Methods, kits and systems for dual labeling of nucleic acids

Abstract

The present disclosure provides a method of introducing a modification at a terminal of a nucleic acid, comprising introducing a modification at the 3 '-terminal, 5'-terminal, or both 3 '-and 5'-terminals of the nucleic acid to label the nucleic acid with a desired moiety The disclosure also provides a kit of introducing a modification at a terminal of a nucleic acid.

Description

References for electronic sequence listing

[0001] The present disclosure relates to modifications at both ends of nucleic acids, and in particular to modifications at the 3'-end and 5'-end of nucleic acids. [Background technology]

[0002] Nucleic acid labeling is commonly performed in biomedical and biological applications, including identification and purification of unknown or target gene fragments, localization of target gene sequences, pinpointing of nucleic acid-protein interactions, and visualization of cell and tissue dynamics. In general, methods for labeling nucleic acids can be divided into chemical methods or enzymatic methods. Chemical labeling methods involve the use of chemically reactive compounds such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), imidazole, hydrazine, sodium periodate, and sodium cyanoborohydride to modify the 5'-phosphate, 3'-hydroxyl, nucleobase, or sugar moiety of the target nucleic acid, thereby modifying the structure of the nucleic acid, and then attaching a chemical or functional moiety to the desired nucleic acid.

[0003] On the other hand, the enzymatic nucleic acid labeling method utilizes enzymes such as alkaline phosphatase, nucleic acid kinase or DNA / RNA polymerase to replace, add or incorporate chemical or functional moieties (such as radioisotopes, biotin groups or fluorescently labeled nucleotides) into the target nucleic acid, thereby attaching the desired label to the nucleic acid.

[0004] Although there are many nucleic acid labeling technologies that can generate nucleic acid probes, such as those labeled with fluorophores, enzymes and radioactive phosphates, or nucleotides modified with digoxigenin or biotin, these methods are usually lengthy and complicated procedures and use multiple types of enzymes, reactive chemicals or radioactive isotopes. They usually require specialized enzymes, chemicals or personnel training to handle toxic or radioactive materials and waste, thus making the nucleic acid labeling process cumbersome and inefficient.

[0005] In addition, the labeling efficiency of existing nucleic acid labeling methods varies depending on the length and type of nucleic acid, the location of the target site to be labeled (e.g., nucleotides inside or at the end of the nucleic acid sequence), and the chemical or functional part to be labeled. Therefore, there is still a need to provide a simple, efficient and environmentally friendly nucleic acid labeling method. [Summary of the invention]

[0006] The present disclosure provides a method for effectively modifying or labeling the 3'-end of a nucleic acid or polynucleotide. The method utilizes a pair of functional moieties or chemical molecules that can be stably or covalently coupled to each other to introduce specific modifications or labels at the 3'-end of a nucleic acid or polynucleotide. For example, a component of the paired functional moiety or chemical molecule is labeled at the 3'-hydroxyl (3'-OH) group of a nucleobase or nucleotide, which is incorporated into the 3'-end of a target nucleic acid or polynucleotide by a polymerase. The resulting target nucleic acid or polynucleotide contains a first functional moiety or chemical molecule at the 3'-end, which can easily react with other components of the paired moiety or molecule carrying the desired modification or label. Therefore, the reaction between the paired functional moieties forms a stable or covalent connection, so that the target nucleic acid or polynucleotide can be modified or labeled with the desired molecule, particularly at the 3'-end of the target nucleic acid or polynucleotide.

[0007] In at least one embodiment, the methods provided herein include modifying natural or synthetic deoxyribonucleic acid (DNA) at the 3'-end. Figure 1 A schematic diagram depicting an exemplary method for introducing a 3'-modification into a target nucleic acid is shown. In some embodiments, the methods provided herein include providing a nucleic acid comprising a reactive portion (e.g., Figure 1 The 3'-terminal nucleotide of "M1" (e.g., Figure 1 and exposing the polynucleotide to a corresponding functional portion (e.g., Figure 1 The desired molecule (e.g., Figure 1 In some embodiments, the desired molecule has a label portion introduced into the polynucleotide (e.g., Figure 1 The "label" shown above) is used to form a labeled polynucleotide.

[0008] In at least one embodiment, the method provided by the present disclosure further includes preparing polynucleotides by template-independent enzymatic nucleic acid synthesis. In some embodiments, the template-independent enzymatic nucleic acid synthesis includes using a DNA polymerase, an RNA polymerase, or an enzyme with equivalent functionality thereof. In some embodiments, the DNA polymerase for the 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 Thermococcaceae DNA polymerase. In at least one embodiment, the B-family DNA polymerase is a Thermococcus DNA polymerase or a Pyrococcus DNA polymerase. In at least one specific embodiment, the B family DNA polymerase is selected from the group consisting of B family DNA polymerase (Kod1) from Thermococcus kodakarensis, B family DNA polymerase (Pfu) from Pyrococcus furiosus, B family DNA polymerase (Vent) from Thermococcus litoralis, B family DNA polymerase (9°N) from Thermococcus sp. 9°N, and B family DNA polymerase (Tgo) from Thermococcus gorgonarius.

[0009] 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, for example, 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.

[0010] In some embodiments, the methods provided by the present disclosure further include preparing the nucleic acid in a solution phase. In some embodiments, the methods provided by the present disclosure include preparing the polynucleotide in a solid phase, for example, providing an initiator bound to a solid support. In some embodiments, the solid support is selected from the group consisting of particles, polymers, beads, resins, slides, chips, array surfaces, membranes, flow cells, wells, matrices, chambers, microfluidic chambers, channels, microfluidic channels, and gels.

[0011] In at least one embodiment, the method provided by the present disclosure further includes providing an endonuclease to enzymatically release the polynucleotide from the initiator. In some embodiments, the endonuclease recognizes the 3'-penultimate nucleotide at the 3'-end of the initiator and cleaves the bond between the 3'-end nucleotide of the initiator and the polynucleotide, between the 3'-penultimate nucleotide at the 3'-end of the initiator and the 3'-penultimate nucleotide at the 3'-end of the initiator, between the 3'-penultimate nucleotide at the 3'-end of the initiator and the 4'-penultimate nucleotide at the 3'-end of the initiator, or between the 4'-penultimate nucleotide at the 3'-end of the initiator and the 5'-penultimate nucleotide at the 3'-end of the initiator.

[0012] In at least one embodiment of the present disclosure, the endonuclease is derived from Thermococcus barophilus (Tba), Pfu, Methanosarcina acetivorans (Mac), Pyrococcus abyssi (Pab), Thermococcus kodakarensis (Tko), Thermococcus gammatolerans (Tga), or Bacillus subtilis (Bsu).

[0013] 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 xenonucleotide. In some embodiments, the reactive moiety is attached to the 2'-carbon or 3'-carbon of the ribose, or to the nucleobase at the 3'-terminus of the nucleotide.

[0014] In at least one embodiment of the present disclosure, the corresponding functional moiety reacts with the reactive moiety via a bioorthogonal reaction. In some embodiments, the bioorthogonal reaction is a click conjugation, an oxime / hydrazone formation, a Staudinger ligation reaction, a tetrazine ligation, or a tetracycloheptane 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), isocyanide-based click reaction, and inverse electron demand Diels-Alder (IEDDA) reaction.

[0015] 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 triarylphosphino group, a cyclooctyne group, a thiol group, an alkenyl group, a nitrone group, an aldehyde group, a keto group, a dienyl group, and a dienophile group.

[0016] In at least one embodiment of the present disclosure, the corresponding functional moiety is selected from the group consisting of an azide group, an alkynyl group, a triarylphosphino group, a cyclooctyne group, a thiol group, an alkenyl group, a nitrone group, an aldehyde group, a keto group, a dienyl group, and a dienophile group.

[0017] In some embodiments, the bioorthogonal reaction is performed at a reaction temperature of 10° C. to 100° C., e.g., 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 performed for, e.g., 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 longer.

[0018] In at least one embodiment, the desired molecule is molecularly identifiable by detecting visible light, fluorescence, photoluminescence, electrochemiluminescence, laser, radiation, fluorescence resonance energy transfer, fluorescence conformational change, or fluorescence quenching. In some embodiments, the desired molecule is a chemical compound, a fluorescent tag, a dye, a marker, a reporter molecule, a quencher, an amine, an antigen, a ligand, a protein, an antibody, an antibody fragment, a peptide, a peptide analog, or a quantum dot.

[0019] In at least one specific embodiment, the method provided by the present disclosure further includes a clean-up or enrichment step to remove unlabeled nucleic acids or polynucleotides, for example, providing a protein with 3' to 5' exonuclease activity to degrade nucleic acids or polynucleotides that fail to successfully synthesize 3'-terminal nucleotides by polymerase or undergo incomplete coupling reaction with the second reactive part.

[0020] In at least one embodiment, the present disclosure further provides a kit for modifying the 3'-end of a polynucleotide, comprising a nucleotide having a reactive portion, a polymerase that incorporates the nucleotide having the reactive portion into the 3'-end of the polynucleotide, and a desired molecule having a corresponding functional portion capable of reacting with the reactive portion. In at least one embodiment, the kit for modifying the 3'-end of a polynucleotide comprises a nucleotide having a reactive portion, a polymerase, a desired labeling molecule, and a 3' to 5' exonuclease, the polynucleotide is coupled to the desired labeling molecule at the 3'-end to form a labeled polynucleotide, which can be further enriched by the removal exonuclease described herein.

[0021] The present disclosure further provides a method for labeling the 5'-end of a nucleic acid, the method comprising: providing a target nucleic acid to be labeled; providing a 5'-end glycosidase to react with the target nucleic acid to generate an intermediate nucleic acid having an abasic site at the 5'-end of the target nucleic acid; and providing an aldehyde-reactive compound carrying a detectable label for coupling with the abasic site of the intermediate nucleic acid to form a labeled nucleic acid with a detectable label attached to the 5'-end.

[0022] In at least one embodiment of the present disclosure, the nucleic acid is single-stranded or comprises a double-stranded region formed by at least 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 synthesized de novo or derived from a biological organism. In some embodiments, the nucleic acid is immobilized on a solid surface or a polymer surface.

[0023] In at least one specific embodiment of the present disclosure, the aldehyde-reactive compound is a compound having at least one primary amine, a hydrazide, an acylhydrazide, a compound having an aminooxy (ONH2) group, 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) bicyclononyne (BCN), fluorescent dye hydroxylamine such as Alexa Fluor 488 hydroxylamine, aldehyde-reactive probe (ARP), aminooxy fluorescent dyes such as aminooxy-5(6)-FAM, aminooxy-5(6)-ROX and aminooxy-5(6)-TAMRA, cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy biotin, naphthalene-containing aminooxy fluorescent dyes, guanidine-containing aminooxy fluorescent dyes, naphthalene and / or guanidine-containing aminooxy-FAM, Cy5-PEG aminooxy, or fluorescent dye hydrazides such as cyanine dye hydrazide or CF dye hydrazide.

[0024] In at least one embodiment of the present disclosure, the nucleic acid comprises 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-carboxycytosine, 3-methyladenine (3-meA), 3-methylguanine, 7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxycytosine, vinylcytosine, vinyladenine, thyminediol, cytosinediol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, formamidopyrimidine derivatives of adenine, and formamidopyrimidine derivatives of guanine.

