Methods, kits and systems for end-labeling of nucleic acids

Abstract

A method for labeling a nucleic acid is provided, comprising: providing a target nucleic acid to be labeled; providing a 5'-terminal glycosylase to react with the target nucleic acid to produce an intermediate nucleic acid having an abasic site at the 5'-terminus; and providing an aldehyde-reactive compound bearing 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. Also provided are kits and systems for labeling nucleic acids.

Description

[Technical Field]

[0001] Reference to Electronic Sequence Listing

[0001] This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on September 14, 2023, is named "05-YDBL-0001PCTUS-Sequence-Listing-20230914" and is 7KB in size. The Sequence Listing contained in this .XML file is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to methods for end-labeling of nucleic acids. More particularly, the present disclosure relates to methods for creating abasic sites and labeling chemical or functional moieties at the abasic sites present at the ends of nucleic acids. [Background technology]

[0003] Nucleic acid labeling is routinely performed in biomedical and biological applications, including the identification and purification of unknown or target gene fragments, the localization of target gene sequences, the identification of nucleic acid-protein interactions, and the visualization of cell and tissue dynamics. Generally, methods for labeling nucleic acids can be classified into chemical approaches or enzymatic approaches. Chemical labeling methods involve modifying the 5'-phosphate group, 3'-hydroxyl group, nucleobase, or sugar moiety of the targeted nucleic acid using chemically reactive compounds such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), imidazole, hydrazine, sodium periodate, and sodium cyanoborohydride to change the structure of the nucleic acid and subsequently attach chemical or functional moieties to the desired nucleic acid. In contrast, enzymatic nucleic acid labeling methods utilize enzymes such as alkaline phosphatase, nucleic acid kinase, or DNA / RNA polymerase to substitute, add, or incorporate chemical or functional moieties, such as radioisotopes, biotin groups, or fluorescently labeled nucleotides, into targeted nucleic acids, thereby attaching the desired label to the nucleic acid. Summary of the Invention [Problem to be solved by the invention]

[0004] Although many nucleic acid labeling techniques are available, these methods usually have very long procedures and utilize many types of enzymes, reactive chemicals, or radioisotopes, and they usually require personal training in handling special enzymes, chemicals, or 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 targeted site to be labeled (for example, the internal or terminal nucleotide(s) of the nucleic acid sequence), and the chemical or functional moiety to be labeled.Therefore, there is still an unmet need for a simple, efficient, and environmentally friendly nucleic acid labeling method. [Means for solving the problem]

[0005] The present disclosure provides a method for 5'-end labeling of a nucleic acid, the method comprising: providing a target nucleic acid to be labeled; providing a 5'-end glycosylase to react with the target nucleic acid to create an intermediate nucleic acid having an abasic site at the 5'-end of the target nucleic acid; and providing an aldehyde-reactive compound bearing a detectable label for coupling to the intermediate nucleic acid at the abasic site to form a labeled nucleic acid having the detectable label attached to the 5'-end.

[0006] In at least one embodiment of the present disclosure, the nucleic acid is single-stranded or comprises at least a double-stranded region formed by two complementary strands of 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 an organism. In some embodiments, the nucleic acid is immobilized on a solid surface or a polymer surface.

[0007] In at least one embodiment of the present disclosure, the aldehyde-reactive compound is a compound having at least one primary amine, a hydrazide, an 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, e.g., Alexa Fluor 488 hydroxylamine, aldehyde-reactive probe (ARP), aminooxy-fluorescent dye, e.g., 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 dye, e.g., Alexa Fluor 488 or Alexa Fluor647, 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 hydrazides or CF dye hydrazides.

[0008] 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-carboxylcytosine, 3-methyladenine (3-meA), 3-methylguanine, 7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxylcytosine, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, formamidopyrimidine derivatives of adenine, and formamidopyrimidine derivatives of guanine.

[0009] In some embodiments, the 5'-terminal glycosylase of the present disclosure can be a monofunctional DNA glycosylase. In at least one embodiment of the present disclosure, the monofunctional DNA glycosylase can be uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG, also known as methylpurine DNA glycosylase (MPG)), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD), abasic site The glycosylase may be selected from the group consisting of 8-oxoguanine glycosylase 1 without abasic site lyase activity (OGG1), endonuclease III-like glycosylase 1 without abasic site lyase activity (NTHL1), endonuclease VIII-like glycosylase 1 without abasic site lyase activity (NEIL1), endonuclease VIII-like glycosylase 2 without abasic site lyase activity (NEIL2), endonuclease VIII-like glycosylase 3 without abasic site lyase activity (NEIL3), enzymatically active fragments thereof, and any combination thereof.

[0010] In at least one embodiment of the present disclosure, the uracil-DNA glycosylase is derived from the Micrococcaceae, Staphylococcaceae, or Caryophanaceae family, which includes the genera Micrococcus, Stomatococcus, Staphylococci, or Planococcus. In some embodiments, the uracil-DNA glycosylase is derived from Micrococcus luteus.

[0011] In at least one embodiment of the present disclosure, the detectable label is selected from the group consisting of an azide, an alkyne, a bicyclononyne (BCN), a dibenzocyclooctyne (DBCO), a maleimide, a peptide, a protein, an antibody, a dendrimer, biotin, a radioisotope, a photochromic dye, a fluorescent dye, a luminescent dye, and any combination thereof.

[0012] In some embodiments, the methods of the present disclosure further comprise providing a 5' to 3' exonuclease to remove unlabeled nucleic acids. In at least one embodiment, the 5' to 3' exonuclease is selected from the group consisting of T5 exonuclease (T5 exo), T7 exonuclease (T7 exo), bacterial alkaline exonuclease, viral alkaline exonuclease, phage lambda exonuclease, such as the 5'-exonuclease of DNA polymerase I from Streptococcus pneumoniae or Helicobacter pylori (ExoVI), Escherichia coli exonuclease VIII (ExoVIII), RecJ from E. coli or Deinococcus radiodurans, RecJf from a RecJ fusion to maltose binding protein, Thermus thermophilus (Tth) RecJ, Mycoplasma pneumoniae (Mycoplasma pneumoniae), and the like. pneumoniae (Mpn) NrnA, human exonuclease 5 (hEXO5), human exonuclease 1 (hEXO1), SNM1 from Saccharomyces cerevisiae, human or bovine SNM1A, human SNM1B / Apollo, bovine SNM1B, SXT-Exo from e.g., Vibrio cholerae, phospholipase D3 (PLD3), phospholipase D4 (PLD4), e.g., Sso1391-Csa1 from Sulfolobus solfataricus, e.g., Sto0027-Csa1 from Sulfolobus tokadaii, e.g., Thermoproteus tenax tenax, e.g., Ttx1248-Csa1 from Sulfolobus solfataricus, e.g., Sso1451-Csa1 from Sulfolobus tokadaii, e.g., Sto2633-Csa1 from Pyrococcus furiosus,Sto2635-Cas4 from Sulfolobus tokadaii, for example, Sso1392-Cas4 from Sulfolobus solfataricus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2) gp19, bacterial AddB, and any combination thereof.

