Methods, kits and systems for nucleic acid terminal labeling

Abstract

The invention provides a method for labeling nucleic acid. The method comprises the following steps: providing target nucleic acid to be labeled; providing a 5 '-terminal glycosidase to react with the target nucleic acid to generate an intermediate nucleic acid having a base-free site at the 5'-terminal; and providing an aldehyde reactive compound carrying a detectable label for coupling with the intermediate nucleic acid to form a labeled nucleic acid having the detectable label attached to the 5 '-terminus The disclosure also provides a kit and system of labeled nucleic acids.

Description

References for electronic sequence listing

[0001] The present disclosure relates to methods for nucleic acid terminus labeling. More particularly, the present disclosure relates to methods for generating abasic sites and labeling the abasic sites present at the nucleic acid termini with chemical or functional moieties. [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. 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.

[0003] Although there are many nucleic acid labeling technologies available, these methods are usually lengthy and use various types of enzymes, reactive chemicals or radioactive isotopes, which generally require specialized enzymes, chemicals or personnel training to handle toxic or radioactive materials and waste. 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]

[0004] The present disclosure 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 the detectable label attached to the 5'-end.

[0005] 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 de novo synthesized or derived from a biological organism. In some embodiments, the nucleic acid is immobilized on a solid surface or a polymer surface.

[0006] 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)-dibenzocyclooctyne (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-hexachloro-fluorescein (HEX), aminooxy-5 (6)-ROX and aminooxy-5 (6)-TAMRA, cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy-Alexa Fluor 488 or Alexa Fluor 647. Fluor dyes, aminooxybiotin, 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.

[0007] 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.

[0008] 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 (methylpurine DNA glycosidase) or methyladenine DNA glycosidase (methylpurine DNA glycosidase). The invention relates to a group consisting of single-stranded selective monofunctional uracil DNA glycosylase 1 (SMUG1), methyl binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homologous DNA glycosylase (MYH), alkylpurine glycosidase C (AlkC), alkylpurine glycosidase D (AlkD), 8-oxoguanine glycosidase 1 without abasic lyase activity (OGG1), endonuclease III glycosidase 1 without abasic lyase activity (NTHL1), endonuclease VIII glycosidase 1 without abasic lyase activity (NEIL1), endonuclease VIII glycosidase 2 without abasic lyase activity (NEIL2), endonuclease VIII glycosidase 3 without abasic lyase activity (NEIL3), enzyme-active fragments thereof, and any combination thereof.

[0009] 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.

[0010] 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, and any combination thereof.

[0011] In some embodiments, the methods of the present disclosure 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), bacterial alkaline exonuclease, viral alkaline exonuclease, bacteriophage lambda exonuclease, 5'-exonuclease (ExoVI) of DNA polymerase I from, for example, Streptococcus pneumoniae or Helicobacter pylori, Escherichia coli exonuclease VIII (ExoVIII), RecJ from, for example, 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) gp19, bacterial AddB, and any combination thereof.

[0012] In some embodiments, the methods of the present disclosure further include methods for synthesizing nucleic acids that have unique 5'-terminal nucleobases. In some embodiments, nucleic acids having unique 5'-terminal nucleobases are synthesized by methods well known in the art, including phosphoramidite-based nucleic acid synthesis methods, and template-dependent and template-independent enzymatic nucleic acid synthesis methods.

[0013] In some specific embodiments, the methods of the present disclosure further include isolating nucleic acid fragments from a sample. For example, nucleic acids can be isolated from samples of intact or destroyed 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 specific embodiments, samples can be obtained from formalin-fixed paraffin embedded (FFPE) tissue samples or other stored cell material samples.

[0014] 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.

[0015] 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.

[0016] In at least one embodiment, the uracil DNA glycosidase in the kit of the present disclosure is derived from a family of Micrococcaceae, Staphylococcaceae, or Nucleococcaceae, which includes the genera of Micrococcus, Oral Coccus, Staphylococcus, or Motile Coccus. In some embodiments, the uracil DNA glycosidase is derived from Micrococcus luteus.

[0017] 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, or a compound having a naphthalene-containing and / or guanidine-containing aminooxy group. 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 hexachlorofluorescein (HEX), aminooxy-5(6)-ROX and aminooxy-5(6)-TAMRA, cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy-Alexa Fluor dyes such as Alexa Fluor 488 or Alexa Fluor 647, aminooxy biotin, naphthalene-containing aminooxy fluorescent dyes, guanidine-containing aminooxy fluorescent dyes, naphthalene- and / or guanidine-containing aminooxy-FAM, Cy5-PEG-aminooxy, or a fluorescent dye hydrazide such as cyanine dye hydrazide or fluorescent CF dye hydrazide.