[0025] In some embodiments, the 5'-end glycosidase of the present disclosure may be a monofunctional DNA glycosidase. In at least one embodiment of the present disclosure, the monofunctional DNA glycosidase may be selected from uracil DNA glycosidase (UDG or UNG), alkyladenine DNA glycosidase (AAG; also known as methylpurine DNA glycosidase (MPG)), single-stranded selective monofunctional uracil DNA glycosidase 1 (SMUG1), methyl binding domain glycosidase 4 (MBD4), thymine DNA glycosidase (TDG), MutY homologous DNA glycosidase (MYH), alkylpurine glycosidase C (AlkC), alkylpurine glycosidase D (AlkD) , 8-oxoguanine glycosidase 1 without abasic lyase activity (OGG1), endonuclease III-like glycosidase 1 without abasic lyase activity (NTHL1), endonuclease VIII-like glycosidase 1 without abasic lyase activity (NEIL1), endonuclease VIII-like glycosidase 2 without abasic lyase activity (NEIL2), endonuclease VIII-like glycosidase 3 without abasic lyase activity (NEIL3), their enzyme-active fragments and any combination thereof.

[0026] In at least one embodiment of the present disclosure, the uracil DNA glycosidase is derived from the family Micrococcaceae, Staphylococcaceae, or Caryophanaceae, which includes the genera Micrococcus, Stomatococcus, Staphylococci, or Planococcus. In some embodiments, the uracil DNA glycosidase is derived from Micrococcus luteus.

[0027] In at least one embodiment of the present disclosure, the detectable label is selected from the group consisting of azide compounds, alkynes, bicyclononyne (BCN), dibenzocyclooctyne (DBCO), maleimide, peptides, proteins, antibodies, dendrimers, biotin, radioisotopes, photochromic dyes, fluorescent dyes, luminescent dyes, or any combination thereof.

[0028] In some embodiments, the disclosed methods further include providing a 5' to 3' exonuclease to remove unlabeled nucleic acids.In at least one embodiment, the 5' to 3' exonuclease is selected from T5 exonuclease (T5 exo), T7 exonuclease (T7 exo), viral alkaline exonuclease, bacterial alkaline exonuclease, bacteriophage lambda exonuclease, 5'-exonuclease of DNA polymerase I from, e.g., Streptococcus pneumoniae or Helicobacter pylori (ExoVI), Escherichia coli exonuclease VIII (ExoVIII), RecJ from, e.g., Escherichia coli or Deinococcus radiodurans, RecJ derived from RecJ fused to maltose binding protein, RecJ from Thermus thermophilus (Tth), RecJ from Mycoplasma pneumoniae (Mycoplasma pneumonia, Mpn), NrnA from human exonuclease 5 (hEXO5), human exonuclease 1 (hEXO1), SNM1 from Saccharomyces cerevisiae, human or bovine SNM1A, human SNM1B / Apollo, bovine SNM1B, SXT-Exo from, for example, Vibrio cholerae, phospholipase D3 (PLD3), phospholipase D4 (PLD4), Sso1391-Csa1 from, for example, Sulfolobus solfataricus, Sto0027-Csa1 from, for example, Sulfolobus tokadaii, SXT-Exo from, for example, Thermoproteus adhering to tenax), Ttx1248-Csa1 from Sulfolobus tenax, Sso1451-Csa1 from Sulfolobus tenax, Sto2633-Csa1 from Sulfolobus tomentosa, Pfu1793-Cas4 from Pyrococcus furiosus, Sto2501 from Sulfolobus tomentosa, Sso0001 from Sulfolobus tenax, Sto2331-Cas4 from Sulfolobus tomentosa, Ttx1245-Cas4 from Thermoproteus adhering, Sso1449-Cas4 from Sulfolobus tenax, Sto2635-Cas4 from Sulfolobus tomentosa, Sso1392-Cas4 from Sulfolobus tenax, Sulfolobus islandus islandicus baculovirus 2 (SIRV2), bacterial AddB, and any combination thereof.

[0029] In some embodiments, the present disclosure further includes methods for synthesizing nucleic acids with unique 5'-terminal nucleobases. In some embodiments, nucleic acids with unique 5'-terminal nucleobases are synthesized by methods known in the art, including phosphoramidite-based nucleic acid synthesis methods, and template-dependent and template-independent enzymatic nucleic acid synthesis methods.

[0030] In some embodiments, the methods disclosed herein further include isolating nucleic acid fragments from a sample. For example, nucleic acids can be isolated from intact or damaged samples of viruses or cells such as bacteria, archaea, and eukaryotic cells (e.g., human cells). Suitable samples include isolated cell and tissue samples, for example, tissue sections including solid tissue or tumor tissue examinations. In some embodiments, samples can be obtained from formalin-fixed paraffin embedded (FFPE) tissue samples or other stored cell material samples.

[0031] The present disclosure also provides a kit for labeling the 5'-end of a nucleic acid, the kit comprising a 5'-end glycosidase and an aldehyde-reactive compound.

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

[0033] In at least one embodiment, the uracil DNA glycosidase in the kit of the present disclosure is derived from Micrococcaceae, Staphylococcaceae or Nucleococcaceae, which includes Micrococcus, Oral Coccus, Staphylococcus or Motile Coccus. In some embodiments, the uracil DNA glycosidase is derived from Micrococcus luteus.

[0034] In at least one embodiment, the aldehyde-reactive compound in the kit of the present disclosure is a compound having at least one primary amine, a hydrazide, an acylhydrazide, a compound having an aminooxy (-ONH2) group, and a compound having an aminooxy group containing naphthalene and / or guanidine. In some embodiments, the aldehyde-reactive compound is hydroxylamine biotin, a fluorescent dye hydroxylamine such as Alexa Fluor 488 hydroxylamine, an aldehyde reactive probe (ARP), an aminooxy fluorescent dye such as aminooxy-5(6)-FAM, aminooxy-5(6)-ROX, and aminooxy-5(6)-TAMRA, cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy biotin, an aminooxy fluorescent dye containing naphthalene, an aminooxy fluorescent dye containing guanidine, an aminooxy-FAM containing naphthalene and / or guanidine, Cy5-PEG-aminooxy, or a fluorescent dye hydrazide such as cyanine dye hydrazide or fluorescent CF dye hydrazide.

[0035] In at least one embodiment, the kit of the present disclosure further comprises a 5' to 3' exonuclease for removing unlabeled nucleic acids. In some embodiments, the 5' to 3' exonuclease is selected from T5 exonuclease (T5 exo), T7 exonuclease (T7 exo), bacteriophage lambda exonuclease, 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 labeling the 5'-end of nucleic acids, the system comprising a reaction reservoir, a reaction chamber or a container, 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 glycosidase and an aldehyde-reactive compound to the nucleic acid in the reaction reservoir, the reaction chamber or the container, and continue for a period of time at a set temperature controlled by the temperature control unit.

[0037] The present disclosure further provides a kit for labeling the 5'-end of nucleic acids, the kit comprising a 5'-end glycosidase and an aldehyde-reactive compound. In some embodiments, the kit further comprises a 5' to 3' exonuclease for removing unlabeled nucleic acids, for example, T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, bacteriophage lambda exonuclease, 5'-exonuclease of DNA polymerase I (ExoVI), exonuclease VIII (Exo VIII), 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'-end glycosidase is selected from uracil DNA glycosidase (UDG or UNG), alkyladenine DNA glycosidase (AAG), single-stranded selective monofunctional uracil DNA glycosidase 1 (SMUG1), methyl binding domain glycosidase 4 (MBD4), thymine DNA glycosidase (TDG), MutY homologous DNA glycosidase (MYH), alkylpurine glycosidase C (AlkC), alkylpurine glycosidase D (AlkD), a baseless cleavage enzyme activity The invention relates to a group consisting of 8-oxoguanine glycosidase 1 (OGG1), endonuclease III glycosidase 1 without abasic cleavage activity (NTHL1), endonuclease VIII glycosidase 1 without abasic cleavage activity (NEIL1), endonuclease VIII glycosidase 2 without abasic cleavage activity (NEIL2), endonuclease VIII glycosidase 3 without abasic cleavage activity (NEIL3), enzyme-active fragments thereof, and any combination thereof. In at least one embodiment, the uracil DNA glycosidase is derived from the family Micrococcaceae, Staphylococcaceae, or Nucleococcaceae.

[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 labeling the 5'-end and 3'-end of a nucleic acid, comprising providing a target nucleic acid to be labeled; adding a 5'-end glycosidase and a nucleotide having a reactive portion 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 the nucleotide having the reactive portion into the 3'-end of the intermediate nucleic acid; providing an aldehyde-reactive compound carrying a detectable label to couple the abasic site of the intermediate nucleic acid at the 5'-end, and a desired molecule having a corresponding functional portion capable of reacting with the reactive portion; and exposing the intermediate nucleic acid to the aldehyde-reactive compound carrying a detectable label and the desired molecule having a corresponding functional portion, thereby forming a labeled nucleic acid having a detectable label, wherein the detectable label is coupled to the intermediate nucleic acid at the abasic site to form a labeled nucleic acid with a detectable label attached to the 5'-end, and a connection is formed between the reactive portion and the corresponding functional portion.

[0041] The present disclosure also provides a kit for labeling the 5'-end and 3'-end of a nucleic acid, the kit comprising a 5'-end glycosidase, an aldehyde-reactive compound, a nucleotide having a reactive portion, a polymerase that incorporates the nucleotide having the reactive portion into the 3'-end of the nucleic acid, and a desired molecule having a corresponding functional portion that can react with the reactive portion.

Brief description of the attached figure

[0042] The present disclosure will be readily appreciated and better understood through the following detailed description when taken in conjunction with the accompanying drawings.

[0043] Figure 1 Schematic diagram depicting exemplary methods for introducing 3'-modifications to nucleic acids or polynucleotides and enriching for labeled nucleic acids or polynucleotides.

[0044] Figure 2A and Figure 2BShown is an example of labeling the 3'-end of a polynucleotide with a Cy5 fluorescent dye / fluorophore by enzymatic synthesis of 3'-O-azidomethyldeoxynucleotides (3'-AZ-dNTPs) to the 3'-end of the polynucleotide, followed by an azide and alkyne click conjugation reaction between the incorporated 3'-O-azidomethyldeoxynucleoside monophosphate (3'-AZ-dNMP) and the alkyne-modified Cy5 fluorescent dye moiety. Figure 2A and Figure 2B are images from the same gel. Figure 2A Depicts the results of gel electrophoresis of unlabeled and labeled polynucleotides visualized by staining with SYBR Gold dye, and Figure 2B The electrophoretic positions of nucleic acids labeled with Cy5 fluorescent dye after the enrichment step are shown. Lane 1: electrophoretic position of target polynucleotide (single-stranded DNA of 45 nucleotides); Lane 2: electrophoretic position of target polynucleotide with 3'-AZ-dNMP incorporated at the 3'-end; Lane 3: electrophoretic position of polynucleotide with 3'AZ-dNMP incorporated at the 3'-end and coupled with Cy5 fluorescent dye after the enrichment step.

[0045] Figure 3 An example of labeling a fluorescent quencher (BHQ1) at the 3'-end of a polynucleotide is shown by enzymatically synthesizing 3'-O-azidomethyldeoxynucleotide (3'-AZ-dNTP) to the 3'-end of the polynucleotide, followed by an azide and alkyne click conjugation reaction between the incorporated 3'-AZ-dNMP and the alkyne-modified fluorescent quencher moiety. Lane 1: electrophoretic position of a polynucleotide (single-stranded DNA of 45 nucleotides); Lane 2: electrophoretic position of a polynucleotide with 3'-AZ-dNMP incorporated at the 3'-end; Lane 3: electrophoretic position of a polynucleotide with 3'-AZ-dNMP incorporated at the 3'-end and coupled or not coupled to a fluorescent quencher; Lane 4: electrophoretic position of a polynucleotide with 3'-AZ-dNMP incorporated at the 3'-end and coupled to a fluorescent quencher after an enrichment step.