[0013] In some embodiments, the methods of the present disclosure further comprise a nucleic acid synthesis process for obtaining a unique 5'-terminal nucleobase. In some embodiments, nucleic acids having a unique 5'-terminal nucleobase are synthesized by processes well known in the art, including phosphoramidite-based nucleic acid synthesis processes and template-dependent and template-independent enzymatic nucleic acid synthesis processes.

[0014] In some embodiments, the method of the present disclosure further comprises isolating nucleic acid fragments from the sample.For example, nucleic acid can be isolated from intact or disrupted virus or cell samples, such as bacterial cells, archaeal cells, and eukaryotic cells, such as human cells.Suitable samples include isolated cells and tissue samples, such as solid tissue or biopsies, including tumor biopsies.In some embodiments, the sample can be obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples or other archived samples of cellular material.

[0015] The present disclosure also provides a kit for 5'-end labeling of a nucleic acid, the kit comprising a 5'-terminal glycosylase and an aldehyde-reactive compound.

[0016] In some embodiments, the 5'-terminal glycosylase in the kit of the present disclosure is selected from the group consisting of uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG, also referred to as methylpurine DNA glycosylase (MPG)), single-strand-selective monofunctional uracil DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD), abasic The glycosylase is selected from the group consisting of 8-oxoguanine glycosylase 1 without abasic site lyase activity (OGG1), endonuclease III-like glycosylase 1 without abasic site lyase activity (NTHL1), endonuclease VIII-like glycosylase 1 without abasic site lyase activity (NEIL1), endonuclease VIII-like glycosylase 2 without abasic site lyase activity (NEIL2), endonuclease VIII-like glycosylase 3 without abasic site lyase activity (NEIL3), enzymatically active fragments thereof, and any combination thereof.

[0017] In at least one embodiment, the uracil-DNA glycosylase in the kit of the present disclosure is derived from the Micrococcaceae, Staphylococcusaceae, or Caryophanaceae family, which include the genera Micrococcus, Stomacoccus, Staphylococcus, or Planococcus. In some embodiments, the uracil-DNA glycosylase is derived from Micrococcus luteus.

[0018] In at least one embodiment, the aldehyde-reactive compounds in the kits of the present disclosure are compounds having at least one primary amine, hydrazides, acylhydrazides, compounds having an aminooxy (-ONH2) group, and compounds having a naphthalene- and / or guanidine-containing aminooxy group. In some embodiments, the aldehyde-reactive compound is hydroxylamine biotin, a fluorescent dye-hydroxylamine, such as Alexa Fluor 488 hydroxylamine, an aldehyde-reactive probe (ARP), an aminooxy-fluorescent dye, 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, an aminooxy-Alexa Fluor dye, such as Alexa Fluor 488 or Alexa Fluor 647, aminooxy-biotin, a naphthalene-containing aminooxy-fluorescent dye, a guanidine-containing aminooxy-fluorescent dye, a naphthalene- and / or guanidine-containing aminooxy-FAM, Cy5-PEG-aminooxy, or a fluorescent dye hydrazide, such as a cyanine dye hydrazide or a fluorescent CF dye hydrazide.

[0019] 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 the group consisting of T5 exonuclease, T7 exonuclease, phage lambda exonuclease, 5'-exonuclease of DNA polymerase I (ExoVI), exonuclease VIII (ExoVIII), RecJ, RecJf, Tth RecJ, Mpn The polypeptide is selected from the 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.

[0020] The present disclosure further provides a system for 5'-end labeling of nucleic acids, comprising a reaction reservoir, chamber or 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 glycosylase and an aldehyde-reactive compound to the nucleic acid in the reaction reservoir, chamber or container for a period of time at a defined temperature controlled by the temperature control unit.

[0021] The present disclosure further provides a kit for 5'-end labeling of nucleic acids, comprising a 5'-terminal glycosylase and an aldehyde-reactive compound. In some embodiments, the kit includes a 5'-to-3' exonuclease for removing unlabeled nucleic acids, such as T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, phage lambda exonuclease, 5'-exonuclease of DNA polymerase I (ExoVI), exonuclease VIII (ExoVIII), RecJ, RecJf, Tth RecJ, Mpn Further including 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.

[0022] In at least one embodiment, the 5'-terminal glycosylase is selected from the group consisting of uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG), single-strand-selective monofunctional uracil DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), MutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD), and 8-oxo- In at least one embodiment, the uracil-DNA glycosylase is selected from the group consisting of guanine glycosylase 1 (OGG1), endonuclease III-like glycosylase 1 without abasic site lyase activity (NTHL1), endonuclease VIII-like glycosylase 1 without abasic site lyase activity (NEIL1), endonuclease VIII-like glycosylase 2 without abasic site lyase activity (NEIL2), endonuclease VIII-like glycosylase 3 without abasic site lyase activity (NEIL3), enzymatically active fragments thereof, and any combination thereof. In at least one embodiment, the uracil-DNA glycosylase is derived from the family Micrococcaceae, Staphylococcusaceae, or Caryophanaceae.In at least one embodiment, the aldehyde-reactive compound is hydroxylamine biotin, fluorescent dye-hydroxylamine, an aldehyde-reactive probe (ARP), such as N-(aminooxyacetyl)-N′-biotinylhydrazine, an 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 amidite) dye, aminooxy-poly(ethylene glycol)-azide (aminooxy-PEG-azide), propargyl, aminooxy-poly(ethylene glycol)-dibenzylcyclooctyne (aminooxy-PEG-DBCO), aminooxy-poly(ethylene glycol)-bicyclononyne (BCN), cyanine 555 aminooxy, cyanine 647 aminooxy, an aminooxy-Alexa Fluor dye, such as Alexa Fluor 488 or Alexa Fluor 647, aminooxy-biotin, naphthalene-containing aminooxy-fluorescent dyes, guanidine-containing aminooxy-fluorescent dyes, fluorescent dye hydrazides, or maleimides.

