Sequence specific cleavage of single-stranded DNA

Abstract

The disclosure relates to ssDNA cleavage using restriction enzymes, as well as its use for sequence specific hydrolysis of oligonucleotides prior to LC-MS / MS analysis resulting in formation of shorter oligonucleotide sequences to enable high sequence coverage by MS / MS fragmentation.

Description

[0001] SEQUENCE SPECIFIC CLEAVAGE OF SINGLE-STRANDED DNA FIELD OF THE INVENTION

[0002] The present invention relates to methods for sequence specific cleavage of single-stranded DNA (ssDNA) using restriction enzymes, for example for verifying a DNA sequence using mass spectrometry.

[0003] BACKGROUND

[0004] The interest in nucleic acid products has increased in recent years, e.g. as diagnostic tools, but also as therapeutics. Their utility was recognized during the latest pandemic caused by SARS-CoV-2, demonstrating that mutations can emerge quickly and, consequently, that tailored medicinal and diagnostic products can become critical. In order to support the development and to ensure the highest quality for these products, their sequence, i.e. the relative position of every incorporated nucleotide, can be confirmed.

[0005] Mass spectrometry (MS) is an analytical technique and allows the analysis of the mass of analyzed molecules or molecular fragments. Mass spectrometry generally has three main purposes: 1. It can identify compounds by their mass-to-charge ratio; 2. It can find out how much there is of a specific compound; and 3. It can help to understand the structure and chemical properties of molecules.

[0006] Nucleic acids can be analyzed with liquid chromatography-mass spectrometry (LC-MS). However, this static technique is limited to intact mass analysis, where the correct sequence of nucleotides is not assessed. The analysis with liquid chromatography-tandem mass spectrometry (LC-MS / MS) by fragmentation of oligonucleotides could deliver information on the sequence context. However, currently, the sequencing of oligonucleotides with LC-MS / MS is restricted to products of shorter length (<35). The fragmentation of longer ssDNA oligonucleotides has not yet been successful, as they have not fragmented in a conclusive way for sequence conformation.

[0007] The characterization of ssDNA oligonucleotides may help confirming the sequence and hence the product quality. It can help analyzing nucleic acid products that are already on the market and drive the development, production and application of new nucleic acid products.

[0008] Restriction enzymes, also termed restriction endonucleases, are enzymes that are capable of cleaving DNA molecules at or near specific recognition motifs within the DNA molecules. Cleavage is achieved by hydrolysis of the sugar-phosphate backbone of the DNA molecule. To cleave double-stranded DNA (dsDNA), restriction enzymes make two incisions, one for each sugar-phosphate backbone of the two strands. Restriction enzymes can be classified into five types (Type I, II, III, IV, and V, respectively), which differ in their structure and whether they cut their DNA substrate at their recognition motif, or if the recognition and cleavage sites are separate from one another. For instance, Type II enzymes (EC 3.1.21.4) cleave within or at short specific distances from a recognition motif.

[0009] Restriction enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. More than 3,600 restriction endonucleases are known which represent over 250 different specificities. Over 3,000 of these have been studied in detail, and more than 800 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a vital tool in molecular cloning.

[0010] Restriction enzymes are commonly known to site-specifically cleave dsDNA. However, there have also been reports on a few restriction enzymes to be active on ssDNA. For example, Nishigaki et al. (Nucleic Acid Research, 1985, 13(16): 5747-5760) reported 13 restriction enzymes to cut ssDNA.

[0011] There have been attempts to perform sequence specific hydrolysis prior to LC-MS / MS analysis for RNA using certain sequence specific ribonucleases on RNA (see e.g. Jiang et al., Anal. Chem., 2019, 91: 8500-8506). However, to the best of our knowledge, the combination of sequence specific hydrolysis of ssDNA, e.g. by using restriction enzymes, and LC-MS / MS analysis for sequence confirmation has not yet been described. This novel approach may provide a powerful tool, enabling full characterization of ssDNA oligonucleotides and may be of great importance for diagnostic and medical advances. However, the known repertoire of means for sequence specific hydrolysis of ssDNA oligonucleotides prior to LC-MS / MS analysis to form shorter oligonucleotide sequences and thereby enable high sequence coverage by MS / MS fragmentation is limited. To enable flexible analysis of ssDNA, e.g. by LC-MS / MS, there is thus a need for further means for sequence specific ssDNA cleavage.