[0046] Figure 4A and Figure 4B An example of labeling the 3'-end of a polynucleotide with a Cy5 fluorescent dye / fluorophore is shown by enzymatic synthesis of 3'-O-azidomethyldeoxyribonucleotides (3'-AZ-dNTPs) to the 3'-end of the polynucleotide, followed by an azide and DBCO conjugation reaction between the incorporated 3'-AZ-dNTP and the DBCO-modified Cy5 fluorescent dye moiety. Figure 4A and Figure 4B are images from the same gel. Figure 4A Depicts the results of gel electrophoresis of unlabeled and labeled polynucleotides visualized by staining with SYBR Gold dye, and Figure 4BThe electrophoretic positions of nucleic acids labeled with Cy5 fluorescent dye at the 3'-end are shown. Lane 1: electrophoretic position of target polynucleotide (single-stranded DNA of 45 nucleotides); Lane 2: electrophoretic position of polynucleotides with 3'-AZ-dNMP conjugated to Cy5 fluorescent dye incorporated at the 3'-end.

[0047] Figure 5 An example of labeling a fluorescent quencher (BHQ1) at the 3'-end of a target polynucleotide is shown by enzymatically synthesizing 3'-O-azidomethyldeoxynucleotide (3'-AZ-dNTP) to the 3'-end of the target polynucleotide, followed by an azide and DBCO conjugation reaction between the incorporated 3'-AZ-dNMP and the DBCO-modified fluorescent quencher moiety. In this figure, lane 1 shows the electrophoretic position of a polynucleotide (single-stranded DNA of 45 nucleotides); lane 2 shows the electrophoretic position of a target polynucleotide with 3'-AZ-dNMP incorporated at the 3'-end; lane 3 shows the electrophoretic position of a target polynucleotide with 3'-AZ-dNMP incorporated at the 3'-end and coupled or not coupled to a fluorescent quencher; lane 4 shows the electrophoretic position of a target polynucleotide with 3'-AZ-dNMP incorporated at the 3'-end and coupled to a fluorescent quencher after an enrichment step.

[0048] Figure 6 The electrophoresis results of an example of labeling the 3'-end of a polynucleotide having a partially double-stranded region formed by a 60-nucleotide chain and a 20-nucleotide chain. Figure 6 In the figure, lane S is the electrophoretic position of a portion of double-stranded polynucleotides before the labeling reaction. Lane 1 shows the formation result of the 61-nucleotide forward strand with an azide group at the 3'-end; lane 2 shows the result after the labeling reaction; and lane 3 shows the result after the clearing reaction.

[0049] Figure 7The electrophoresis results are shown for polynucleotides that were enzymatically synthesized with different 3'-O-azidomethyldeoxynucleotides and labeled with Cy5 fluorescent dye / fluorophore at the 3'-end. Lane S1: electrophoretic position of the polynucleotide before incorporation of Cy5-labeled 3'-AZ-dATP; Lane 1: electrophoretic position of the polynucleotide incorporating Cy5-labeled 3'-AZ-dATP after the labeling reaction; Lane 1-1: electrophoretic position of the polynucleotide labeled with Cy5 after the clearing reaction; Lane S2: electrophoretic position of the polynucleotide before incorporation of Cy5-labeled 3'-AZ-dGTP; Lane 2: electrophoretic position of the polynucleotide incorporating Cy5-labeled 3'-AZ-dGTP after the labeling reaction; Lane 2-1: electrophoretic position of the polynucleotide labeled with Cy5 after the clearing reaction; Lane S3: electrophoretic position of the polynucleotide before incorporation of IF700-labeled 3'-AZ-dCTP; Lane 3: electrophoretic position of the polynucleotide incorporating IF700-labeled 3'-AZ-dCTP after the labeling reaction; Lane 3-1: electrophoretic position of the polynucleotide labeled with IF700 after the clearing reaction.

[0050] Figure 8 The results of urea PAGE using uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus and aldehyde reactive probe (ARP) to label the 5'-ends of single-stranded DNA (ssDNA) and double-stranded DNA. "S" indicates a lane containing only unlabeled DNA.

[0051] Fig. 9 The results of urea PAGE using uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus and aminooxy-5(6)-FAM to label the 5'-ends of single-stranded and double-stranded DNA. "S" indicates a lane containing only unlabeled DNA.

[0052] Fig.10 The results of urea PAGE of 5'-phosphorylated single-stranded DNA and 5'-end of double-stranded DNA labeled with uracil DNA glycosidase derived from Micrococcus luteus (MluUDG) and naphthalene- and guanidine-containing aminooxy-FAM. "S" indicates the lane containing only unlabeled DNA.

[0053] Fig.11 Urea PAGE results showing labeling of the 5'-end of single-stranded DNA using uracil DNA glycosidase from Micrococcus luteus (MluUDG) and aldehyde reactive probe (ARP), followed by a cleanup step using bacteriophage lambda exonuclease (λexo) to eliminate unlabeled DNA. "S" indicates a lane containing only unlabeled DNA.

[0054] Fig.12Urea PAGE results showing labeling of the 5'-ends of single-stranded and double-stranded DNA using uracil DNA glycosidase derived from Micrococcus luteus (MluUDG) and naphthalene- and guanidine-containing aminooxy-FAM, followed by a cleanup step using bacteriophage lambda exonuclease (λexo) to eliminate unlabeled DNA. "S" indicates a lane containing only unlabeled DNA.

[0055] Fig.13 The electrophoresis results of 5'-end labeling, 3'-end labeling and dual labeling of double-stranded DNA are shown. Lane 1 shows the electrophoresis position of ssDNA containing 21 nucleotides of uracil; Lane 2 shows the electrophoresis position of the 5'-end FAM-labeled product; Lane 3 shows the electrophoresis position of the 3'-end labeled product; Lane 4 shows the electrophoresis position of the dual-labeled product.

[0056] Fig.14 The electrophoresis results of 3'-end labeling and dual labeling of double-stranded DNA are shown. Lane 1 and lane 2 show the electrophoretic positions of ssDNA containing 26 nucleotides of uracil and the complementary strand of 27 nucleotides, respectively; lane 3 shows the electrophoretic position of the 3'-end labeled product; lane 4 shows the electrophoretic position of the 3'-end labeled product after exonuclease degradation to remove unlabeled DNA; lane 5 shows the electrophoretic position of the dual-labeled product; lane 6 shows the electrophoretic position of the dual-labeled product after exonuclease degradation to remove unlabeled DNA.

[0057] Fig.15 The electrophoresis results of double-stranded DNA labeled sequentially or simultaneously are shown. Lane 1 and lane 2 show the electrophoretic positions of ssDNA containing 25 nucleotides of uracil and the complementary strand of 26 nucleotides, respectively; Lane 3 shows the electrophoretic positions of the products of double-stranded DNA labeled sequentially after exonuclease degradation; Lane 4 shows the electrophoretic positions of the products of double-stranded DNA labeled simultaneously after exonuclease degradation; Lane 5 shows the electrophoretic positions of the products of double-stranded DNA having azide and DBCO linked at the 3'-end after exonuclease degradation; Lane 6 shows the electrophoretic positions of the products of double-stranded DNA having azide and DBCO linked at the 3'-end after exonuclease degradation. [Implementation Method]

[0058] All terms used herein including descriptive or technical terms should be interpreted as having obvious meanings to those with ordinary knowledge in the art. However, these terms may have different meanings according to the intentions, precedents or new technologies of those with ordinary knowledge in the art. In addition, some terms can be selected arbitrarily by the applicant, in which case the meaning of the selected terms will be described in detail in the narration of this disclosure. Therefore, the terms used herein are defined based on the meaning of the terms and the description of the entire specification.

[0059] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques within the scope of molecular biology, microbiology, cell biology, biochemistry, and immunology, within the skill of one of ordinary skill in the art. Such techniques are fully explained in the literature, for example, "Molecular Cloning: A Laboratory Manual" 2nd Edition (Sambrook, et al., 1989), Cold Spring Harbor Press; "Oligonucleotide Synthesis" (MJ Gait, 1984); "Methods in Molecular Biology", Humana Press; "Cell Biology: A Laboratory Notebook" (JE Cellis, ed., 1998), Academic Press; "Animal Cell Culture" (RI Freshney, ed., 1987); "Handbook of Experimental Immunology" (Weir, 1996); "Introduction to Cell and Tissue Culture" (JP Thayer and PE Roberts, 1998); "Celland Tissue Culture: Laboratory Procedures" (A. Doyle, JB Griffiths and DG Newell, eds., 1993-8); "Methods in Enzymology" (Academic Press, 1993-1999); "Cell Biology: A Laboratory Notebook" (JE Cellis, ed., 1998), Academic Press; "Animal Cell Culture" (RI Freshney, ed., 1987); "Handbook of Experimental Immunology" (Weir, 1996); "Introduction to Cell and Tissue Culture" (JP Thayer and PE Roberts, 1998); "Cell and Tissue Culture: Laboratory Procedures" (A. Doyle, JB Griffiths and DG Newell, eds., 1993-1999); "Methods in Enzymology" (Academic Press, 1993-1999); "Cell Biology: A Laboratory Notebook" (JE Cellis, ed., 1998), Academic Press; "Cell Biology: A Laboratory Notebook" (JE Cellis, ed., 1998), Academic Press; "Cell Biology: A Laboratory Notebook" (JE Press, Inc.); "Handbook of Experimental Immunology" (DMWeir and CC Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (JMMiller and MPCalos, eds., 1987); "Current Protocols in Molecular Biology" (FMAusubel, et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis, et al., 1987); al., eds., 1994); "Current Protocols in Immunology" (JEColigan etal., eds., 1991); "Short Protocols in Molecular Biology" (Wiley & Sons, 1999); "Immunobiology" (C.A. Janeway and P. Travers, 1997); "Antibodies" (P. Finch, 1997); "Antibodies: a practical approach" (D. Catty., ed., IRL Press, 1988 - 1989); "Monoclonal antibodies: a practical approach" (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); "Using antibodies: a laboratory manual" (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); and "The Antibodies" (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995). The specific use techniques for specific specific embodiments will be described in the following paragraphs. Without further elaboration, it is believed that those of ordinary skill in the art can utilize this disclosure to the greatest extent based on the above description. Therefore, the following specific embodiments are for illustrative explanation only and do not limit the rest of this disclosure in any way. All publications cited herein are incorporated herein by reference for the purposes and subject matter cited.

[0060] As used in this disclosure, unless otherwise specified herein, the singular forms "a" or "an" and "the" include plural forms. The terms "includes" or "including", "has", "comprises" or "comprising", and "contains" used in the detailed description and / or claims of this disclosure are intended to include, without excluding other terms, such as other components, materials, steps, etc. The terms "sec", "min", and "hr" used herein are abbreviations for "second", "minute", and "hour", respectively.

[0061] When providing a range, the endpoints are included. In addition, unless otherwise indicated or obvious from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges may be used in different embodiments of the present disclosure for any specific value or sub-range within that range, unless the context clearly indicates otherwise.