[0023] The present disclosure can be better understood from the following description of embodiments, taken in conjunction with one or more of the accompanying drawings, in which: [Brief explanation of the drawings]

[0024] [Figure 1] This figure shows the results of urea-PAGE of 5'-end labeled single-stranded DNA (ssDNA) and double-stranded DNA using uracil-DNA glycosylase (MluUDG) from Micrococcus luteus and an aldehyde-reactive probe (ARP). "S" indicates a lane containing only unlabeled DNA. [Figure 2]This figure shows the results of urea-PAGE of 5'-end labeled single-stranded and double-stranded DNA using uracil-DNA glycosylase (MluUDG) from Micrococcus luteus (MLUUDG) and aminooxy-5(6)-FAM. "S" indicates a lane containing only unlabeled DNA. [Figure 3] This figure shows the results of urea-PAGE of 5'-end-labeled 5'-phosphorylated single-stranded DNA and double-stranded DNA using uracil-DNA glycosylase from Micrococcus luteus (MluUDG) and naphthalene- and guanidine-containing aminooxy-FAM (guanidine-FAM). "S" indicates a lane containing only unlabeled DNA. [Figure 4] Urea-PAGE results of 5'-end labeling of single-stranded DNA with uracil-DNA glycosylase (MluUDG) from Micrococcus luteus and an aldehyde-reactive probe (ARP), followed by a cleanup step to eliminate unlabeled DNA using phage lambda exonuclease (lambda exo). "S" indicates a lane containing only unlabeled DNA. [Figure 5] This figure shows the results of urea-PAGE of 5'-end labeling of single-stranded and double-stranded DNA using uracil-DNA glycosylase from Micrococcus luteus (MluUDG) and naphthalene- and guanidine-containing aminooxy-FAM (guanidine-FAM), followed by a cleanup step to eliminate unlabeled DNA using phage lambda exonuclease (lambda exo). "S" indicates a lane containing only unlabeled DNA. [Figure 6]This figure shows the results of urea-PAGE of 5'-end labeling of single-stranded DNA (ssDNA) with fluorescent dyes or biotin moieties. "S" indicates the lane containing unlabeled ssDNA. Lanes 1 through 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 a biotin moiety. [Figure 7] This figure shows the results of urea-PAGE of 5'-end labeling of single-stranded DNA (ssDNA) with dibenzylcyclooctyne (DBCO) or azide functional groups. Lane 1 shows the ssDNA before labeling, lane 2 shows the 5'-abasic ssDNA created by enzymatic excision, and lanes 3 and 4 show the ssDNA samples labeled with DBCO or azide, respectively. [Figure 8] This figure shows the results of urea-PAGE of single-stranded DNA (ssDNA) 5'-end labeled with a maleimide group. Lane 1 shows the ssDNA before labeling, and lane 2 shows the ssDNA labeled with a maleimide group. [Figure 9] Figure 1 shows the results of HPLC purification of 5'-labeled ssDNA with 5(6)-FAM dye. The estimated purity of the labeled ssDNA is approximately 98.2% as determined by the peak area at λ = 260 nm. [Figure 10] Figure 1 shows the results of HPLC purification of 5'-labeled ssDNA with dibenzylcyclooctyne (DBCO). The estimated purity of the labeled ssDNA is approximately 99.9% as determined by peak area at λ = 260 nm. [Figure 11] Figure 1 shows the results of HPLC purification of 5'-labeled ssDNA bearing an azide group. The estimated purity of the labeled ssDNA is approximately 98.9% as determined by the peak area at λ = 260 nm. [Figure 12]Figure 1 shows the results of MALDI-TOF mass spectrometry of HPLC-purified 5'-labeled ssDNA with 5(6)-FAM dye. In the spectrum, both the singly and doubly charged ions of the 5'-FAM-labeled oligonucleotide are identified at m / z 8539.3 and 4265.3, respectively. [Figure 13] Figure 1 shows the results of MALDI-TOF mass spectrometry of HPLC-purified 5'-labeled ssDNA with dibenzylcyclooctyne (DBCO). In the spectrum, singly, doubly, triply, and quadruply charged ions of the 5'-DBCO-labeled oligonucleotide are identified at m / z 14520.3, 7265.0, 4840.6, and 3631.5, respectively. [Figure 14] 1 shows the results of MALDI-TOF mass spectrometry of HPLC-purified 5′-labeled ssDNA bearing an azide group. In the spectrum, the singly, doubly, triply, and quadruply charged ions of the 5′-azido-labeled oligonucleotide are identified at m / z 14158.0, 7081.1, 4718.3, and 3538.8, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following examples are used to illustrate the present disclosure. Those skilled in the art can easily conceive other advantages of the present disclosure based on the disclosure herein. It is clear that one or more embodiments can be implemented without specific details. The present disclosure can also be implemented or applied as described in various examples. For various purposes, the following examples can be modified or changed to implement the present disclosure without violating its scope. Titles or subtitles may be used in this disclosure for the convenience of readers, but they shall not affect the scope of the present disclosure.

[0026] In this disclosure, all terms, including descriptive terms or technical terms, used herein should be interpreted as having a meaning that is clear to those skilled in the art. However, terms may have various meanings in light of the intentions of those skilled in the art, precedents, or the emergence of new technology. In addition, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected term will be described in detail in the description of this disclosure. Therefore, the terms used herein are defined based on the meaning of the terms along with the explanation throughout this specification.

[0027] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, and biochemistry, which are well within the skill of the art. Such techniques are described in "Molecular Cloning: A Laboratory Manual," 2nd ed. (Sambrook et al., 1989), Cold Spring Harbor Press; "Oligonucleotide Synthesis" (M.J. Gait, 1984); "Methods in Molecular Biology," Humana Press; "Cell Biology: A Laboratory Notebook" (J.E. Cellis, ed., 1998), Academic Press; "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Introduction to Cell and Tissue Culture" (J.P. Mather and P.E. Roberts, 1998); "Cell and Tissue Culture: Laboratory Procedures" (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-98); "Methods in Enzymology" (Academic Press, Inc.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P. Calos, eds., 1987); "Current Protocols in "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); "Short Protocols in Molecular Biology" (Wiley and Sons, 1999), and other publications. Techniques particularly useful for particular embodiments are discussed in the following sections. Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present disclosure to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.All publications cited herein are incorporated by reference for the purpose or subject matter mentioned herein.

[0028] As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. The term "or" is used interchangeably with the term "and / or" unless the context clearly dictates otherwise.

[0029] Also, when an element "comprises" or "includes" a component or step, unless specifically stated to the contrary, the element may further include other components or steps, and not exclude others.