[0018] 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, T7 exonuclease, bacteriophage lambda exonuclease, 5'-exonuclease of DNA polymerase I (ExoVI), exonuclease VIII (Exo VIII), RecJ, RecJf, Tth RecJ, Mpn A group consisting of 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.

[0019] The present disclosure further provides a system for labeling the 5'-end of a nucleic acid, 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 to continue for a period of time at a set temperature controlled by the temperature control unit.

[0020] 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. 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 A group consisting of 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.

[0021] 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. In at least one embodiment, the aldehyde-reactive compound is hydroxylamine biotin, fluorescent dye hydroxylamine, aldehyde-reactive probe (ARP) such as N-(aminooxyacetyl)-N'-biotin hydrazine, aminooxy fluorescent dye such as aminooxy-PEG-TAMRA (5-carboxytetramethylrhodamine), aminooxy-PEG-Cy3 (cyanine 3) dye, aminooxy-PEG-Cy5 (cyanine 5) dye, aminooxy-PEG-FAM (fluorescein amide) dye, aminooxy poly (ethylene glycol) azide (aminooxy-PEG-azide), propargyl, aminooxy poly (ethylene glycol)-dibenzocyclooctyne (aminooxy-PEG-DBCO), aminooxy poly (ethylene glycol) bicyclononyne (BCN), cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy-Alexa Fluor 488 or Alexa Fluor 647. Fluor dyes, aminooxybiotin, naphthalene-containing aminooxy fluorescent dyes, guanidine-containing aminooxy fluorescent dyes, fluorescent dye hydrazides or maleimides.

Brief description of the attached figure

[0022] The present disclosure may be more fully understood by reading the following description of specific embodiments and by referring to one or more of the accompanying drawings.

[0023] Figure 1 The results of urea PAGE using uracil DNA glycosidase (MluUDG) derived from Micrococcus luteus and an 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.

[0024] Figure 2 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.

[0025] Figure 3 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 (guanidine-FAM). "S" indicates a lane containing only unlabeled DNA.

[0026] Figure 4 Urea PAGE results showing labeling of the 5'-end of single-stranded DNA using uracil DNA glycosidase from Micrococcus luteus (MluUDG) and an 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.

[0027] Figure 5 Urea 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 (guanidine-FAM), followed by a cleanup step using bacteriophage lambda exonuclease (λexo) to eliminate unlabeled DNA. "S" indicates a lane containing only unlabeled DNA.

[0028] Figure 6 Shows the results of urea PAGE of 5'-end labeling of single-stranded DNA (ssDNA) with fluorescent dye or biotin moiety. "S" indicates a lane containing unlabeled ssDNA. Lanes 1 to 4 show ssDNA samples labeled with 5-TAMRA (5-carboxytetramethylrhodamine), Cy3 (cyanine 3) dye, Cy5 (cyanine 5) dye, and FAM (fluorescein) dye, respectively. Lane 5 shows ssDNA labeled with biotin moiety.

[0029] Figure 7 The urea PAGE results of labeling the 5'-end of single-stranded DNA (ssDNA) with a dibenzocyclooctyne (DBCO) functional group or an azide functional group are shown. Lane 1 shows the ssDNA before labeling; Lane 2 shows the 5'-basic ssDNA produced by enzyme excision; and Lanes 3 and 4 show the ssDNA samples labeled with DBCO or azide, respectively.

[0030] Figure 8The urea PAGE results of labeling the 5'-end of single-stranded DNA (ssDNA) with a maleimide group are shown. Lane 1 shows the ssDNA before labeling, and lane 2 shows the ssDNA labeled with a maleimide group.

[0031] Fig. 9 The results of HPLC purification of 5'-end labeled ssDNA using 5(6)-FAM dye are shown. The estimated purity of the labeled ssDNA was about 98.2% as determined by the peak area at λ=260 nm.

[0032] Fig.10 The results of HPLC purification of 5'-end labeled ssDNA using dibenzocyclooctyne (DBCO) are shown. The estimated purity of the labeled ssDNA was about 99.9% as determined by the peak area at λ=260 nm.