[0062] As used herein, the terms "about", "approximately" and "approximately" generally mean within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the terms "about", "approximately" and "approximately" mean within the standard error of the mean value acceptable to those with ordinary knowledge in the art. Unless otherwise indicated, all numerical ranges, amounts, values ​​and percentages disclosed herein, such as the amount of material, the duration of a time period, temperature, operating conditions, the ratio of amounts, etc., should be understood as modified by the terms "about", "approximately" or "approximately" in all cases.

[0063] As used herein, the term "derived" when referring to a biological sample, refers to a sample obtained from the source at a certain point in time. For example, a biological sample derived from an organism represents a primary biological sample obtained directly from the organism (i.e., unmodified), or may be modified, such as by introduction of a recombinant vector, by culturing under specific conditions, or immortalization.

[0064] As used herein, the term "at least one" in relation to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the presence of elements other than the elements referred to in the list of elements referred to by the term "at least one", whether related or unrelated to the referred elements. Thus, as a 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") in one embodiment may refer to at least one, optionally including more than one, A, and B is absent (and optionally including elements other than B); in another embodiment may refer to at least one, optionally including more than one, B, and A is absent (and optionally including elements other than A); in yet another embodiment may refer to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements).

[0065] As used herein, an abasic position is also referred to as an apurinic / apyrimidinic (AP) position, which encompasses any chemical structure after the base portion (including the entire base) is removed by an agent capable of cleaving the base portion of a nucleotide, for example, by treating a nucleotide (present in a polynucleotide chain) with an agent capable of cleaving the base portion of a nucleotide (e.g., an enzyme, acidic conditions, or a chemical agent). In some embodiments, an AP position is a position in a nucleic acid backbone such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that lacks a nucleobase, that is, the deoxyribose of the DNA backbone or the ribose of the RNA backbone is not covalently linked to a purine base such as adenine (A) or guanine (G) or a pyrimidine base such as cytosine (C), uracil (U), or thymine (T). The AP position can be located within the nucleotide sequence of a nucleic acid, at both the 5'-end and the 3'-end of a nucleic acid, or at one end such as the 5'-end or the 3'-end of a nucleic acid.

[0066] As used herein, the terms "nucleic acid", "nucleic acid sequence" and "nucleic acid fragment" refer to a nucleotide sequence in single-stranded or double-stranded form, which is derived from the present invention without limitation, and generally includes naturally occurring nucleotides or artificial chemical mimetics. As used herein, the term "nucleotide" refers to a monomer unit of a nucleic acid or polynucleotide described herein, which has a glycoside with or without a nucleobase, and one or more internucleotide bonds, such as a phosphodiester bond. In some embodiments, the nucleobase includes naturally occurring bases such as adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U); non-naturally occurring bases such as xanthine, hypoxanthine, isoguanine and isocytosine; and any analogs or derivatives thereof. In some embodiments, nucleosides with abasic positions (nucleobase loss) are also included within the scope of the present disclosure. In some embodiments, sugars in glycosides include naturally occurring sugars such as pentoses (such as deoxyribose and ribose), non-naturally occurring sugars and their analogs. In some embodiments, the nucleotides are linked via internucleotide bonds, such as, but not limited to, phosphate, boranophosphate, phosphorothioate, phosphodiester, phosphotriester, H-phosphate, phosphamidate, methylphosphonate, phosphoacetate, thiophosphoacetate, or other variants of the phosphate backbone of natural nucleic acids. As used herein, the term "nucleotide" also encompasses structural analogs that replace natural or non-natural nucleotides, such as modified nucleotides. For example, the term "xenonucleotide" refers to a nucleotide that has been modified to have a sugar moiety that is different from that contained in natural DNA or RNA. Exemplary nucleic acids with xenonucleotide labels, i.e., xenogeneic nucleic acids (XNA), include, but are not limited to, peptide nucleic acids (PNA), locked nucleic acids (LNA), 1,5-anhydrohexitol nucleic acids (HNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), cyclohexene nucleic acids (CeNA), and fluoroarabinose nucleic acids (FANA).

[0067] As used herein, the term "polynucleotide" refers to a polymer of nucleotides and generally refers to any type of nucleic acid, for example, natural or non-natural DNA or RNA, and modified nucleic acids, such as xenogeneic nucleic acids (XNA) described herein. Polynucleotides may also include any combination of glycosides with or without nucleobases and internucleotide linkages. Unless otherwise indicated, the polynucleotides characterized herein have an intrinsic directionality between the 5'-end of one nucleotide and the 3'-end of its adjacent nucleotide, and the template-independent synthesis of the polynucleotides provided herein is carried out in a direction from the 5'-end to the 3'-end.

[0068] The term "polynucleotide" as used herein is not intended to distinguish between the lengths of the nucleotide units, and the term refers only to the polymeric molecular structure. In other words, the polynucleotides used herein are interchangeable with the term "oligonucleotides" and may range in size from a few monomeric nucleotide units to thousands of monomeric nucleotide units, for example, 2 to 5 nucleotides, 5 to 20 nucleotides, 20 to 100 nucleotides, 100 to 1,000 nucleotides or longer. A polynucleotide may be composed entirely of naturally or non-naturally occurring, modified or unmodified deoxyribonucleotides, or entirely of naturally or non-naturally occurring, modified or unmodified ribonucleotides, or chimeric mixtures 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. In addition, a polynucleotide may contain one or more abasic sites (apurinic / apyrimidinic sites), also known as AP sites.

[0069] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. These terms encompass nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, and / or have similar chemical properties to reference nucleic acids, and / or are metabolized in vivo in a manner similar to reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequences explicitly indicated. In some embodiments, nucleotides are linked via internucleotide bonds, such as, but not limited to, phosphates, boranophosphates, phosphorothioates, phosphodiesters, phosphotriesters, H-phosphates, aminophosphoesters, methylphosphonates, phosphoacetates, thiophosphoacetates, or other variants of the phosphate backbone of natural nucleic acids. The term "nucleotide" as used herein also encompasses structural analogs, such as modified nucleotides, that replace natural or non-natural nucleotides. For example, the term "heterologous nucleotide" refers to a nucleotide that has been modified to have a sugar moiety different from that contained in natural DNA or RNA. Exemplary nucleic acids having heterologous nucleotides, i.e., heterologous nucleic acids (XNA), include but are not limited to peptide nucleic acids (PNA), locked nucleic acids (LNA), 1,5-anhydrohexitol nucleic acids (HNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), cyclohexene nucleic acids (CeNA), and fluoroarabinose nucleic acids (FANA).

[0070] As used herein, nucleic acids may be 5 bases to 10,000 bases in length, e.g., 10 to 3,000 bases in length, 10 to 1,000 bases in length, or 10 to 100 bases in length. Nucleic acids isolated from biological sources may be greater than 1,000 bases in length and may be fragmented, e.g., by ultrasonic treatment, for use as described herein.

[0071] 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 can 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 some embodiments, the nucleic acid can 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 should be understood that the length of a nucleic acid can represent the average size in a population.

[0072] As used herein, glycosidases are enzymes capable of removing the base portion of a nucleotide and generating an AP site in a nucleic acid, including N-glycosidases, and also referred to as "DNA glycosidases" or "glycosidases", including but not limited to uracil-N-glycosidase (UNG) that specifically cleaves dUTP, which is interchangeably referred to as "uracil DNA glycosidase" (UDG); hypoxanthine-N-glycosidase; hydroxymethylcytosine-N-glycosidase; 3-methyladenine DNA glycosidase; 3- or 7-methylguanine DNA glycosidase; hydroxymethyluracil DNA glycosidase; and T4 endonuclease V. Glycosidases cleave the base portion of a nucleotide in the middle, at either end, or at both ends of a nucleic acid. As used herein, 5'-terminal glycosidases remove the base portion of a nucleotide at the 5'-end of a nucleic acid.

[0073] The term "initiator" as used herein refers to a nucleoside monomer, nucleotide monomer, oligonucleotide, polynucleotide or a modified analog thereof that is synthesized de novo by a nucleic acid polymerase. The term "initiator" may also refer to an XNA having a 3'-hydroxyl group, such as a 3'-hydroxy PNA.

[0074] According to the present disclosure, the initiator can also be linked or immobilized to a solid support, and the linking nucleotide is the 3'-terminal nucleotide coupled to the initiator and the 5'-terminal nucleotide of the synthesized nucleic acid. The initiator can be directly attached to the solid support, attached to the support via a linker, or immobilized via physical interactions such as adsorption, electrostatic interactions, and hydrogen bonding. Examples of solid supports include, but are not limited to, microarrays, beads (coated or uncoated), columns, optical fibers, tissues, nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, magnetic particles, plastics (e.g., polyethylene, polypropylene, and polystyrene), gel-forming materials (e.g., proteins (such as gelatin), lipopolysaccharides, silicates, agarose, polyacrylamide, or methyl methacrylate polymers), sol-gels, porous polymers, hydrogels, nanostructured surfaces, nanotubes (e.g., carbon nanotubes), and nanoparticles (e.g., gold nanoparticles or quantum dots).

[0075] The term "polymerase" as used herein refers to an enzyme / protein capable of synthesizing nucleic acids, which is generally a DNA polymerase, an RNA polymerase, or a functionally equivalent enzyme, including naturally occurring enzymes, modified enzymes, enzyme subunits, and derivatives thereof. For example, for the desired purpose, these enzymes may be subjected to amino acid sequence modification (e.g., mutation and functional group substitution), for example, removal of 5' to 3' exonuclease activity and enhancement of polymerase activity to obtain enzymes with improved properties, for example, thermal stability / thermostability and catalytic efficiency.

[0076] According to the present disclosure, the term "polymerase" may be a template-dependent polymerase or a template-independent polymerase. The polymerase may include A family DNA polymerases (e.g., T7 DNA polymerase, Pol I, Pol γ, θ, and ν), B family DNA polymerases (e.g., Pol II, Pol B, Pol ζ, Pol α, δ, and ε), C family DNA polymerases (e.g., Pol III), D family DNA polymerases (e.g., Pol D), X family DNA polymerases (e.g., Pol β, Pol σ, Pol λ, Pol μ, and terminal deoxynucleotidyl transferase), Y family DNA polymerases (e.g., Pol ι, Pol κ, Pol η, Din B, Pol IV, and Pol V), reverse transcriptases (e.g., telomerase and hepatitis B virus), and enzyme-active fragments thereof.

[0077] Non-limiting examples of widely used template-dependent polymerases include T7 DNA polymerase of T7 bacteriophage and T3 DNA polymerase of T3 phage, which are DNA-dependent DNA polymerases; T7 RNA polymerase of T7 phage and T3 RNA polymerase of T3 phage, which are DNA-dependent RNA polymerases; DNA polymerase I or a fragment thereof called Klenow fragment of Escherichia coli, which is a DNA-dependent DNA polymerase; Thermophilus aquaticus DNA polymerase, Tth DNA polymerase and Vent DNA polymerase, which are all thermostable 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, for example, modified RNA (ribozymes; Unrau & Bartel, 1998) and DNA having template-dependent polymerase activity.

[0078] Non-limiting examples of template-independent polymerases include reverse transcriptase, polyadenylate (poly A) polymerase, DNA polymerase θ, terminal deoxynucleotidyl transferase (TdT), and DNA polymerase μ. Since polymerases suitable for nucleic acid synthesis, nucleotide addition / incorporation, and nucleic acid synthesis processes are common knowledge and conventional techniques of those with ordinary knowledge in the art, further details thereof are omitted herein for the sake of brevity. In addition, the B family DNA polymerases previously provided by the inventors are also suitable for use under template-independent conditions, and U.S. Patent No. 11,591,629B2 is incorporated herein by reference in its entirety.