[0030] When ranges are given, the endpoints are included. Furthermore, unless otherwise stated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any specific value or subrange within the ranges set forth in various embodiments of the present disclosure, unless the context clearly dictates otherwise.

[0031] 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 an acceptable standard error of the mean, as considered by one of ordinary skill in the art. Unless expressly stated otherwise, all numerical ranges, amounts, values, and percentages of materials, amounts, periods, temperatures, operating conditions, ratios of amounts, and the like disclosed herein should be understood to be modified in all instances by the terms "about," "approximately," or "approximately."

[0032] As used herein, the term "derived from," when referring to a biological sample, indicates that the sample is obtained from the stated source at a certain point in time. For example, a biological sample derived from an organism may represent a primary biological sample obtained directly from that organism (i.e., unmodified), or may be modified, for example, by the introduction of a recombinant vector, by culturing under specific conditions, or by immortalization.

[0033] As used herein, the phrase "at least one" in connection with 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 every element listed within the list of elements, nor does it exclude any combination of elements in the list of elements. This definition also allows for elements other than those identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the identified elements, may optionally be present. 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") can refer in one embodiment to at least one A, optionally including more than one A but no B (and optionally including elements other than B); in another embodiment to at least one B, optionally including more than one B but no A (and optionally including elements other than A); and in yet another embodiment to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements).

[0034] As used herein, also referred to as apurinic / apyrimidinic (AP) sites be calledAn abasic site encompasses any chemical structure resulting from the removal of a base portion (including the entire base) by treating a nucleotide (present in a polynucleotide chain) with an agent capable of cleaving the base portion of a nucleotide, for example, with an agent (e.g., an enzyme, acidic conditions, or chemical reagent) capable of causing cleavage of the base portion of a nucleotide. In one embodiment, an AP site is a backbone of a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), that lacks a nucleobase, i.e., a deoxyribose in a DNA backbone or a ribose in an RNA backbone that is not covalently bound to a purine base, such as adenine (A) or guanine (G), or a pyrimidine base, such as cytosine (C), uracil (U), or thymine (T). An AP site can be present at both the 5'-end and 3'-end of a nucleic acid, or within the nucleotide sequence of a nucleic acid, at one end of the nucleic acid, such as the 5'-end or 3'-end.

[0035] As used herein, nucleic acid may be single-stranded, double-stranded, or a mixture of single- and double-stranded nucleic acids. A double-stranded nucleic acid may be a nucleic acid having at least a duplex region formed by two complementary strands of nucleic acid. For example, the nucleic acid may be a DNA molecule such as a plasmid, synthetic DNA, or viral DNA. In other embodiments, the nucleic acid may be an RNA molecule such as synthetic RNA, mRNA, tRNA, rRNA, and non-coding RNA. The term also encompasses analogs of either DNA or RNA made from nucleotide analogs, and, where applicable, single-stranded (sense or antisense) and double-stranded polynucleotides. The term further encompasses modified polynucleotides, including modified DNA and modified RNA, for example, DNA and RNA containing one or more non-natural nucleotides or nucleosides. The terms "nucleic acid" and "polynucleotide" may be used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. These terms encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which may be synthetic, naturally occurring, or non-naturally occurring, and / or which have similar chemical properties to the reference nucleic acid, and / or which are metabolized in a manner similar to the reference nucleotide. Unless otherwise specified, a particular nucleic acid sequence also encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. In some embodiments, nucleotides are linked via internucleotide linkages, such as, but not limited to, phosphate, borane phosphate, phosphorothioate, phosphodiester, phosphotriester, H-phosphonate, aminophosphonate, methylphosphonate, phosphonoacetate, sulfur phosphonoacetate, 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 "xenonucleotide" 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 xenonucleotides, i.e., xenonucleic acids (XNAs), include, but are not limited to, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), 1,5-anhydrohexitol nucleic acids (HNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), cyclohexene nucleic acids (CeNAs), and fluoro-arabinonucleic acids (FANAs).

[0036] Nucleic acids, as used herein, can be from 5 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 sonication, for uses as described herein.

[0037] In some embodiments, nucleic acids to be labeled by the disclosed methods can 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 other embodiments, the nucleic acids can be less than about 20, 25, 30, 35, 40, 50, 65, 75, 85, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides in length. It will be understood that the length of a nucleic acid can represent the average size in a population.

[0038] As used herein, glycosylase refers to an enzyme capable of excising the base portion of a nucleotide and creating an AP site in a nucleic acid, including N-glycosylases, also known as "DNA glycosylases" or "glycosidases," which specifically cleave dUTP and include, but are not limited to, uracil N-glycosylase (UNG), interchangeably referred to as "uracil DNA glycosylase" (UDG), hypoxanthine-N-glycosylase, hydroxymethylcytosine-N-glycosylase, 3-methyladenine DNA glycosylase, 3- or 7-methylguanine DNA glycosylase, hydroxymethyluracil DNA glycosylase, and T4 endonuclease V. Glycosylases cleave the base portion of a nucleotide in the middle of a nucleic acid or at either or both ends. As used herein, 5'-terminal glycosylases excise the base portion of a nucleotide at the 5' end of a nucleic acid.

[0039] As used herein, the term "exonuclease" refers to any wild-type or variant enzyme capable of cleaving the phosphodiester bond(s) linking the terminal nucleotides of an oligonucleotide or polynucleotide, such as a 5' to 3' exonuclease, a 3' to 5' exonuclease, and a poly(A)-specific 3' to 5' exonuclease. Non-limiting examples of exonucleases include exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VIII, Xm1, and Rat1.

[0040] As used herein, the term "5' to 3' exonuclease" refers to an exonuclease that cleaves a phosphodiester bond at the 5' end of an oligonucleotide or polynucleotide. Non-limiting examples of 5' to 3' exonucleases include T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, phage lambda exonuclease, 5'-exonuclease of DNA polymerase I, exonuclease VIII, RecJ, RecJf, Tth RecJ, Mpn These include 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.

[0041] As used herein, the term "3' end" generally refers to a region or position in a polynucleotide or oligonucleotide that is downstream from a 5' region or position in the same polynucleotide or oligonucleotide.

[0042] As used herein, the term "5' end" generally refers to a region or position in a polynucleotide or oligonucleotide that is upstream from a 3' region or position in the same polynucleotide or oligonucleotide.

[0043] As used herein, an aldehyde-reactive compound is a class of compounds that react with or form bonds with aldehyde groups. In one embodiment, the aldehyde-reactive compound is a compound having at least one primary amine in its structure, a hydrazide, an acylhydrazide, a compound having an aminooxy (-ONH) group, a compound having a naphthalene-containing aminooxy group, and / or a compound having a guanidine-containing aminooxy group.