[0033] Fig.11 The results of HPLC purification of 5'-end labeled ssDNA using an azide group are shown. The estimated purity of the labeled ssDNA was about 98.9% as determined by the peak area at λ=260 nm.

[0034] Fig.12 The MALDI-TOF mass spectrometry results of HPLC-purified 5'-end labeled ssDNA using 5(6)-FAM dye are shown. In the spectrum, the singly charged ions and doubly charged ions of the 5'-FAM labeled oligonucleotide are identified at m / z 8539.3 and 4265.3, respectively.

[0035] Fig.13 The MALDI-TOF mass spectrometry results of HPLC-purified 5'-end labeled ssDNA with dibenzocyclooctyne (DBCO) are shown. In the spectrum, the singly charged ions, doubly charged ions, triply charged ions, and quadruple charged ions of the 5'-end DBCO labeled oligonucleotides were identified at m / z 14520.3, 7265.0, 4840.6, and 3631.5, respectively.

[0036] Fig.14 The MALDI-TOF mass spectrometry results of 5'-end labeled ssDNA with azide group purified by HPLC are shown. In the spectrum, the singly charged ion, doubly charged ion, triply charged ion and quadruple charged ion of the 5'-end azide labeled oligonucleotide are identified at m / z 14158.0, 7081.1, 4718.3 and 3538.8, respectively. [Specific implementation method]

[0037] The following examples are used to illustrate the present disclosure. Based on the disclosure of this specification, a person with ordinary knowledge in the art can easily think of other effects of the present disclosure. Obviously, one or more specific embodiments can be implemented without specific details. The present disclosure can also be implemented or applied as described in different embodiments. For different applications, the following embodiments can be modified or changed to implement the present disclosure without violating the scope of the present disclosure. The titles or subtitles used in the present disclosure are for ease of reading and should not affect the scope of the present disclosure.

[0038] In the present disclosure, all terms used herein (including descriptive terms or technical terms) should be interpreted as having obvious meanings for those with ordinary knowledge in the art. However, according to the intention of those with ordinary knowledge in the art, precedents or the emergence of new technologies, these terms may have different meanings. In addition, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected terms will be described in detail in the embodiments of the present disclosure. Therefore, the terms used herein are defined based on the meaning of the terms and the description of the entire specification.

[0039] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques within the scope of molecular biology, microbiology, cell biology, and biochemistry 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); "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., 1993-8); "Methods in Enzymology" (Academic Press, Inc.); "Gene Transfer Vectors for Mammalian Cell ...Cell and Tissue Culture: Laboratory Procedures" (A. Doyle, JB Griff Cells" (JM Miller and MP Calos, eds., 1987); "Current Protocols in Molecular Biology" (FM Ausubel, et al., eds., 1987); "PCR: The Polymerase Chain Reaction (Mullis, et al., ed., 1994); "Short Protocols in Molecular Biology" (Wiley and Sons, 1999). Specific usage techniques for specific embodiments will be described in the following paragraphs. Without further elaboration, it is believed that a person with ordinary knowledge in the art can utilize the present disclosure to the greatest extent based on the above description. Therefore, the following specific embodiments are only used for illustrative explanations and are not intended to limit the rest of the present disclosure in any way. All publications cited herein are incorporated herein by reference to the purposes and subjects cited.

[0040] As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent. The term "or" can be used interchangeably with the term "and / or," unless the context clearly indicates otherwise.

[0041] In addition, when a component “includes” or “comprises” components or steps, unless there is a specific description to the contrary, the component may also include other components or steps, rather than excluding other components or steps.

[0042] 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 employ any specific value or sub-range within that range in different embodiments of the present disclosure, unless the context clearly indicates otherwise.

[0043] 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 of ordinary skill 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" and "approximately" in all cases.

[0044] 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.

[0045] As used herein, the term "at least one" with respect 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, i.e., A, while B is not present (and optionally including elements other than B); in another embodiment may refer to at least one, optionally including more than one, i.e., B, while A is not present (and optionally including elements other than A); in yet another embodiment may refer to at least one, optionally including more than one, i.e., A, and at least one, optionally including more than one, i.e., B (and optionally including other elements).

[0046] 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 one embodiment, 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 may 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.