[0079] The term "modification" as used herein refers to a change in the chemical structure of a reactant molecule. When a nucleic acid is used as a reactant molecule, regardless of the source of the nucleic acid, the means of modification include, but are not limited to, introducing additional chemical groups / parts into the nucleic acid, removing or replacing the original chemical groups / parts from the nucleic acid, or a combination thereof. Alternatively, modifications can be introduced into a specific nucleic acid sequence during de novo synthesis of the nucleic acid, resulting in direct modification or multiple modifications to the nucleic acid. For example, fluorophore-labeled nucleotide analogs can be incorporated into nucleic acids together with their natural counterparts to become "fluorescently labeled" nucleic acids. Similarly, one or more site-specific modifications can also be enzymatically inserted into nucleic acids by incorporating nucleotides carrying the desired modifications. For example, a nucleoside triphosphate with a 3'-O-azidomethyl group can be enzymatically introduced into the 3'-end of the nucleic acid, thereby directly adding the azidomethyl modification to the 3'-end of the nucleic acid. Such modifications add nucleotides to the target nucleic acid together with site-specific chemical groups.

[0080] As used herein, the term "detecting" or "detection" refers to both a specified amount and a qualitative determination, so the term "detecting" or "detection" can be used interchangeably with "determination", "measurement" and similar terms herein. When a specified amount determination is intended, the term "determine the amount" or its similar terms are used. When a qualitative or quantitative determination is intended, the term "determine the level" or "detect the level" can be used.

[0081] As used herein, the term "exonuclease" refers to any wild-type or variant enzyme capable of cleaving the phosphodiester bond connecting the terminal nucleotides of an oligonucleotide or polynucleotide, for example, a 5' to 3' exonuclease, a 3' to 5' exonuclease, and a polyadenylic acid (poly A)-specific 3' to 5' exonuclease. Non-limiting examples of exonucleases include exonucleases I, II, III, IV, V, VI, VII, VIII, Xm1, and Rat1.

[0082] As used herein, the term "5' to 3' exonuclease" refers to an exonuclease that breaks the phosphodiester bond at the 5' end of an oligonucleotide or polynucleotide. Non-limiting examples of 5' to 3' exonucleases include T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, bacteriophage lambda exonuclease, 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.

[0083] As used herein, the term "3'-end" generally refers to a region or position in a polynucleotide or oligonucleotide that is downstream from the 5'-region or position in the same polynucleotide or oligonucleotide.

[0084] As used herein, the term "5'-end" generally refers to a region or position in a polynucleotide or oligonucleotide that is upstream of the 3'-region or position in the same polynucleotide or oligonucleotide.

[0085] As used herein, an aldehyde-reactive compound is a class of compounds that react or form a bond with an aldehyde group. In some embodiments, the aldehyde-reactive compound is a compound having at least one primary amine, a hydrazide, an acylhydrazide, a compound having an aminooxy (-ONH2) group, a compound having a naphthalene-containing aminooxy group, and / or a compound having a guanidine-containing aminooxy group.

[0086] As used herein, the term "label" (interchangeably referred to as "detectable label") refers to a chemical group or functional moiety that is bound or linked to a polynucleotide (interchangeably referred to as "labeling"). The labeled polynucleotide is generally detectable directly or indirectly by a detectable signal. Detectable labels can be attached (or bound) directly or through a non-interfering linking group to other moieties that are capable of specifically binding to one or more sites to be labeled. Detectable labels can be covalently or non-covalently bound and directly or indirectly bound.

[0087] As used herein, the term "monofunctional DNA glycosidase" refers to a naturally occurring monofunctional glycosidase that essentially contains only DNA glycosidase activity. The term "monofunctional DNA glycosidase" may also refer to a monofunctional glycosidase derived from a bifunctional DNA glycosidase that naturally has DNA glycosidase and abasic lyase (AP lyase) activity by eliminating or inactivating the AP lyase domain of a bifunctional DNA glycosidase.

[0088] As used herein, the term "enzymatically active fragment" refers to a fragment of a catalytic or enzymatically active protein or polypeptide that contains at least 10%, e.g., 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.

[0089] Signal detection methods are known in the art. Signal detection can be visual or utilize a suitable instrument suitable for the label used, for example, a spectrometer, a fluorimeter, a photometer, a phosphorimager, a Geiger counter, a scintillation counter or a microscope. For example, when the label is a radioisotope, detection can be achieved by using a photosensitive film such as a scintillation counter or autoradiography. When a fluorescent label is used, detection can be achieved by using light of an appropriate wavelength to excite the fluorophore and detecting the emitted fluorescence, for example, by a fluorescent microscope, visual inspection, a photosensitive photograph, a fluorimeter, a photometer, a charge coupled device (CCD) camera and a scanner. When an enzyme label is used, detection can be achieved by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. For example, many substrates of horseradish peroxidase (e.g., o-phenylenediamine) produce colored products. Instruments suitable for high-sensitivity detection are known in the art. Otherwise, a signal amplification strategy can be selected to facilitate the detection of low-abundance molecular targets.

[0090] Although the present disclosure is described by specific embodiments and optional features, it should be understood that those skilled in the art may make modifications and variations to the concepts disclosed herein, and such modifications and variations still fall within the scope of the present disclosure. Example

[0092] Some specific embodiments of the present disclosure are further disclosed in the following examples, but should not be construed as limiting the scope of the present disclosure. The materials and methods used in the present disclosure are described in detail below, and those used in the present disclosure but not described herein are commercially available materials.

[0093] Provided herein are examples of modifying nucleic acids using the methods of the present disclosure, and demonstrate that several exemplary modifications of polynucleotides result in labeled nucleic acids having a fluorophore or quencher at their 3'-end.

[0094] As shown in Figure 1, the methods used for such modifications can actually be divided into three consecutive steps: (1) using a polymerase to incorporate a modified nucleotide carrying a reactive portion into the 3'-end of the target polynucleotide; (2) joining a desired molecule having a labeling portion such as a fluorescent dye or a quencher and a corresponding functional portion to the 3'-end of the target polynucleotide; (3) degrading unlabeled or incompletely labeled polynucleotides by a 3' to 5' nuclease to enrich the desired labeled polynucleotides.

[0095] In some embodiments, the reactive portion is introduced into the target polynucleotide using an enzymatic synthesis method. For example, one or more nucleotide analogs are enzymatically added to the 3'-hydroxyl (3'-OH) end of a single-stranded nucleic acid initiator (the target polynucleotide) in a template-independent synthesis manner to produce a polynucleotide having the desired reactive portion. In at least one embodiment, the desired reactive portion is an azide (azide, N3) or an azido group, and a suitable reagent / compound containing an azido moiety such as a 3'-O-azidomethyl group (e.g., a nucleotide analog) can be used to introduce the modification to the 3'-end of the polynucleotide. Based on this situation, the following embodiments are further provided.

[0096] Example 1: Preparation of polynucleotides containing 3'-O-azidomethyl groups

[0097] A DNA polynucleotide containing 45 nucleotides labeled with fluorescein at the 5'-end (5'-FAM-45 nucleotides DNA) is used as a 3'-modified or labeled target polynucleotide. To introduce an azidomethyl group into the 3'-end of the 5'-FAM-45 nucleotides DNA, 3'-O-azidomethyldeoxynucleoside triphosphate (3'-AZ-dNTP) is used as a template-independent DNA synthesis substrate for a selective B family DNA polymerase or a variant thereof. An exemplary polymerase used herein may be an internal polymerase variant derived from Vent DNA polymerase, which can effectively incorporate 3'-AZ-dNTP into the 3'-end of the target polynucleotide. The nucleotide incorporation reaction is carried out in a reaction mixture (10 μL) containing 100 nM of the 5'-FAM-45 nucleotides DNA target polynucleotide, 0.25 mM of manganese chloride (MnCl2) and 200 nM of the polymerase. The reaction was initiated by adding 25 μM of 3'-O-azidomethyl-dTTP (3'-AZ-dTTP), followed by reaction at 60°C for a specified time. The time period used was 2 minutes to 30 minutes. The reaction was then terminated by adding 10 μL of a 2x quenching solution (95% deionized formamide and 25 mM EDTA). The reaction mixture was further denatured at 95°C for 10 minutes, and the reaction products were analyzed by 20% polyacrylamide gel electrophoresis (Urea-PAGE) containing 8 M urea. The gel was then imaged by an Amersham Typhoon laser scanner (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the reaction products. At the end of the polymerase-dependent incorporation of 3'-AZ-dTMP into 5'-FAM-45 nucleotide DNA, a 5'-FAM-46 nucleotide DNA carrying an azide group at the 3'-end was formed (respectively as Figure 2A , Figure 3 and Figure 5 The 5'-FAM-46 nucleotide DNA was further purified using a DNA QIAquick Nucleotide Removal Kit (Qiagen, Germantown, MD) prior to the subsequent labeling reaction. The preparation of a 3'-azide labeled target polynucleotide containing a 3'-O-azidomethyl group is shown in Scheme 1 below. Solution 1

[0098] Example 2: 3'-labeling of the 3'-end of a polynucleotide carrying an azidomethyl group by coupling reaction of an azide with an alkyne

[0099] As described in Example 1, after obtaining a polynucleotide containing an azidomethyl group at the 3'-end, the 3'-end azide (N3) group can be directly used to label any desired tag or functional molecule, such as a fluorescent dye or quencher, via an azide-alkyne coupling reaction.

[0100] For example, when it is desired to label the 3'-end of the target polynucleotide with a cyanine 5 (Cy5) fluorophore, a Cy5 alkyne molecule can be selected to perform an azide-alkyne coupling reaction with a polynucleotide carrying an azide (N3) group at the 3'-end. In at least one embodiment, a polynucleotide having a 3'-azide group can be directly reacted with a Cy5 alkyne in the presence of a tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand, copper ions, and sodium ascorbate to trigger 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 is typically performed at 37° C. for 1 hour. Unreacted Cy5 alkyne molecules are then removed using a DNA QIAquick Nucleotide Removal Kit (Qiagen, Germantown, MD, USA). The clearing reaction product is further treated with a 3' to 5' exonuclease (e.g., 200 nM 3' to 5' exonuclease) at 37°C for 1 hour. The final reaction product is analyzed using 20% ​​urea PAGE and the gel is imaged by an Amersham Typhoon laser scanner (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the final reaction product. The result of the azide and alkyne cycloaddition and enzymatic degradation reaction is a homogeneous target polynucleotide containing a Cy5 fluorophore at the 3'-end (see Figure 2A and Figure 2B Lane 3). The following scheme 2 shows the labeling of the 3'-end of the target polynucleotide with Cy5 fluorophore. Solution 2

[0101] Similarly, when the 3'-end of the target polynucleotide needs to be labeled with a fluorescence quencher such as Black Hole Quencher 1 (BHQ1), the BHQ1 alkyne molecule can be selected to perform an azide-alkyne coupling reaction with the target polynucleotide carrying an azide (N3) group at the 3'-end. For example, the polynucleotide with a 3'-azide group can be reacted with the BHQ1 alkyne in the presence of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand, copper ions and sodium ascorbate to trigger a Cu-catalyzed cycloaddition reaction between the 3'-azide group and the alkyne group of the BHQ1 alkyne molecule. The azide-alkyne coupling reaction is usually carried out at 37°C for 1 hour. The unreacted BHQ1 alkyne molecule is then removed using the DNA QIAquick Nucleotide Removal Kit (Qiagen, Germantown, MD, USA). The cleanup reaction product is further treated with a 3' to 5' exonuclease (e.g., 200 nM 3' to 5' exonuclease) at 37°C for 1 hour. The final reaction product is analyzed using 20% ​​urea PAGE and the gel is imaged by an Amersham Typhoon laser scanner (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the final reaction product. The sequential azide and alkyne cycloaddition and enzymatic degradation reactions produce a homogeneous target polynucleotide containing a BHQ1 tag at the 3'-end (see Figure 3 Lane 4). The following Scheme 3 shows the labeling of the 3'-end of the target polynucleotide with a fluorescent quencher. Solution 3

[0102] The components of the BHQ1 labeling reaction and the experimental groups are listed in Table 1 below. The labeling reaction results corresponding to each experimental group are shown in Figure 3 In Table 1, 4 experimental groups are designed and the reaction components are shown. The symbol "+" indicates that the specified reagent is added to the reaction of each experimental group, and the symbol "-" indicates that the specified reagent is not added to the reaction of the experimental group.