[0044] As used herein, the term "label" (interchangeably referred to as a "detectable label" or "modification") refers to a chemical group or functional moiety associated or linked (interchangeably referred to as "labeling" or "modified") with a polynucleotide. Labeled polynucleotides can generally be detected directly or indirectly through a detectable signal. Detectable labels can be attached (or associated) directly or through a non-interfering linking group to another moiety capable of specifically associating with the site or sites to be labeled. Detectable labels may be associated by covalent or non-covalent bonds, and may be associated directly or indirectly.

[0045] As used herein, the term "monofunctional DNA glycosylase" refers to a naturally occurring monofunctional glycosylase that originally contains only DNA glycosylase activity. The term "monofunctional DNA glycosylase" may also refer to a monofunctional glycosylase derived from a bifunctional DNA glycosylase that naturally possesses both DNA glycosylase activity and abasic site lyase (AP lyase) activity by eliminating or inactivating the AP lyase domain of the bifunctional DNA glycosylase.

[0046] As used herein, the term "enzymatically active fragment" refers to a catalytically or enzymatically active fragment of a 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.

[0047] Processes for signal detection are known in the art. Signal detection can be visual or can utilize instruments appropriate for the label used, such as a spectrometer, fluorometer, luminometer, phosphorimager, Geiger counter, scintillation counter, or microscope. For example, if the label is a radioisotope, detection can be achieved using, for example, a scintillation counter or photographic film as in autoradiography. When a fluorescent label is used, detection can be achieved by exciting the fluorescent dye with light of the appropriate wavelength and detecting the emitted fluorescence, for example, by a fluorescence microscope, visual inspection, photographic film, a fluorometer, a luminometer, a charge-coupled device (CCD) camera, or 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 for horseradish peroxidase, such as o-phenylenediamine, give colored products. Instruments suitable for sensitive detection are known in the art. In other situations, signal amplification strategies may optionally be used to facilitate detection of low abundance molecular targets.

[0048] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Also intended are packages for use in combination with devices or systems. Optionally, the kits may provide additional components, such as buffers and interpretive information. In some embodiments, the present disclosure provides an article of manufacture containing the contents of the kits described herein. [Example]

[0049] Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed as limiting the scope of the present disclosure. The materials and methods used in the following examples are described in detail below. Materials used in this disclosure but not annotated herein are commercially available.

[0050] Example 1. 5'-end labeling of nucleic acids using aldehyde-reactive compounds A 45-mer single-stranded DNA (ssDNA, 5'- / deoxyU / CTCGGCCTGGCACAGGTCCGTCTCAGTGCTGCGGCGACCACCGA-3' (SEQ ID NO: 1)) containing a uracil residue at the 5' end and fluorescein (FAM) dye at the 3' end was synthesized. To perform uracil excision and subsequent labeling of the abasic sites, 100 nM of the uracil-containing 45-mer ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Micrococcus luteus (MluUDG) and 5 mM aldehyde-reactive probe (N-(aminooxyacetyl)-N'-biotinylhydrazine). The reaction was initiated by the addition of MluUDG and the aldehyde-reactive probe (ARP) for 15 min at 37 °C. 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 results were visualized by scanning the gel with an Amersham Typhoon Imager (Cytiva Life Sciences, Marlborough, MA, USA) and are shown in Figure 1. As shown in Figure 1, the addition of MluUDG and the aldehyde-reactive probe resulted in an additional band with a higher molecular weight in the gel image, indicating the presence of the labeled ssDNA product.

[0051] Similarly, in another example, partially double-stranded DNA molecules were labeled and analyzed using an aldehyde-reactive probe. The double-stranded DNA was prepared by annealing a 45-mer uracil-containing ssDNA (SEQ ID NO: 1) to a 15-mer complementary strand (5'-TGTGCCAGGCCGAGA-3' (SEQ ID NO: 2)) at a molar ratio of 1:1.5 in 1x Tris-EDTA (TE) buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98°C for 3 minutes in a thermal cycler, followed by gradual cooling to 4°C (e.g., 30-second intervals of 5°C). The resulting double-stranded DNA was subjected to uracil excision with MluUDG and subsequent labeling of the abasic sites, as described above in the steps and conditions for labeling ssDNA. The reaction was terminated by adding an equal volume of 2x quench solution, followed by denaturation at 95°C for 10 minutes. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon Imager and are shown in Figure 1. In Figure 1, addition of MluUDG and an aldehyde-reactive probe to the double-stranded DNA produced an additional band with a higher molecular weight in the gel, indicating the presence of the labeled DNA product.

[0052] Thus, both single-stranded and double-stranded DNA could be labeled with aldehyde-reactive probes by the provided method.

[0053] Example 2. 5'-end labeling of nucleic acids using aminooxy-5(6)-FAM A 45-mer single-stranded DNA (ssDNA, SEQ ID NO: 1) containing a uracil residue at the 5' end and cyanine 5 (Cy5) dye at the 3' end was synthesized. To perform uracil excision and subsequent labeling of the abasic sites, 100 nM ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Micrococcus luteus (MluUDG) and 2 mM aminooxy-5(6)-FAM. The reaction was initiated by the addition of MluUDG and aminooxy-5(6)-FAM and incubated at 37°C for 60 minutes. The reaction was terminated by the addition of an equal volume of 2x quench 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 20% urea-PAGE. Results were visualized by scanning the gel with an Amersham Typhoon Imager. As shown in Figure 2, addition of MluUDG and aminooxy-5(6)-FAM resulted in an additional band with a higher molecular weight in the gel, indicating the presence of the labeled FAM-ssDNA product.

[0054] Similarly, in another example, partially double-stranded DNA molecules were labeled with aminooxy-5(6)-FAM and analyzed. The double-stranded DNA was prepared by annealing a 45-mer uracil-containing ssDNA (SEQ ID NO: 1) to a 15-mer complementary strand (5'-TGTGCCAGGCCGAGA-3' (SEQ ID NO: 2)) at a molar ratio of 1:1.5 in 1x TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98°C for 3 minutes in a thermal cycler, followed by gradual cooling to 4°C (e.g., 30-second intervals of 5°C). The resulting double-stranded DNA was subjected to uracil excision with MluUDG and subsequent labeling of the abasic sites, as described above in the steps and conditions for labeling ssDNA. The reaction was terminated, and the reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon Imager. As also shown in Figure 2, the addition of MluUDG and aminooxy-5(6)-FAM to the double-stranded DNA produced an additional band with a higher molecular weight in the gel, indicating the presence of the labeled FAM-DNA product.