[0047] The nucleic acid used herein may be a single-stranded, double-stranded, or a mixture of single-stranded and double-stranded nucleic acids. Double-stranded nucleic acids may be double-stranded regions having at least two complementary strands of nucleic acid. For example, nucleic acids are DNA molecules, such as plasmids, synthetic DNA, or viral DNA. In other specific embodiments, nucleic acids may be RNA molecules, such as synthetic RNA, mRNA, tRNA, rRNA, and non-coding RNA. The term also includes analogs of DNA or RNA made from nucleotide analogs, and, if applicable, single-stranded (sense strand or antisense strand) and double-stranded polynucleotides. The term also includes modified polynucleotides, including modified DNA and modified RNA, such as DNA and RNA containing one or more non-natural nucleotides or nucleosides. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein, meaning 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 linkages, which are synthetic, naturally occurring, and non-naturally occurring, and / or have similar chemical properties to reference nucleic acids, and / or are metabolized in a manner similar to reference nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. In some embodiments, nucleotides are linked via internucleotide bonds, such as, but not limited to, phosphates, boranophosphates, thiophosphates, 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 that replace natural or non-natural nucleotides, such as modified nucleotides. For example, the term "heteronucleotide" refers to a nucleotide that has been modified to have a sugar portion 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 acid (PNA), locked nucleic acid (LNA), 1,5-anhydrohexitol nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), cyclohexene nucleic acid (CeNA), and fluoro-arabino nucleic acid (FANA).

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] As used herein, the term "label" (interchangeably referred to as "detectable marker" or "modification") refers to a chemical group or functional moiety that is bound or linked to a polynucleotide (interchangeably referred to as "labeling" or modified). Labeled polynucleotides are typically 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.

[0057] 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.

[0058] 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.

[0059] Methods for signal detection 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 fluorometer, 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 photographic 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, photographic film, a fluorometer, 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 strategy for signal amplification can be selected to facilitate detection of low-abundance molecular targets.

[0060] The kits provided herein are packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Packaging used in conjunction with a device or system is also contemplated. The kits may optionally provide additional components, such as buffers and explanatory information. In some embodiments, the present disclosure provides an article comprising the contents of the kits described herein. Example

[0061] 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 examples will be described in detail below. Materials used in the present disclosure but not noted herein are all commercially available.

[0062] Example 1: 5'-end labeling of nucleic acids using aldehyde-reactive compounds

[0063] 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'-biotin hydrazine). The reaction was initiated by adding MluUDG and aldehyde-reactive probe (ARP) at 37°C for 15 minutes. The reaction was terminated by adding an equal volume of 2x quenching solution (30 mM EDTA and 95% (v / v) deionized formamide), followed by denaturation at 95°C for 10 minutes. The reaction products were analyzed by denaturing 20% ​​polyacrylamide gel electrophoresis (urea-PAGE) containing 8 M urea. The gel was scanned by Amersham Typhoon Imager (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the results and are shown in Figure 2. Figure 1 In. Figure 1 As shown, the addition of MluUDG and the aldehyde-reactive probe produced additional bands with higher molecular weights in the gel image, indicating the presence of labeled ssDNA products.

[0064] Similarly, in another embodiment, a portion of double-stranded DNA molecules is labeled and analyzed with an aldehyde-reactive probe. Double-stranded DNA is prepared by ligating 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 ligation 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 to terminate the reaction, followed by denaturation at 95° C. for 10 minutes. The reaction products were analyzed by 20% urea PAGE. The gel was scanned by Amersham Typhoon imager to visualize the results and are shown in Figure 1 In Figure 1 In the assay, MluUDG and an aldehyde-reactive probe were added to the double-stranded DNA, resulting in an additional band with a higher molecular weight in the gel, indicating the presence of the labeled DNA product.

[0065] Therefore, both single-stranded and double-stranded DNA can be labeled with aldehyde-reactive probes by the provided methods.

[0066] Example 2: 5'-end labeling of nucleic acids using aminooxy-5(6)-FAM

[0067] 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 to terminate the reaction, followed by denaturation at 95°C for 10 min. The reaction products were analyzed by 20% urea PAGE. The gel was scanned using an Amersham Typhoon imager to visualize the results. Figure 2 As shown, the addition of MluUDG and aminooxy-5(6)-FAM produced additional bands with higher molecular weight in the gel, indicating the presence of labeled FAM-ssDNA products.