[0103] Table 1 5'-FAM-45 nucleotide DNA polynucleotide + + + + DNA polymerase - + + + 3'-AZ-dTTP - + + + BHQ1 Alkyne - - + + 3' to 5' exonuclease - - - +

[0104] Example 3: Labeling of polynucleotides carrying an azide group at the 3'-end by azide-DBCO ligation reaction

[0105] Alternatively, after obtaining a polynucleotide containing an azidomethyl group at the 3'-end, the terminal azide group can be used to label any desired tag or functional molecule, such as a fluorescent dye or a quencher, via azide-dibenzocyclooctyne (DBCO) ligation reaction.

[0106] For example, when it is desired to label the cyanine 5 (Cy5) fluorophore at the 3'-end of the target polynucleotide, the Cy5-DBCO molecule can be selected to carry out an azide-DBCO ligation reaction with a polynucleotide carrying an azide (N3) 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 1x TE buffer. The azide-DBCO ligation reaction is usually carried out at 37°C for 1 hour, followed by removal of unreacted Cy5-DBCO molecules using a DNA QIAquick Nucleotide Removal Kit (Qiagen, Germantown, MD, USA). The final reaction products were analyzed using 20% ​​urea PAGE, and the gel was imaged by an Amersham Typhoon laser scanner (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the final reaction products. The addition of azide to DBCO and enzymatic degradation reaction generates a target polynucleotide containing a Cy5 fluorophore at the 3'-end (see Figure 4A and Figure 4B The following scheme 4 shows the labeling of the 3'-end of the target polynucleotide with the Cy5 fluorophore. Solution 4

[0107] Similarly, when the 3'-end of the target polynucleotide needs to be labeled with a fluorescence quencher such as Black Hole Quencher 1 (BHQ1), the BHQ1-DBCO molecule can be selected to perform an azide-to-DBCO ligation reaction with a target polynucleotide carrying an azide (N3) group at the 3'-end. For example, a target polynucleotide having a 3'-azide group can be directly reacted with BHQ1-DBCO in 1x TE buffer. The azide-to-DBCO ligation reaction is usually carried out at 37°C for 1 hour, followed by removal of unreacted BHQ1 alkyne molecules using a DNA QIAquick Nucleotide Removal Kit (Qiagen, Germantown, MD, USA). The removal reaction product is further treated with a 3' to 5' exonuclease (e.g., 200 nM 3' to 5' exonuclease) at 37°C for 1 hour. The final reaction products were analyzed by 20% urea PAGE and visualized by imaging the gel with an Amersham Typhoon laser scanner (Cytiva Life Sciences, Marlborough, MA, USA). Sequential azide and DBCO ligation and enzymatic degradation reactions produced homogeneous target polynucleotides containing a BHQ1 tag at the 3'-end (see Figure 5 Lane 4). Scheme 5 below shows labeling of the 3'-end of a target polynucleotide with a fluorescent quencher. Solution 5

[0108] The components of the BHQ1 labeling reaction and the experimental groups are listed in Table 2 below. The labeling reaction results corresponding to each experimental group are shown in Figure 5 Lanes 1 to 4 of Table 2. In Table 2, 4 experimental groups are designed and the reaction components are shown. The symbol "+" indicates that the specified reagent is added to the reaction of each experimental group, and the symbol "-" indicates that the specified reagent is not added to the reaction of the experimental group.

[0109] Table 2 DNA polymerase - + + + 3'-AZ-dTTP - + + + BHQ1-DBCO - - + + 3' to 5' exonuclease - - - +

[0110] Example 4: 3'-labeling of polynucleotides in partially double-stranded regions

[0111] A polynucleotide having a partially double-stranded region can also be used as a 3'-modified or labeled target polynucleotide by adding 3'-AZ-dNTP to the 3'-end of the target polynucleotide. For example, a partially double-stranded polynucleotide consisting of a forward strand of 60 nucleotides and a reverse strand of 20 nucleotides is used as a target polynucleotide. To introduce an azidomethyl group into the 3'-end of the partially double-stranded DNA, 3'-O-azidomethyldeoxynucleoside triphosphate (3'-AZ-dNTP) is used as a template-independent DNA synthesis substrate for a selective B-family DNA polymerase or a variant thereof. The nucleotide incorporation reaction is carried out in a reaction mixture (10 μL) containing 100 nM of the partially double-stranded target polynucleotide, 0.25 mM of manganese chloride (MnCl2) and 200 nM of polymerase. The reaction is initiated by adding 25 μM of 3'-O-azidomethyl-dTTP (3'-AZ-dTTP) and reacted at 60°C for 10 minutes. The reaction was then terminated by adding 10 μL of a 2x quenching solution (95% deionized formamide and 25 mM EDTA). The reaction mixture was further denatured at 95°C for 10 minutes and then analyzed by 20% polyacrylamide gel electrophoresis (Urea-PAGE) containing 8 M urea. The gel was then imaged by an Amersham Typhoon laser scanner (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the reaction products. After the polymerase-dependent incorporation of 3'-AZ-dTMP into the partially double-stranded polynucleotide, a 61-nucleotide forward strand carrying an azide group at the 3'-end was formed (e.g. Figure 6 As shown in lane 1, where lane S shows the electrophoretic position of the partially double-stranded region polynucleotide before the 3'-terminal nucleotide is incorporated into the reaction), which can be used for subsequent labeling reactions.

[0112] In this embodiment, the partially double-stranded polynucleotide containing an azidomethyl group at the 3'-end obtained as above is subjected to a subsequent labeling reaction via the aforementioned azide and dibenzocyclooctyne (DBCO) ligation reaction.

[0113] The results of the labeling reaction of partially double-stranded polynucleotides are shown in Figure 6 , wherein lane S is the electrophoretic position of the partial double-stranded polynucleotide before the labeling reaction; lane 1 shows the result of the forward chain of 61 nucleotides carrying an azide group at the 3'-end; lane 2 shows the result after the labeling reaction; lane 3 shows the result after the cleaning reaction, and the labeled product is further treated with a 3' to 5' exonuclease (e.g., 200 nM 3' to 5' exonuclease) at 37° C. for 1 hour.

[0114] This example demonstrates that the 3'-labeling method disclosed herein is applicable to polynucleotides having partially double-stranded regions or DNA composed of strands of different lengths.

[0115] Example 5: 3'-labeling of polynucleotides with different 3'-O-azidomethyl deoxynucleotides

[0116] A DNA polynucleotide containing 38 nucleotides labeled with fluorescein at the 5'-end (5'-FAM-38 nucleotides DNA) was used as a 3'-modified or labeled target polynucleotide. To introduce an azidomethyl group to the 3'-end of the 5'-FAM-38 nucleotides DNA, 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 a selective B-family DNA polymerase. An exemplary polymerase used herein is Vent polymerase, which can efficiently incorporate various 3'-AZ-dNTPs into the 3'-end of the target polynucleotide. The nucleotide incorporation reaction was performed in a reaction mixture (10 μL) containing 100 nM 5'-FAM-38 nucleotides, 0.25 mM manganese chloride (MnCl2), and 200 nM polymerase. The reaction was initiated by adding 25 μM of Cy5-labeled 3'-AZ-dATP, Cy5-labeled 3'-AZ-dGTP, and IF700-labeled 3'-AZ-dCTP, respectively, and then reacted at 60°C for 20 minutes. After that, 10 μL of 2x quenching solution (95% deionized formamide and 25 mM EDTA) was added to each reaction mixture to terminate the reaction, and further denatured at 95°C for 10 minutes. The reaction products were analyzed by 20% polyacrylamide gel electrophoresis (Urea-PAGE) containing 8 M urea, and the gel was imaged on an Amersham Typhoon laser scanner (Cytiva LifeSciences, Marlborough, MA, USA) to visualize the reaction products. After the reaction in which Cy5-labeled 3'-AZ-dATP, Cy5-labeled 3'-AZ-dGTP, and IF700-labeled 3'-AZ-dCTP are incorporated into the 5'-FAM-38-nucleotide polynucleotide, a 5'-FAM-39-nucleotide polynucleotide having an azide group at the 3'-end is formed (respectively as Figure 7 1, 2 and 3).

[0117] In this example, the 5'-FAM-39 nucleotide polynucleotides containing different azidomethyl groups at the 3' end obtained above were subjected to subsequent labeling reaction by directly incorporating fluorescently labeled 3'-O-azidomethyl deoxynucleoside triphosphate.

[0118] The results of the 5'-FAM-39 nucleotide polynucleotide labeling reaction are shown in Figure 7 Lane S1, Lane S2, and Lane S3 respectively show the electrophoretic positions of 5'-FAM-38 nucleotides and 5'-FAM-39 nucleotides polynucleotides before the labeling reaction; Lane 1, Lane 2, and Lane 3 respectively show the electrophoretic positions of fluorescently labeled 5'-FAM-38 nucleotides and 5'-FAM-39 nucleotides polynucleotides after the labeling reaction; and Lane 1-1, Lane 2-1, and Lane 3-1 respectively show the results after the cleaning reaction, wherein the labeled product is further treated with a 3' to 5' exonuclease (e.g., 200 nM 3' to 5' exonuclease) at 37°C for 1 hour.

[0119] This example demonstrates that the 3'-labeling method of the present disclosure is applicable to introducing an azidomethyl group into the 3'-end of a target polynucleotide using different 3'-O-azidomethyl deoxynucleoside triphosphates.

[0120] Example 6: 5'-end labeling of nucleic acids using aldehyde-reactive compounds

[0121] A 45-nucleotide single-stranded DNA (ssDNA; 5'- / deoxyU / CTCGGCCTGGCACAGGTCCGTCTCAGTGCTGCGGCGACCACCGA-3') containing a uracil residue at the 5'-end and a fluorescein (FAM) dye at the 3'-end was synthesized. (SEQ ID NO: 1)). For uracil excision and subsequent abasic labeling, 100 nM ssDNA containing 45 nucleotides of uracil was mixed with 100 ng of uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus and 5 mM of aldehyde reactive probe (N-(aminooxyacetyl)-N'-biotinylhydrazine). MluUDG and aldehyde reactive probe were added at 37°C for 15 minutes to initiate the reaction. An equal volume of 2-fold quenching solution (30 mM EDTA and 95% (v / v) deionized formamide) was added, followed by denaturation at 95°C for 10 minutes to terminate the reaction. The reaction products were analyzed by denaturing 20% ​​polyacrylamide gel electrophoresis (Urea-PAGE) containing 8 M urea, and the gel was scanned with an Amersham Typhoon laser imager (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the results. Figure 8 In. Figure 8 As shown, the addition of MluUDG and the aldehyde reactive probe produced an additional band with a higher molecular weight in the gel image, indicating the presence of the labeled ssDNA product.

