 3 -dCTP-BHQ1, 100 μM of N 3The labeling of dsDNA was performed again, either sequentially or simultaneously, following the same procedure as above except replacing -dCTP and 100 μM of DBCO-BHQ1. After exonuclease digestion, the labeled products were analyzed. As shown in lanes 5 and 6, which show sequential and simultaneous dual labeling at the 5'-end and 3'-end respectively, the simultaneous dual labeling reaction, including azide-DBCO ligation at the 3'-end, has a similar labeling efficiency compared to the sequential labeling where 5'-end labeling is performed first and then 3'-end labeling.

[0122] While the present disclosure has been described in conjunction with its embodiments, it will be understood that various changes may be made thereto without departing from the scope of the present disclosure. Accordingly, the described embodiments are not intended to limit the present disclosure, but are intended to embrace changes within the scope of the present disclosure. Therefore, the claims should be given the broadest interpretation so as to encompass such changes.

Claims

**Claim 1** A method for labeling the 5'-end and 3'-end of a nucleic acid, comprising: preparing a target nucleic acid to be labeled; adding a 5'-terminal glycosylase to the target nucleic acid; adding a nucleotide having a reactive moiety to the target nucleic acid; creating an intermediate nucleic acid having an abasic site at the 5'-end of the target nucleic acid; incorporating the nucleotide having the reactive moiety at the 3'-end of the intermediate nucleic acid; preparing an aldehyde-reactive compound having a detectable label for binding to the intermediate nucleic acid at the abasic site at the 5'-end; preparing a desired molecule having a corresponding functional moiety capable of reacting with the reactive moiety; exposing the intermediate nucleic acid to the aldehyde-reactive compound having the detectable label; and exposing the intermediate nucleic acid to the desired molecule having the corresponding functional moiety, thereby forming a labeled nucleic acid having the detectable label bound to the intermediate nucleic acid at the abasic site, forming a labeled nucleic acid having the detectable label bound to the 5'-end and a bond formed between the reactive moiety and the corresponding functional moiety. **Claim 2** The method according to claim 1, further comprising preparing at least one of a molecule having 5'→3' exonuclease activity and a molecule having 3'→5' exonuclease activity, and removing the unlabeled target nucleic acid and the intermediate nucleic acid. **Claim 3** The method according to claim 2, wherein the 5'→3' exonuclease is selected from the group consisting of T5 exonuclease, T7 exonuclease, phage lambda exonuclease, exonuclease VIII, RecJ, RecJf, Tth RecJ, Mpn NrnA, human exonuclease 5, human exonuclease 1, SNM1, SNM1A, human SNM1B / Apollo, bovine SNM1B, SXT-Exo, phospholipase D3, phospholipase D4, Sso1391-Csa1, Sto0027-Csa1, Ttx1248-Csa1, Sso1451-Csa1, Sto2633-Csa1, Pfu1793-Cas4, Sto2501, Sso0001, Sto2331-Cas4, Ttx1245-Cas4, Sso1449-Cas4, Sto2635-Cas4, Sso1392-Cas4, SIRV2 gp19, bacterial AddB, and any combination thereof.

4. The method according to claim 1, further comprising a labeling moiety configured to form a labeled nucleic acid when the desired molecule is bound to the 3' end of the nucleic acid.

5. The method according to claim 1, wherein the nucleotide having the reactive moiety is incorporated into the 3' end of the nucleic acid by template-independent enzymatic nucleic acid synthesis.

6. The method according to claim 5, wherein the template-independent enzymatic nucleic acid synthesis comprises using a DNA polymerase, an RNA polymerase, or an enzyme functionally equivalent thereto.

7. The method according to claim 6, wherein the DNA polymerase is a family A DNA polymerase, a family B DNA polymerase, or a family X DNA polymerase.

8. The method according to claim 7, wherein the family B DNA polymerase is a Thermococcaceae DNA polymerase.

9. The method according to claim 8, wherein the family B DNA polymerase is a Thermococcus DNA polymerase or a Pyrococcus DNA polymerase.

10. The method according to claim 9, wherein the Family B DNA polymerase is selected from the group consisting of the Family B DNA polymerase of Thermococcus kodakarensis, the Family B DNA polymerase of Pyrococcus furiosus, the Family B DNA polymerase of Thermococcus litoralis, the Family B DNA polymerase of Thermococcus sp. 9°N, and the Family B DNA polymerase of Thermococcus gorgonarius.

11. The method according to claim 6, wherein the DNA polymerase is a modified DNA polymerase.

12. The method according to claim 1, further comprising preparing the nucleic acid in a solution phase.

13. The method according to claim 1, wherein the nucleic acid is bound to an initiator attached to a solid support.

14. The method according to claim 13, further comprising enzymatically releasing the nucleic acid from the initiator with an endonuclease.

15. The method according to claim 1, wherein the nucleotide incorporated at the 3' end of the nucleic acid is a natural nucleotide, a nucleotide analog, or an abasic nucleotide.

16. The method according to claim 1, wherein the corresponding functional moiety reacts with the reactive moiety by a bioorthogonal reaction.

17. The method according to claim 16, wherein the bioorthogonal reaction is click conjugation, oxime / hydrazone formation, Staudinger ligation, tetrazine ligation, or quadricyclane ligation.

18. The method according to claim 17, wherein the click conjugation is selected from the group consisting of copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, isocyanide-based click reaction, and inverse electron demand Diels-Alder reaction.

19. The method according to claim 1, wherein the desired molecule is molecularly recognizable by detection of visible light, fluorescence, photoluminescence, electrochemiluminescence, laser, irradiation, fluorescence resonance energy transfer, fluorescence structural change, or fluorescence quenching.

20. The method according to claim 19, wherein the desired molecule is a chemical compound, a fluorescent tag, a dye, a marker, a reporter, a quencher, an amine, an antigen, a ligand, a protein, an antibody, an antibody fragment, a peptide, a peptide analog, or a quantum dot.

21. The method according to claim 1, further comprising preparing a 3'→5' exonuclease to digest nucleic acids or unmodified nucleic acids for which the binding was unsuccessful.

22. The method according to claim 1, wherein the labeling of the 5' end and the 3' end is performed sequentially or simultaneously.

23. A kit for dual-labeling a nucleic acid at the 5' end and the 3' end, comprising a 5' end glycosylase, an aldehyde-reactive compound, a nucleotide having a reactive moiety, a polymerase for incorporating the nucleotide having the reactive moiety at the 3' end of the nucleic acid, and a desired molecule having a corresponding functional moiety capable of reacting with the reactive moiety.

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