[0055] Thus, both single-stranded and double-stranded DNA were shown to be labeled with aminooxy-5(6)-FAM by the provided method.

[0056] Example 3. 5'-end labeling of DNA with naphthalene and guanidine-containing aminooxy-FAM A 47-mer single-stranded DNA (ssDNA, 5'- / deoxyU / CTCGGCCTGGCACAGGTCCGTCTCAGTGCTGCGGCGACCACCGAGG-3' (SEQ ID NO: 3)) containing a uracil residue at the 5' end was synthesized. To perform uracil excision and subsequent labeling of the abasic sites, 100 nM ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Micrococcus luteus (MluUDG) and 2 mM naphthalene- and guanidine-containing aminooxy-FAM. The reaction was initiated by the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAM and incubated at 37°C for 60 min. The reaction was terminated by the addition of an equal volume of 2x quench solution (30 mM EDTA and 95% (v / v) deionized formamide), followed by denaturation at 95°C for 10 min. The reaction products were analyzed by 20% urea-PAGE. Results were visualized by scanning the gel with an Amersham Typhoon Imager. As shown in Figure 3, addition of MluUDG and the naphthalene- and guanidine-containing aminooxy-FAM produced an additional band with a higher molecular weight in the gel, indicating the presence of the labeled FAM-ssDNA product.

[0057] Similarly, in another example, partially double-stranded DNA molecules were also labeled and analyzed using naphthalene- and guanidine-containing aminooxy-FAM. Double-stranded DNA was prepared by annealing a 47-mer uracil-containing ssDNA (SEQ ID NO: 3) to a 15-mer complementary strand (SEQ ID NO: 2) at a molar ratio of 1:1.5 in 1x TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98°C for 3 minutes in a thermal cycler, followed by gradual cooling to 4°C (e.g., 5°C intervals for 30 seconds). The resulting double-stranded DNA was subjected to uracil excision and subsequent labeling of abasic sites, as described above in the steps and conditions for labeling ssDNA. The reaction was terminated, and the reaction products were analyzed by 20% urea-PAGE. Results were visualized by scanning the gel with an Amersham Typhoon Imager. Also shown in Figure 3, addition of MluUDG and the 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 product.

[0058] Thus, both single-stranded and double-stranded DNA could be labeled with naphthalene- and guanidine-containing aminooxy-FAM by the provided method.

[0059] Example 4. 5'-End Labeling of 5'-Phosphorylated DNA with Aldehyde-Reactive Compounds and Concentration of Labeled DNA Using Phage Lambda Exonuclease A 45-mer single-stranded DNA (ssDNA, SEQ ID NO: 1) containing a uracil residue at the 5' end and fluorescein (FAM) at the 3' end was synthesized. The DNA was first 5'-phosphorylated with T4 polynucleotide kinase in the presence of adenosine triphosphate (ATP) for 10 min at 37 °C. To perform uracil excision and subsequent labeling of abasic sites, 100 nM 5'-phosphorylated ssDNA was mixed with 100 ng of uracil-DNA glycosylase from Micrococcus luteus (MluUDG) and 5 mM aldehyde-reactive probe (N-(aminooxyacetyl)-N'-biotinylhydrazine). The reaction was initiated by the addition of MluUDG and the aldehyde-reactive probe simultaneously for 15 min at 37 °C. To eliminate unlabeled ssDNA, 2 units of phage lambda exonuclease were added, followed by an additional 3.5 h of incubation at 37 °C. The reaction was stopped by adding an equal volume of 2x quench 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 20% urea-PAGE. Results were visualized by scanning the gel with an Amersham Typhoon Imager. As shown in Figure 4, addition of MluUDG and an 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 phage lambda exonuclease to degrade unlabeled DNA (i.e., a cleanup step), the portion of the labeled ssDNA product was further enriched.

[0060] Thus, DNA can be labeled with an aldehyde-reactive probe by the provided method, and the labeled DNA fraction can be further enriched by treatment with phage lambda exonuclease.

[0061] Example 5. 5'-End Labeling of 5'-Phosphorylated DNA with Naphthalene and Guanidine-Containing Aminooxy-FAM and Purification and Concentration of FAM-Labeled DNA Using Phage Lambda Exonuclease A 47-mer single-stranded DNA (ssDNA, SEQ ID NO: 3) containing a uracil residue at the 5' end was synthesized. The DNA was first 5'-phosphorylated with T4 polynucleotide kinase in the presence of adenosine triphosphate (ATP) at 37°C for 30 minutes. To perform uracil excision and subsequent labeling of the abasic sites, 100 nM ssDNA was mixed with 115 ng of uracil-DNA glycosylase from Micrococcus luteus (MluUDG) and 1 mM naphthalene- and guanidine-containing aminooxy-FAM. The reaction was initiated by the addition of MluUDG and naphthalene- and guanidine-containing aminooxy-FAM and incubated at 37°C for 30 minutes. To eliminate unlabeled ssDNA, 2 units of phage lambda exonuclease were added, followed by an additional 30-minute incubation at 37°C. The reaction was stopped by adding an equal volume of 2x quench 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 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon Imager. As shown in Figure 5, the addition of MluUDG and the naphthalene- and guanidine-containing aminooxy-FAM produced an additional band with a higher molecular weight in the gel, indicating the presence of the FAM-labeled ssDNA product. When the reaction mixture was further treated with phage lambda exonuclease to degrade unlabeled DNA, the FAM-labeled ssDNA product was enriched.

[0062] Similarly, in another example, 5'-phosphorylated double-stranded DNA molecules were also labeled with naphthalene and guanidine-containing aminooxy-FAM, followed by phage lambda exonuclease treatment to enrich for FAM-labeled double-stranded DNA. Double-stranded DNA was prepared by annealing a 47-mer uracil-containing ssDNA (SEQ ID NO: 3) to a 15-mer complementary strand (SEQ ID NO: 2) at a molar ratio of 1:1.5 in 1x TE buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. The DNA annealing reaction was carried out by heating the DNA mixture to 98°C for 3 minutes in a thermal cycler, followed by gradual cooling to 4°C (e.g., 5°C intervals for 30 seconds). The resulting double-stranded DNA was first 5'-terminally phosphorylated with T4 polynucleotide kinase in the presence of ATP, then subjected to uracil excision and subsequent labeling of abasic sites, as described above in the steps and conditions for labeling ssDNA, followed by phage lambda exonuclease treatment. The reaction products were analyzed by 20% urea-PAGE. The results were visualized by scanning the gel with an Amersham Typhoon Imager. As shown in Figure 5, the addition of MluUDG and the naphthalene- and guanidine-containing aminooxy-FAM to the double-stranded DNA produced an additional band with a higher molecular weight in the gel, indicating the presence of the FAM-labeled DNA product. When the reaction mixture was further treated with phage lambda exonuclease to degrade unlabeled DNA, the FAM-labeled DNA product was enriched.