[0122] Similarly, in another example, a portion of double-stranded DNA molecules is labeled and analyzed with an aldehyde-reactive probe. Double-stranded DNA is prepared by annealing the 45-nucleotide ssDNA containing uracil (SEQ ID NO: 1) and the 15-nucleotide complementary strand (5'-TGTGCCAGGCCGAGA-3' (SEQ ID NO: 2)) at a molar ratio of 1:1.5 in 1x Tris-EDTA (TE) buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA mixture is heated to 98° C. for 3 minutes in a thermal cycler, and then gradually cooled (e.g., 30 seconds per 5° C.) to 4° C. to perform a DNA annealing reaction. As described in the steps and conditions for labeling ssDNA above, the resulting double-stranded DNA is subjected to uracil excision by MluUDG, followed by abasic labeling. An equal volume of 2x quenching solution is added, followed by denaturation at 95° C. for 10 minutes to terminate the reaction. The reaction products were analyzed by 20% urea PAGE and the gel was scanned with an Amersham Typhoon imager to visualize the results and are shown in Figure 8 In. Figure 8 As shown, the addition of MluUDG and the aldehyde-reactive probe to double-stranded DNA produced an additional band with a higher molecular weight in the gel, indicating the presence of the labeled DNA product.

[0123] Therefore, both single-stranded and double-stranded DNA can be labeled with aldehyde-reactive probes by the provided methods.

[0124] Example 7: 5'-end labeling of nucleic acids using aminooxy-5(6)-FAM

[0125] A 45-nucleotide 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. For uracil excision and subsequent abasic labeling, 100 nM of ssDNA was mixed with 100 ng of uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus and 2 mM of aminooxy-5(6)-FAM. MluUDG and aminooxy-5(6)-FAM were added to initiate the reaction, which was reacted at 37°C for 60 min. An equal volume of 2x quenching solution (30 mM EDTA and 95% (v / v) deionized formamide) was added, followed by denaturation at 95°C for 10 min to terminate the reaction. The reaction products were analyzed by 20% urea PAGE, and the gel was scanned with an Amersham Typhoon imager to visualize the results. Fig. 9As shown, the addition of MluUDG and aminooxy-5(6)-FAM produced an additional band with a higher molecular weight in the gel, indicating the presence of the FAM-labeled single-stranded DNA product.

[0126] Similarly, in another example, a portion of double-stranded DNA molecules was labeled with aminooxy-5(6)-FAM and analyzed. Double-stranded DNA was prepared by annealing ssDNA (SEQ ID NO: 1) containing 45 nucleotides of uracil and a complementary strand of 15 nucleotides (5'-TGTGCCAGGCCGAGA-3' (SEQ ID NO: 2)) at a molar ratio of 1:1.5 in 1x TE buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA mixture was heated to 98° C. for 3 minutes in a thermal cycler, and then gradually cooled (e.g., 30 seconds per 5° C.) to 4° C. to perform DNA annealing reaction. As described above for the steps and conditions for labeling ssDNA, the resulting double-stranded DNA was subjected to uracil excision by MluUDG, followed by subsequent abasic labeling. The reaction was then terminated and the products were analyzed by 20% urea PAGE and the gel was scanned with an Amersham Typhoon imager to visualize the results. Fig. 9 As shown, the addition of MluUDG and aminooxy-5(6)-FAM to double-stranded DNA produced an additional band with a higher molecular weight in the gel, indicating the presence of the labeled FAM-DNA product.

[0127] It can be seen that both single-stranded and double-stranded DNA can be labeled with aminooxy-5(6)-FAM using the provided method.

[0128] Example 8: 5'-end labeling of DNA with naphthalene- and guanidine-containing aminooxy-FAM

[0129] A single-stranded DNA of 47 nucleotides containing a uracil residue at the 5'-end (ssDNA; 5'- / deoxyU / CTCGGCCTGGCACAGGTCCGTCTCAGTGCTGCGGCGACCACCGAGG-3' (SEQ ID NO: 3)) was synthesized. For uracil excision and subsequent abasic labeling, 100 nM of ssDNA was mixed with 100 ng of uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus and 2 mM of naphthalene- and guanidine-containing aminooxy-FAM. MluUDG and naphthalene- and guanidine-containing aminooxy-FAM were added to initiate the reaction, and the reaction was allowed to proceed at 37° C. for 60 minutes. An equal volume of a 2-fold quenching solution (30 mM EDTA and 95% (v / v) deionized formamide) was added, followed by denaturation at 95° C. for 10 minutes to terminate the reaction. The reaction products were analyzed by 20% urea PAGE and the gel was scanned with an Amersham Typhoon imager to visualize the results. Fig.10 As shown, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAMs produced an additional band with a higher molecular weight in the gel image, indicating the presence of the labeled FAM-ssDNA product.

[0130] Similarly, in another example, a portion of double-stranded DNA molecules were labeled and analyzed with aminooxy-FAM containing naphthalene and guanidine. Double-stranded DNA was prepared by annealing a 47-nucleotide ssDNA containing uracil (SEQ ID NO: 3) and a 15-nucleotide complementary strand (SEQ ID NO: 2) at a molar ratio of 1:1.5 in 1x TE buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA mixture was heated to 98°C for 3 minutes in a thermal cycler, and then gradually cooled (e.g., 30 seconds per 5°C) to 4°C to perform a DNA annealing reaction. As described in the steps and conditions for labeling ssDNA above, the resulting double-stranded DNA was subsequently subjected to uracil excision and subsequent abasic labeling. The reaction was then terminated, and the product was analyzed by 20% urea PAGE, and the gel was scanned with an Amersham Typhoon imager to visualize the results. Similarly, Fig.10 As shown, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAM to double-stranded DNA produced an additional band with a higher molecular weight in the gel, indicating the presence of FAM-labeled double-stranded DNA products.

[0131] Therefore, both single-stranded and double-stranded DNA can be labeled with naphthalene- and guanidine-containing aminooxy-FAM by the provided methods.

[0132] Example 9: 5'-end labeling of 5'-phosphorylated DNA with aldehyde-reactive compounds and enrichment of labeled DNA using bacteriophage lambda exonuclease

[0133] A 45-nucleotide 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. The DNA was first 5'-terminally phosphorylated by T4 polynucleotide kinase in the presence of adenosine triphosphate (ATP) at 37°C for 10 minutes. For uracil excision and subsequent abasic site labeling, 100 nM of the 5'-terminally phosphorylated ssDNA was mixed with 100 ng of uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus 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 for 15 minutes. To remove unlabeled ssDNA, 2 units of bacteriophage lambda exonuclease were added and reacted at 37°C for an additional 3.5 hours. An equal volume of 2-fold quenching solution (30 mM EDTA and 95% (v / v) deionized formamide) was added, followed by denaturation at 95°C for 10 minutes to terminate the reaction. The reaction products were analyzed by 20% urea PAGE, and the gel was scanned with an Amersham Typhoon imager to visualize the results. Fig.11 As shown, the addition of MluUDG and the aldehyde-reactive probe produced an additional band with a higher molecular weight in the gel, indicating the presence of the labeled ssDNA product. When the reaction mixture was further treated with bacteriophage lambda exonuclease to degrade the unlabeled DNA (i.e., cleanup step), the fraction of the labeled ssDNA product was further enriched.

[0134] Thus, DNA can be labeled with an aldehyde-reactive probe by the provided methods, and the labeled DNA fraction can be further enriched by treatment with bacteriophage lambda exonuclease.

[0135] Example 10: 5'-end labeling of 5'-phosphorylated DNA with naphthalene- and guanidine-containing aminooxy-FAM and purification and enrichment of FAM-labeled DNA using bacteriophage lambda exonuclease

[0136] A 47-nucleotide single-stranded DNA (ssDNA; SEQ ID NO: 3) containing a uracil residue at the 5'-end was synthesized. The DNA was first 5'-terminally phosphorylated by T4 polynucleotide kinase in the presence of adenosine triphosphate (ATP) at 37°C for 30 minutes. For uracil excision and subsequent abasic labeling, 100 nM of ssDNA was mixed with 115 ng of uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus and 1 mM of naphthalene- and guanidine-containing aminooxy-FAM. MluUDG and naphthalene- and guanidine-containing aminooxy-FAM were added to initiate the reaction, and the reaction was allowed to proceed at 37°C for 30 minutes. To remove unlabeled ssDNA, 2 units of bacteriophage lambda exonuclease were added, and the reaction was allowed to proceed at 37°C for an additional 3.5 hours. An equal volume of 2-fold quenching solution (30 mM EDTA and 95% (v / v) deionized formamide) was added, followed by denaturation at 95°C for 10 minutes to terminate the reaction. The reaction products were analyzed by 20% urea PAGE, and the gel was scanned with an Amersham Typhoon imager to visualize the results. Fig.12 As shown, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAMs produced an additional band with a higher molecular weight in the gel, indicating the presence of FAM-labeled ssDNA products. When the reaction mixture was further treated with bacteriophage lambda exonuclease to degrade unlabeled DNA, the FAM-labeled ssDNA products were enriched.

[0137] Similarly, in another example, 5'-phosphorylated double-stranded DNA was also labeled with aminooxy-FAM containing naphthalene and guanidine, followed by treatment with bacteriophage lambda exonuclease to enrich the FAM-labeled double-stranded DNA. Double-stranded DNA was prepared by annealing ssDNA (SEQ ID NO: 3) containing 47 nucleotides of uracil and a complementary strand (SEQ ID NO: 2) of 15 nucleotides at a molar ratio of 1:1.5 in 1x TE buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA mixture was heated to 98°C for 3 minutes in a thermal cycler, and then gradually cooled (e.g., 30 seconds per 5°C) to 4°C to perform a DNA annealing reaction. As described above for the steps and conditions for labeling ssDNA, the resulting double-stranded DNA was first phosphorylated at the 5'-end by T4 polynucleotide kinase in the presence of adenosine triphosphate (ATP), followed by uracil excision and subsequent abasic labeling, and then treated with bacteriophage lambda exonuclease. The results were analyzed by 20% urea PAGE, and the gel was scanned with an Amersham Typhoon imager to visualize the results. Fig.12As shown, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAM to double-stranded DNA produces an additional band with a higher molecular weight in the gel, indicating the presence of FAM-labeled double-stranded DNA products. When the reaction mixture is further treated with bacteriophage lambda exonuclease to degrade unlabeled DNA, the FAM-labeled DNA product is enriched.

[0138] Thus, both single-stranded and double-stranded DNA can be labeled with naphthalene- and guanidine-containing aminooxy-FAM by the provided methods, and can be treated with bacteriophage lambda exonuclease to further enrich the labeled DNA fraction.

[0139] Example 11: Dual Labeling of Double-Stranded DNA with Guanidine-FAM at the 5'-end and Directly Incorporated at the 3'-end

[0140] The ssDNA containing 21 nucleotides of uracil was obtained, and its electrophoretic position was as follows Fig.13 As shown in lane 1 in FIG. Double-stranded DNA was prepared from ssDNA by annealing ssDNA of 21 nucleotides containing uracil and a complementary strand of 22 nucleotides at a molar ratio of 1:1 in 1×TE buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA mixture was heated to 98° C. for 3 minutes in a thermal cycler and then gradually cooled (e.g., 30 seconds per 5° C.) to 4° C. for DNA annealing reaction.