[0068] Similarly, in another embodiment, a portion of double-stranded DNA molecules were labeled with aminooxy-5(6)-FAM and analyzed. Double-stranded DNA was prepared by ligating 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 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 ligation reaction. As described in the steps and conditions for labeling ssDNA above, the resulting double-stranded DNA was subjected to uracil excision by MluUDG and then abasic labeling. The reaction was then terminated and the reaction products were analyzed by 20% urea PAGE. The gel was scanned using an Amersham Typhoon imager to visualize the results. Figure 2 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.

[0069] It can be seen that both single-stranded and double-stranded DNA can be labeled with aminooxy-5(6)-FAM using the provided method.

[0070] Example 3: 5'-end labeling of DNA with aminooxy-FAM containing naphthalene and guanidine

[0071] 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 carried out 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 to terminate the reaction, followed by denaturation at 95° C. for 10 minutes. The reaction products were analyzed by 20% urea PAGE. The gel was scanned using an Amersham Typhoon imager to visualize the results. Figure 3As shown, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAMs produced additional bands with higher molecular weights in the gel, indicating the presence of labeled FAM-ssDNA products.

[0072] Similarly, in another embodiment, a portion of double-stranded DNA molecules were also labeled and analyzed with aminooxy-FAM containing naphthalene and guanidine. Double-stranded DNA was prepared by bonding the 47-nucleotide ssDNA containing uracil (SEQ ID NO: 3) and the 15-nucleotide complementary chain (SEQ ID NO: 2) at a molar ratio of 1:1.5 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 every 5°C) to 4°C to perform a DNA bonding reaction. As described in the steps and conditions for labeling ssDNA above, the resulting double-stranded DNA was subjected to uracil excision and then abasic labeling. The reaction was then terminated and the product was analyzed by 20% urea PAGE. The gel was scanned using an Amersham Typhoon imager to visualize the results. Similarly, Figure 3 As shown, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAM to double-stranded DNA produced additional bands with higher molecular weights in the gel, indicating the presence of FAM-labeled double-stranded DNA products.

[0073] Therefore, both single-stranded and double-stranded DNA can be labeled with naphthalene- and guanidine-containing aminooxy-FAM by the provided methods.

[0074] Example 4: 5'-end labeling of 5'-phosphorylated DNA with aldehyde-reactive compounds and enrichment of labeled DNA using bacteriophage lambda exonuclease

[0075] 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 labeling, 100 nM of the 5'-terminally phosphorylated ssDNA was mixed with 100 ng of uracil DNA glycosidase derived from Micrococcus luteus (MluUDG) and 5 mM of an aldehyde-reactive probe (N-(aminooxyacetyl)-N'-biotin hydrazine). 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 2x quenching solution (30 mM EDTA and 95% (v / v) deionized formamide) was added to terminate the reaction, followed by denaturation at 95°C for 10 minutes. The reaction products were analyzed by 20% urea PAGE. The gel was scanned by Amersham Typhoon imager to visualize the results. Figure 4 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.

[0076] 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.

[0077] Example 5: 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

[0078] A single-stranded DNA of 47 nucleotides containing a uracil residue at the 5'-end (ssDNA; SEQ ID NO: 3) 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 site labeling, 100 nM of ssDNA was mixed with 115 ng of uracil DNA glycosidase derived from Micrococcus luteus (MluUDG) 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 30 minutes. An equal volume of 2x quenching solution (30 mM EDTA and 95% (v / v) deionized formamide) was added to terminate the reaction, followed by denaturation at 95°C for 10 minutes. The reaction products were analyzed by 20% urea PAGE. The gel was scanned by Amersham Typhoon imager to visualize the results. Figure 5 As shown, the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAMs produced additional bands with higher molecular weights 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.

[0079] Similarly, in another embodiment, 5'-phosphorylated double-stranded DNA molecules were 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 ligating the 47-nucleotide ssDNA containing uracil (SEQ ID NO: 3) and the 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 ligation 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 ATP, followed by uracil excision and subsequent abasic labeling, and then treated with bacteriophage lambda exonuclease. The reaction products were analyzed by 20% urea PAGE. The gel was scanned using an Amersham Typhoon imager to visualize the results. Figure 5As 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.

[0080] 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.