[0063] Thus, both single-stranded and double-stranded DNA can be labeled with the naphthalene- and guanidine-containing aminooxy-FAM by the provided method, and the labeled DNA fraction can be further enriched by treatment with phage lambda exonuclease.

[0064] Example 6. 5'-end labeling of ssDNA with fluorescent dyes, biotin moieties, azide, or dibenzylcyclooctyne (DBCO) functional groups 5'-end labeling or modification of a 45-mer single-stranded DNA (ssDNA, SEQ ID NO: 1) containing a uracil residue at the 5' end was performed using a reaction buffer containing 1 mM Tris-HCl (pH 8.0), 5 mM NaCl, 10 μM EDTA, and 0.02% PEG 4000, with 100 nM ssDNA, 1 μM uracil-DNA glycosylase, and 200 μM PEG-TAMRA (5-carboxytetramethylrhodamine), aminooxy-PEG-Cy3 (cyanine 3) dye, aminooxy-PEG-Cy5 (cyanine 5) dye, aminooxy-PEG-FAM (fluorescein amidite) dye, ARP (aldehyde-reactive probe, N-(aminooxyacetyl)-N'-biotinylhydrazine), aminooxy-PEG-azide, and aminooxy-PEG-dibenzylcyclooctyne (DBCO). This was performed with the desired molecule to be added to the 5' end of the ssDNA.

[0065] The DNA labeling reaction was incubated at 37°C for 60 minutes and then stopped by adding an equal volume (10 μL) of 2× quenching solution (30 mM EDTA and 95% deionized formamide). The 5′-end labeled ssDNA reaction products were analyzed by 20% polyacrylamide gel electrophoresis (PAGE) containing 8 M urea. The gel was first stained with 1× SYBR Gold Nucleic Acid Gel Stain solution (Waltham, MA, USA) and visualized by scanning the gel with an Amersham Typhoon Biomolecular Imager (Marlborough, MA, USA).

[0066] The results of 5'-end labeling of ssDNA with fluorescent dyes or biotin moieties are shown in Figure 6, where lane S indicates the position of unlabeled ssDNA, and lanes 1 to 5 indicate the higher electrophoretic positions of the 5'-end labeled products of ssDNA with 5-TAMRA (5-carboxytetramethylrhodamine), Cy3 (cyanine 3) dye, Cy5 (cyanine 5) dye, FAM (fluorescein) dye, and biotin moiety (ARP), respectively.

[0067] The results of 5′-end labeling of ssDNA with azide or DBCO are shown in Figure 7, where lane 1 shows the ssDNA substrate before modification, lane 2 shows the 5′-abasic ssDNA created by enzymatic excision, and lanes 3 and 4 show the ssDNA samples modified with DBCO and azide, respectively.

[0068] Example 7. 5'-end labeling of ssDNA with a maleimide group 5'-end labeling of a 45-mer single-stranded DNA (ssDNA, SEQ ID NO: 1) with a maleimide group was first carried out using 100 nM ssDNA, 1 μM uracil-DNA glycosylase, 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% PEG-4000. The DNA labeling reaction was carried out at 37°C for 60 min and then terminated by the addition of an equal volume (10 μL) of 2x quench solution. The 5'-azide-labeled ssDNA was purified using a QIAquick Nucleotide Removal Kit (Qiagen, Waltham, MA, USA). To label the maleimide group, the purified 5'-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 stopped by adding an equal volume (10 μL) of 2× quenching solution. The 5′-labeled ssDNA reaction products were analyzed by 20% polyacrylamide gel electrophoresis (PAGE) containing 8 M urea. The gel was first stained with 1× SYBR Gold Nucleic Acid Gel Stain solution (ThermoFisher Scientific, Waltham, MA, USA) and visualized by scanning the gel with an Amersham Typhoon Biomolecular Imager (Marlborough, MA, USA).

[0069] The results of 5'-end labeling of ssDNA with a maleimide group are shown in Figure 8, where lane 1 shows the ssDNA substrate before modification and lane 2 shows the ssDNA sample labeled with a maleimide group.

[0070] Example 8. Purification of 5'-end-labeled ssDNA by ion-pair reversed-phase high-performance liquid chromatography (IPRP-HPLC) 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'-labeled ssDNA was first cleaned up using a QIAquick Nucleotide Removal Kit (Qiagen, Waltham, MA, USA). The cleaned-up sample was then purified on an AdvanceBio Oligonucleotide (2.1 x 50 mm) column using an Agilent 1260 Infinity II Bio-Inert LC System (Santa Clara, CA, USA). The desired 5'-labeled ssDNA was eluted from the column using a varying gradient of mobile phase formed by mixing two separate components: solvent A (50 mM TEAA dissolved in deionized water) and solvent B (100 mM TEAA dissolved in acetonitrile). The mobile phase gradient was run according to the following program: increasing the solvent B concentration from 8% to 15% from 0 to 9 min; increasing the solvent B concentration from 15% to 30% from 9 to 15 min; and decreasing the solvent B concentration from 30% to 8% from 15 to 18 min. The column flow rate was maintained at 0.6 mL / min, and the column temperature was kept at 65°C. The sample injection volume ranged from 10 μL to 20 μL. Sample fractions were monitored by absorbance values ​​at both 260 nm and 498 nm wavelengths with peak widths greater than 0.05 min (5 Hz). Column fractions containing 5′-labeled ssDNA were pooled together and further concentrated using a centrifugal vacuum concentrator. The results, shown in Figures 9 to 11, demonstrate that the 5′-end labeling method of the present disclosure yields highly pure 5′-end-labeled ssDNA products using FAM, DBCO, and azide, respectively.