[0141] 100 nM of the double-stranded DNA obtained above was reacted 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 5' label the double-stranded DNA. To remove unlabeled DNA, 1 μM of bacteriophage λ exonuclease was added and reacted at 37°C for an additional 60 minutes. An equal volume of 2-fold quenching solution (25 mM EDTA and 95% (v / v) deionized formamide) was added, followed by denaturation at 95°C for 10 minutes to terminate the reaction. The reaction products were analyzed by 20% urea PAGE, and the gel was scanned with an Amersham Typhoon imager to visualize the results. Fig.13 As shown in lane 2, the addition of MluUDG and guanidine-FAM to double-stranded DNA causes the band to move to a position with a higher molecular weight in the gel, indicating the presence of the labeled FAM-DNA product.

[0142] In a separate reaction, 3' labeling of the resulting double-stranded DNA was performed by directly incorporating nucleotides carrying fluorescent dyes. The reaction was carried out at 37°C for 60 minutes in a 10 μL mixture containing 100 nM double-stranded DNA, 200 nM Vent polymerase, and 20 μM N3-dTTP-Cy3. The results of 3' labeling are shown in Fig.13 As shown in lane 3.

[0143] The double labeling reaction was performed by preparing a 10 μL mixture containing 100 nM double-stranded DNA, 100 ng of MluUDG, 1 mM of guanidine-FAM, 20 mM of p-phenylenediamine, 200 nM of Vent polymerase, and 20 μM of N3-dTTP-Cy3. MluUDG and Vent polymerase were added to initiate the reaction and reacted at 37°C for 60 minutes. The double labeling results of double-stranded DNA at both the 5'-end and the 3'-end are shown in Fig.13 In lane 4, the shift in electrophoretic position indicates that the double-labeled product has a higher molecular weight than the 5'-end labeled product and the 3'-end labeled product.

[0144] Example 12: Dual Labeling of Double-Stranded DNA by Ligation of 5'-Terminal Guanidine-FAM and 3'-Terminal Azide-DBCO

[0145] The ssDNA containing 26 nucleotides of uracil was obtained, and its electrophoretic position was as follows: Fig.14 As shown in lane 1 in the figure. By annealing the 26-nucleotide ssDNA containing uracil and the 27-nucleotide complementary chain (such as Fig.14 The DNA mixture was heated to 98°C for 3 minutes in a thermal cycler, and then gradually cooled (eg, 30 seconds per 5°C) to 4°C to perform a DNA annealing reaction.

[0146] 100 nM of the double-stranded DNA obtained above was reacted with 200 nM Vent polymerase, 100 μM N3-dTTP and 100 μM DBCO-Cy5 at 37°C for 60 minutes to perform 3' labeling on the double-stranded DNA. The results of 3' labeling are shown in Fig.14The electrophoretic position of the 3' labeled product shifted compared to the position in lanes 1 and 2, indicating successful labeling with Cy5. The resulting labeled product was further enriched by 3'-exonuclease degradation. To remove the unlabeled DNA strand, 1 μM hTREX1 was added to the labeling mixture at 37°C to initiate the reaction. After 60 minutes, 10 μL of 2x quenching solution (95% deionized formamide and 25 mM EDTA) was added to terminate the degradation reaction. The results are shown in Fig.14 Lane 4 shows a thicker band compared to the same position in lane 3.

[0147] A 10 μL mixture containing 100 nM double-stranded DNA of this example, 100 ng of MluUDG, 1 mM of guanidine-FAM, 20 mM of p-phenylenediamine, 200 nM of Vent polymerase, 100 μM of N3-dTTP and 100 μM of DBCO-Cy5 was prepared at 37° C. and double labeling reaction was performed for 60 minutes. MluUDG and Vent polymerase were added to initiate the reaction and reacted at 37° C. for 60 minutes. The double labeling results of double-stranded DNA at both the 5'-end and the 3'-end are shown in Fig.14 The shift in the electrophoretic position of lane 5 compared to lanes 3 and 4 indicates that the double-labeled product has a higher molecular weight.

[0148] The double-labeled product was further degraded by 5'-exonuclease and 3'-exonuclease to remove the unlabeled DNA strand, also known as the cleanup step. 1 μM λ exonuclease and 1 μM hTREX1 were added to the above labeling mixture at 37°C to initiate the reaction. After 60 minutes, 10 μL of 2x quenching solution (95% deionized formamide and 25 mM EDTA) was added to terminate the reaction. The results are shown in Fig.14 In lane 6, it shows a thicker band compared to the band at the same position in lane 5, indicating that the labeled product has been effectively cleared.

[0149] Example 13: Dual labeling of double-stranded DNA with guanidine-FAM at the 5'-end and BHQ1 at the 3'-end

[0150] The ssDNA containing 25 nucleotides of uracil was obtained, and its electrophoretic position was as follows: Fig.15 As shown in lane 1 in the figure. By annealing the 25-nucleotide ssDNA containing uracil and the 26-nucleotide complementary chain (such as Fig.15The DNA mixture was heated to 98°C for 3 minutes in a thermal cycler, and then gradually cooled (eg, 30 seconds per 5°C) to 4°C to perform a DNA annealing reaction.

[0151] 100nM of the double-stranded DNA obtained above was reacted with 100ng of MluUDG, 1mM of guanidine-FAM and 20mM of p-phenylenediamine in a 10μL reaction mixture at 37°C for 60 minutes to 5'-label the double-stranded DNA. The 5'-labeled mixture was then 3'-labeled by adding 200nM Vent polymerase and 100μM N3-dCTP-BHQ1 to the 5'-labeled mixture at 37°C and reacting for an additional 60 minutes. Next, to remove the unlabeled DNA chain, 1μM of λ exonuclease and 1μM of hTREX1 were added to the resulting labeled mixture at 37°C. After 60 minutes, 10μL of a 2x quenching solution (95% deionized formamide and 25mM EDTA) was added to terminate the reaction. The results are shown in Fig.15 Lane 3 of the assay showed a shift in the band position compared to lanes 1 and 2.

[0152] In separate experiments, simultaneous labeling of the 5' and 3' ends was performed by mixing a 10 μL reaction volume containing 100 nM double-stranded DNA, 100 ng of MluUDG, 1 mM guanidine-FAM, 20 mM p-phenylenediamine, 200 nM Vent polymerase, and 100 μM N3-dCTP-BHQ1. The reaction was initiated by the addition of MluUDG and Vent polymerase at 37°C and allowed to react for 60 minutes before exonuclease degradation. To remove unlabeled DNA strands, 1 μM λ exonuclease and 1 μM hTREX1 were added to the labeling mixture and reacted for 60 minutes at 37°C. The reaction was terminated by adding 10 μL of 2x quenching solution (95% deionized formamide and 25 mM EDTA). The results of simultaneous dual labeling and exonuclease degradation are shown in Fig.15 The results show that the simultaneous reaction does not affect the labeling efficiency of double-stranded DNA, and lanes 3 and 4 show similar band thickness, which indicates the amount of labeled products.

[0153] The same procedure as described above for sequential or simultaneous labeling of dsDNA was performed again, except that 100 μM N3-dCTP and 100 μM DBCO-BHQ1 were used to replace 100 μM N3-dCTP-BHQ1 in the 3'-end labeling reaction. The labeled products were analyzed after exonuclease degradation. As shown in lanes 5 and 6, which respectively show the sequential and simultaneous dual labeling of 5'-end and 3'-end, the simultaneous dual labeling reaction involving 3'-end azide and DBCO ligation has similar labeling efficiency compared to the sequential labeling of first 5'-end labeling and then 3'-end labeling.

[0154] The present disclosure has been described by its embodiments, and it should be understood that various modifications are consistent with the embodiments of the present disclosure without departing from the scope of the present disclosure. Therefore, the embodiments described herein are intended to cover modifications within the scope of the present disclosure, but not to limit the present disclosure. Therefore, the scope of the claims should be interpreted in the broadest sense to cover all such modifications.

Claims

1. A method for labeling the 5'-end and 3'-end of a nucleic acid, comprising: Providing a target nucleic acid to be labeled; adding 5'-terminal glycosidase to the target nucleic acid; adding nucleotides having reactive moieties 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 into the 3'-end of the intermediate nucleic acid; providing an aldehyde-reactive compound carrying a detectable label for coupling to the abasic site at the 5'-end of the intermediate nucleic acid; providing 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 carrying the detectable label; as well as exposing the intermediate nucleic acid to the desired molecule having the corresponding functional portion, thereby forming a labeled nucleic acid having the detectable label coupled to the abasic position of the intermediate nucleic acid, the labeled nucleic acid having the detectable label attached to the 5'-end, and having a linkage formed between the reactive moiety and the corresponding functional moiety. 2 . The method of claim 1 , further comprising providing at least one of a molecule having 5′ to 3′ exonuclease activity and a molecule having 3′ to 5′ exonuclease activity to remove unlabeled target nucleic acid and the intermediate nucleic acid.

3. The method of claim 2, wherein: The 5' to 3' exonuclease is selected from the group consisting of T5 exonuclease, T7 exonuclease, bacteriophage lambda exonuclease, exonuclease VIII, RecJ, RecJf, Tth RecJ, Mpn A group consisting of 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 of claim 1, wherein: The desired molecule further comprises a labeling moiety for forming the labeled nucleic acid when the desired molecule is coupled to the 3'-end of the nucleic acid.

5. The method of 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 of claim 5, wherein: The template-independent enzymatic nucleic acid synthesis includes the use of DNA polymerase, RNA polymerase or their functional equivalents.

7. The method of claim 6, wherein: The DNA polymerase is an A family DNA polymerase, a B family DNA polymerase or an X family DNA polymerase.

8. The method of claim 7, wherein: The B family DNA polymerase is a Thermococcaceae DNA polymerase.

9. The method of claim 8, wherein: The B family DNA polymerase is a Thermococcus DNA polymerase or a Pyrococcus DNA polymerase.

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

11. The method of claim 6, wherein: The DNA polymerase is a modified DNA polymerase.

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

13. The method of claim 1, wherein: The nucleic acid is linked to an initiator attached to a solid support.

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

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

16. The method of claim 1, wherein: The corresponding functional moiety reacts with the reactive moiety via a bioorthogonal reaction.

17. The method of claim 16, wherein: The bioorthogonal reaction is click ligation, oxime / hydrazone formation, Staudinger ligation, tetrazine ligation or tetracycloheptane ligation.

18. The method of claim 17, wherein: The click conjugation is selected from the group consisting of copper-catalyzed cycloaddition of azides and alkynes, strain-promoted cycloaddition of azides and alkynes, isocyanide-based click reactions, and reverse electron-demanding Diels-Alder reactions.

19. The method of claim 1, wherein: The desired molecule is molecularly identifiable by detection of visible light, fluorescence, photoluminescence, electrochemiluminescence, laser, radiation, fluorescence resonance energy transfer, fluorescence conformational change, or fluorescence quenching.

20. The method of claim 19, wherein: The desired molecule is a chemical compound, a fluorescent tag, a dye, a label, a reporter molecule, 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 of claim 1, further comprising providing a 3' to 5' exonuclease to degrade unsuccessfully coupled or unmodified nucleic acids.

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

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

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