[0081] Example 6: 5'-end labeling of ssDNA with fluorescent dyes, biotin moieties, azides or dibenzocyclooctyne (DBCO) functional groups

[0082] The 5'-end labeling or modification of a single-stranded DNA (ssDNA; SEQ ID NO: 1) of 45 nucleotides containing uracil residues was performed in a reaction buffer containing 1 mM Tris-HCl (pH 8.0), 5 mM NaCl, 10 μM EDTA and 0.02% PEG4000 using 100 nM ssDNA, 1 μM uracil DNA glycosidase and 200 μM of a desired molecule to be added to the 5'-end of ssDNA, including aminooxypolyethylene glycol (PEG)-TAMRA (5-carboxytetramethylrhodamine), aminooxy-PEG-Cy3 (cyanine 3) dye, aminooxy-PEG-Cy5 (cyanine 5) dye, aminooxy-PEG-FAM (fluorescein amide) dye, ARP (aldehyde reactive probe, N-(aminooxyacetyl)-N'-biotin hydrazine), aminooxy-PEG-azide and aminooxy-PEG-dibenzocyclooctyne (DBCO).

[0083] The DNA labeling reaction was carried out at 37°C for 60 minutes and then terminated by adding an equal volume (10 μL) of 2x quenching solution (30 mM EDTA and 95% deionized formamide). The reaction products of 5'-end labeled ssDNA were analyzed by 20% polyacrylamide gel electrophoresis (PAGE) containing 8 M urea. The gel was first stained with 1x SYBR Gold nucleic acid gel staining solution (Waltham, MA, USA) and the gel was scanned by Amersham Typhoon Biomolecular Imager (Marlborough, MA, USA) to visualize the results.

[0084] The results of labeling the 5'-end of ssDNA with fluorescent dye or biotin are as follows Figure 6As shown, lane S shows the position of unlabeled ssDNA, and lanes 1 to 5 respectively show that the electrophoretic positions of the 5'-end labeled products of ssDNA are higher, and the labels are 5-TAMRA (5-carboxytetramethylrhodamine), Cy3 (cyanine 3) dye, Cy5 (cyanine 5) dye, FAM (fluorescein) dye and biotin part (ARP), respectively.

[0085] The results of labeling the 5'-end of ssDNA with azide or DBCO are as follows Figure 7 As shown, lane 1 shows the ssDNA substrate before modification, lane 2 shows the 5'-basic ssDNA generated by enzyme excision, and lanes 3 and 4 show ssDNA samples modified with DBCO or azide, respectively.

[0086] Example 7: 5'-end labeling of ssDNA with maleimide groups

[0087] First, 5'-end labeling of 45-nucleotide single-stranded DNA (ssDNA; SEQ ID NO: 1) with maleimide groups was performed using 100 nM ssDNA, 1 μM uracil DNA glycosidase, and 200 μM aminooxy-PEG-azide in a reaction buffer containing 1 mM Tris-HCl (pH 8.0), 5 mM NaCl, 10 μM EDTA, and 0.02% PEG4000. The DNA labeling reaction was carried out at 37°C for 60 minutes and then terminated by adding an equal volume (10 μL) of 2x quenching solution. The 5'-end azide-labeled ssDNA was purified using a QIAquick Nucleotide Removal Kit (Qiagen, Waltham, MA, USA). To label the maleimide group, the purified 5'-end azide-labeled ssDNA was further reacted with 200 μM DBCO-PEG-maleimide. The reaction was carried out at 37°C for 60 minutes and then terminated by adding an equal volume (10 μL) of 2x quenching solution. The reaction products of 5'-end labeled ssDNA were analyzed by 20% polyacrylamide gel electrophoresis (PAGE) containing 8 M urea. The gel was first stained with 1x SYBR Gold nucleic acid gel staining solution (ThermoFisherScientific, Waltham, MA, USA) and scanned by Amersham Typhoon Biomolecular Imager (Marlborough, MA, USA) to visualize the results.

[0088] The results of labeling the 5'-end of ssDNA with maleimide groups are as follows Figure 8As shown, lane 1 shows the ssDNA substrate before modification, and lane 2 shows the ssDNA sample labeled with a maleimide group.