[0071] Example 9. Analysis of 5'-labeled ssDNA by MALDI-TOF mass spectrometry To further confirm the identity of the 5'-end-labeled product from the above reaction, the HPLC-purified 5'-end-labeled ssDNA was further analyzed by MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry. For sample preparation, the purified and dried 5'-labeled ssDNA was reconstituted in deionized water. After confirming that the instrument's vacuum level had reached a certain threshold and performing mass calibration, 1 μL of matrix solution was dispensed onto an anchor chip plate (Bruker Daltonics, Billerica, MA, USA). The matrix solution was prepared by combining 200 μL of 50 mg / mL 3-hydroxypicolinic acid (3-HPA) solution with 10 μL of 100 mg / mL diammonium hydrogen citrate (DAC) solution, followed by dilution with 790 μL of deionized water to achieve a final concentration of 4 mg / mL 3-HPA and 0.2 mg / mL DAC. The plate was allowed to air dry at room temperature. Subsequently, 1 μL of the 5′-labeled ssDNA sample was applied to the matrix spot and allowed to air-dry 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 set to operate in positive ion linear mode, allowing for precise analysis of molecular ions. The selected mass range was set from 2,000 to 18,000 to cover a wide range of molecular mass spectra. The results, shown in Figures 12 to 14, demonstrate that the disclosed 5′-end labeling method yields 5′-end labeled ssDNA products of high purity and accurate mass using FAM, DBCO, and azide, respectively.

[0072] Although some embodiments of the present disclosure have been described in detail, those skilled in the art may make various modifications and variations to the illustrated embodiments without substantially departing from the teachings of the present disclosure, and such modifications and variations are encompassed within the scope of the present disclosure as set forth in the appended claims.

Claims

1. A kit for 5'-end labeling of nucleic acids, comprising: a 5'-terminal glycosylase configured to generate an intermediate having an abasic site at the 5'-terminus; an aldehyde-reactive compound carrying a detectable label and configured to couple with the intermediate at the abasic site to form a labeled nucleic acid having the detectable label attached to its 5' end; and a 5' to 3' exonuclease configured to remove unlabeled nucleic acid and said intermediate.

2. The 5' to 3' exonuclease is selected from the group consisting of T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, phage lambda exonuclease, exonuclease VIII, RecJ, RecJf, Tth RecJ, and 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 2. The kit of claim 1, wherein the antibody is selected from the group consisting of gp19, bacterial AddB, and any combination thereof.

3. 2. The kit of claim 1, wherein the 5' to 3' exonuclease comprises the 5'-exonuclease domain of DNA polymerase I.

4. 2. The kit of claim 1, wherein the 5'-terminal glycosylase is selected from the group consisting of uracil-DNA glycosylase, alkyladenine DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase 1, methyl-binding domain glycosylase 4, thymine DNA glycosylase, MutY homolog DNA glycosylase, alkylpurine glycosylase C, alkylpurine glycosylase D, 8-oxo-guanine glycosylase 1 without abasic site lyase activity, endonuclease III-like glycosylase 1 without abasic site lyase activity, endonuclease VIII-like glycosylase 1 without abasic site lyase activity, endonuclease VIII-like glycosylase 2 without abasic site lyase activity, endonuclease VIII-like glycosylase 3 without abasic site lyase activity, enzymatically active fragments thereof, and any combination thereof.

5. 5. The kit of claim 4, wherein the uracil-DNA glycosylase is derived from the family Micrococcaceae, Staphylococcusae, or Caryophanaceae.

6. 2. The kit of claim 1, wherein the detectable label is selected from the group consisting of an azide, an alkyne, a bicyclononyne, a dibenzocyclooctyne, a maleimide, a peptide, a protein, an antibody, a dendrimer, biotin, a radioisotope, a photochromic dye, a fluorescent dye, a luminescent dye, and any combination thereof.

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

8. 2. The kit 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, aminooxy-biotin, naphthalene-containing aminooxy-fluorescent dye, guanidine-containing aminooxy-fluorescent dye, fluorescent dye hydrazide, or maleimide.

9. 1. A method for 5′-end labeling of a nucleic acid, comprising: providing a target nucleic acid to be labeled; providing a 5'-terminal glycosylase and reacting it with the target nucleic acid to produce an intermediate having an abasic site at the 5'-terminus of the target nucleic acid; providing an aldehyde-reactive compound bearing a detectable label for coupling with the intermediate at the abasic site to form a labeled nucleic acid having the detectable label attached to the 5' terminus; and providing a 5' to 3' exonuclease to remove both the unlabeled nucleic acid and the intermediate; A method comprising:

10. 10. The method of claim 9, wherein the 5'-terminal glycosylase is selected from the group consisting of uracil-DNA glycosylase, alkyladenine DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase 1, methyl-binding domain glycosylase 4, thymine DNA glycosylase, MutY homolog DNA glycosylase, alkylpurine glycosylase C, alkylpurine glycosylase D, 8-oxo-guanine glycosylase 1 without abasic site lyase activity, endonuclease III-like glycosylase 1 without abasic site lyase activity, endonuclease VIII-like glycosylase 1 without abasic site lyase activity, endonuclease VIII-like glycosylase 2 without abasic site lyase activity, endonuclease VIII-like glycosylase 3 without abasic site lyase activity, enzymatically active fragments thereof, and any combination thereof.

11. 10. The method of claim 9, 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, aminooxy-biotin, naphthalene-containing aminooxy-fluorescent dye, guanidine-containing aminooxy-fluorescent dye, or fluorescent dye hydrazide.

12. The 5' to 3' exonuclease is selected from the group consisting of T5 exonuclease, T7 exonuclease, bacterial alkaline exonuclease, viral alkaline exonuclease, phage 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 10. The method of claim 9, wherein the antibody is selected from the group consisting of gp19, bacterial AddB, and any combination thereof.

13. 10. The method of claim 9, wherein the target nucleic acid is single-stranded or comprises at least a double-stranded region formed by two complementary strands of nucleic acid.

14. 10. The method of claim 9, wherein the target nucleic acid is DNA or RNA.

15. The method of claim 9 , wherein the target nucleic acid is synthesized de novo or derived from an organism.

16. 10. The method of claim 9, wherein the target nucleic acid comprises a uracil residue at the 5' end.

17. A system for 5'-end labeling of nucleic acids, comprising a reaction reservoir, a liquid handling device, a temperature control unit, and a time control unit, the liquid treatment device is configured to transport a 5'-end glycosylase and an aldehyde-reactive compound to react with the nucleic acid in the reaction reservoir for a certain period of time at a predetermined temperature controlled by the temperature control unit, the 5'-end glycosylase is configured to generate an intermediate having an abasic site at the 5'-end of the nucleic acid, and the aldehyde-reactive compound has a detectable label and is configured to couple with the intermediate at the abasic site to form a labeled nucleic acid having the detectable label attached to its 5'-end; The liquid handling device is configured to deliver a 5' to 3' exonuclease to the reaction reservoir for removing unlabeled nucleic acids and the intermediates.

Images