[0089] Example 8: Purification of 5'-end labeled single-stranded DNA using ion-pair reversed-phase high performance liquid chromatography (IPRP-HPLC)

[0090] To confirm the purity of the 5'-end labeled product, the 5'-end labeled ssDNA was further purified by high performance liquid chromatography (HPLC). Briefly, the 5'-end labeled ssDNA was first cleaned up by QIAquick Nucleotide Removal Kit (Qiagen, Waltham, MA, USA). The cleaned up sample was then purified using an Agilent 1260 Infinity II Bio-inert LC System (Santa Clara, CA, USA) with an AdvanceBio Oligonucleotide (2.1 × 50 mm) column. The desired 5'-end labeled ssDNA was eluted from the column using a gradient of mobile phase formed by mixing two separate components: solvent A (50 mM triethylammonium acetate (TEAA) dissolved in deionized water) and solvent B (100 mM TEAA dissolved in acetonitrile). The mobile phase gradient was implemented according to the following program: from 0 to 9 minutes, the concentration of solvent B increased from 8% to 15%; from 9 to 15 minutes, the concentration of solvent B increased from 15% to 30%; and from 15 to 18 minutes, the concentration of solvent B decreased from 30% to 8%. The column flow rate was maintained at 0.6 mL / min and the column temperature was maintained at 65°C. The sample feed volume ranged from 10 μL to 20 μL. The sample fractions were monitored by absorbance values ​​at wavelengths of 260 nm and 498 nm, with peak widths greater than 0.05 min (5 Hz). The column fractions containing 5'-end labeled ssDNA were combined and further concentrated by centrifugal vacuum concentrator. The results are shown in Figures 9 to 11 , it is shown that the 5'-end labeling method of the present disclosure delivers high purity 5'-end labeled ssDNA products with FAM, DBCO and azide compounds, respectively.

[0091] Example 9: Analysis of 5'-end labeled ssDNA by MALDI-TOF mass spectrometry

[0092] To further confirm the identity of the 5'-end-labeled product of the above reaction, the 5'-end-labeled ssDNA purified by HPLC was further analyzed by MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry. For sample preparation, the purified and dried 5'-end-labeled ssDNA was reconstituted in deionized water. After confirming that the instrument vacuum level reached a specific threshold and mass calibration, 1 μL of matrix solution was dispensed onto the anchor wafer plate (Bruker Daltonics, Billerica, MA, USA). The matrix solution was prepared by mixing 200 μL of 50 mg / mL 3-hydroxypicolinic acid (3-HPA) solution with 10 μL of 100 mg / mL diammonium hydrogen citrate (DAC) solution, and then diluting with 790 μL of deionized water to obtain a final concentration of 4 mg / mL 3-HPA and 0.2 mg / mL DAC. The wafer plate was air-dried at room temperature, and then 1 μL of the 5'-end labeled ssDNA sample was applied to the matrix spot and air-dried at room temperature. Mass spectra were acquired using a highly sensitive BRUKER microflex LRF20 MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The instrument was configured to operate in positive ion linear mode, allowing accurate analysis of molecular ions. The selected mass range was set from 2,000 to 18,000, covering a wide range of molecular masses. The results are shown in Figure 12 to Figure 14 , it is shown that the 5'-end labeling method of the present disclosure delivers ssDNA products of high purity and correct quality with FAM, DBCO and azide compounds, respectively.

[0093] Although some specific embodiments of the present disclosure have been described in detail above, a person skilled in the art may make various modifications and changes to the specific embodiments shown without departing substantially from the teachings of the present disclosure. Such modifications and changes are within the scope of the present disclosure, as set forth in the appended claims.

Claims

1. A method for labeling the 5'-end of a nucleic acid, 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; as well as An aldehyde-reactive compound carrying a detectable label is provided for coupling with the abasic site of the intermediate nucleic acid to form a labeled nucleic acid having the detectable label attached to the 5'-end. 2 . The method of claim 1 , further comprising providing a 5′ to 3′ exonuclease 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 2, wherein: The 5' to 3' exonuclease comprises the 5'-exonuclease domain of DNA polymerase I.

5. The method of claim 1, wherein: The nucleic acid comprises a 5'-terminal nucleobase selected from the group consisting of hypoxanthine, cytosine, 3-alkyladenine, 8-oxoguanine, uracil, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 5-fluorouracil, dihydroxyuracil, 5-formylcytosine, 5-carboxycytosine, 3-methyladenine, 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.

6. The method of claim 1, wherein: The 5'-end glycosidase is selected from the group consisting of uracil DNA glycosidase, alkyladenine DNA glycosidase, single-stranded selective monofunctional uracil DNA glycosidase 1, methyl binding domain glycosidase 4, thymine DNA glycosidase, MutY homologous DNA glycosidase, alkylpurine glycosidase C, alkylpurine glycosidase D, 8-oxoguanine glycosidase 1 without abasic lyase activity, endonuclease III glycosidase 1 without abasic lyase activity, endonuclease VIII glycosidase 1 without abasic lyase activity, endonuclease VIII glycosidase 2 without abasic lyase activity, endonuclease VIII glycosidase 3 without abasic lyase activity, enzyme active fragments thereof, and any combination thereof.

7. The method of claim 6, wherein: The uracil DNA glycosidase is derived from the family Micrococcaceae, Staphylococcaceae or Caryophanaceae.

8. The method of claim 1, wherein: The detectable label is selected from the group consisting of azide compounds, alkynes, bicyclononyne, dibenzocyclooctyne, maleimide, peptides, proteins, antibodies, dendrimers, biotin, radioisotopes, photochromic dyes, fluorescent dyes, luminescent dyes, and any combination thereof.

9. The method of claim 1, wherein: The aldehyde-reactive compound is a compound having at least one primary amine, hydrazide, acylhydrazide, a compound having an aminooxy group, a compound having a naphthalene-containing aminooxy group, or a compound having a guanidine-containing aminooxy group.

10. The method of claim 1, wherein: The aldehyde-reactive compound is hydroxylamine biotin, aminooxy poly (ethylene glycol) azide, propargyl, aminooxy poly (ethylene glycol) -dibenzocyclooctyne, aminooxy poly (ethylene glycol) bicyclononyne, fluorescent dye hydroxylamine, aldehyde-reactive probe, aminooxy fluorescent dye, cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy-Alexa Fluor dye, Alexa Fluor 488, Alexa Fluor 647, aminooxy biotin, naphthalene-containing aminooxy fluorescent dye, guanidine-containing aminooxy fluorescent dye, Cy5-PEG-aminooxy, fluorescent dye hydrazide or maleimide.

11. The method of claim 1, wherein: The target nucleic acid is single-stranded or comprises a double-stranded region formed by at least two complementary strands of nucleic acid.

12. The method of claim 1, wherein: The target nucleic acid is DNA or RNA.

13. The method of claim 1, wherein: The target nucleic acid is de novo synthesized or derived from a biological organism.

14. The method of claim 1, wherein: The target nucleic acid comprises a uracil residue at the 5'-end.

15. A kit for labeling the 5'-end of a nucleic acid, comprising a 5'-end glycosidase and an aldehyde-reactive compound.

16. The set of claim 15, wherein: The 5'-end glycosidase is selected from the group consisting of uracil DNA glycosidase, alkyladenine DNA glycosidase, single-stranded selective monofunctional uracil DNA glycosidase 1, methyl binding domain glycosidase 4, thymine DNA glycosidase, MutY homologous DNA glycosidase, alkylpurine glycosidase C, alkylpurine glycosidase D, 8-oxoguanine glycosidase 1 without abasic lyase activity, endonuclease III glycosidase 1 without abasic lyase activity, endonuclease VIII glycosidase 1 without abasic lyase activity, endonuclease VIII glycosidase 2 without abasic lyase activity, endonuclease VIII glycosidase 3 without abasic lyase activity, enzyme active fragments thereof, and any combination thereof.

17. The set of claim 15, wherein: The aldehyde-reactive compound is hydroxylamine biotin, aminooxy poly (ethylene glycol) azide, propargyl, aminooxy poly (ethylene glycol) -dibenzocyclooctyne, aminooxy poly (ethylene glycol) bicyclononyne, fluorescent dye hydroxylamine, aldehyde-reactive probe, aminooxy fluorescent dye, cyanine 555 aminooxy, cyanine 647 aminooxy, aminooxy-Alexa Fluor dye, Alexa Fluor 488, Alexa Fluor 647, aminooxy biotin, naphthalene-containing aminooxy fluorescent dye, guanidine-containing aminooxy fluorescent dye, Cy5-PEG-aminooxy or fluorescent dye hydrazide.

18. The kit of claim 15, further comprising a 5' to 3' exonuclease for removing unlabeled nucleic acids.

19. The set of claim 18, wherein: The 5' to 3' exonuclease is selected from the group consisting of 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 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.

20. A system for labeling the 5'-end of a nucleic acid, comprising a reaction reservoir, a liquid handling device, a temperature control unit and a time control unit, wherein: The liquid handling device is configured to transfer 5'-end glycosidase and aldehyde-reactive compound to react with the nucleic acid in the reaction reservoir for a period of time at a set temperature controlled by the temperature control unit.

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