[0012] SUMMARY OF THE INVENTION

[0013] Facing the limited repertoire of means for sequence specific hydrolysis of ssDNA oligonucleotides prior to LC-MS / MS analysis resulting in formation of shorter oligonucleotide sequences to enable high sequence coverage by MS / MS fragmentation.

[0014] To our knowledge, there have been no attempts yet in combining the sequence specific hydrolysis of ssDNA and subsequent LC-MS / MS analysis for sequence conformation, possibly due to the insufficient repertoire of enzymes with known specific cleavage of ssDNA.

[0015] Restriction enzymes are commonly known to perform sequence specific hydrolysis on doublestranded DNA (dsDNA). A few Type II enzymes have been described to cleave also ssDNA (Nishigaki et al., supra). However, many of these enzymes bind to the same recognition motifs on the oligonucleotide substrate, limiting the generic and broad applicability of ssDNA cleavage using restriction enzymes.

[0016] In pursuit of new ways of ssDNA analysis, the present application shows validation of the activity of the reported enzymes on ssDNA and testing of numerous other restriction enzymes on ssDNA. As a result, a number of additional enzymes could be shown to cleave ssDNA as well that were previously be thought to be active on dsDNA only. Thereby, the number of possible cleavage positions could be improved, enabling a more broadly applicable approach for specific cleavage of ssDNA, e.g. in LC-MS / MS analysis of ssDNA.

[0017] Moreover, the use of more than one restriction enzyme was investigated in a “one pot reaction” to find out if all expected oligonucleotides of shorter length were formed according to the recognition motifs of the utilized restriction enzymes.

[0018] The restriction enzymes newly recognized to cleave ssDNA offer a sophisticated way to analyze the sequence of ssDNA products with LC-MS / MS, and other applications.

[0019] Exemplary embodiments of the disclosure are as follows:

[0020] [1] A method for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule, comprising incubating a sample comprising the ssDNA molecule with a restriction enzyme capable of cleaving the ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0021] [2] The method of [1], wherein the ssDNA molecule comprises a recognition motif for the restriction enzyme.

[0022] [3] The method of [1] or [2], wherein the restriction enzyme has a sequence selected from SEQ ID NOs: 1-6.

[0023] [4] The method of any one of

[0001] -[3], wherein the sample comprising the ssDNA molecule is incubated with two or more different restriction enzymes.

[0024] [5] The method of [4], wherein the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0025] [6] The method of [4] or [5], wherein the ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes. [7] The method of any one of [l]-[6], wherein the sample comprises two or more different ssDNA molecules.

[0026] [8] The method of [7], wherein at least one of the two or more different ssDNA molecules comprises a recognition motif for the restriction enzyme, or, when the sample is incubated with two or more different restriction enzymes, at least one of the two or more different ssDNA molecules comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0027] [9] The method of any one of [l]-[8], wherein at least 80% of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0028]

[0010] The method of any one of [l]-[9], wherein at least 90%, at least 95% or at least 99%, of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0029]

[0011] A method for analyzing a single-stranded DNA (ssDNA) molecule, comprising the steps:

[0030] (a) incubating a sample comprising the ssDNA molecule with a restriction enzyme capable of cleaving the ssDNA molecule, thereby sequence-specifically cleaving the ssDNA molecule; and

[0031] (b) analyzing the cleavage products of step (a) by mass spectrometry.

[0032]

[0012] The method of

[0011] , wherein the restriction enzyme is a Type II restriction enzyme.

[0033]

[0013] The method of

[0011] or

[0012] , wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I, wherein optionally the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0034]

[0014] The method of any one of

[0011] -

[0013] , wherein the ssDNA molecule comprises a recognition motif for the restriction enzyme.

[0035]

[0015] The method of any one of

[0011] -

[0014] , wherein the restriction enzyme has a sequence selected from SEQ IDNOs: 1-6.

[0016] The method of any one of

[0011] -

[0015] , wherein the sample comprising the ssDNA molecule is incubated with two or more different restriction enzymes.

[0036]

[0017] The method of

[0016] , wherein the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I, wherein optionally the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0037]

[0018] The method of

[0016] or

[0017] , wherein the ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0038]

[0019] The method of any one of

[0011] -

[0018] , wherein the sample comprises two or more different ssDNA molecules.

[0039]

[0020] The method of

[0019] , wherein at least one of the two or more different ssDNA molecules comprises a recognition motif for the restriction enzyme, or, when the sample is incubated with two or more different restriction enzymes, at least one of the two or more different ssDNA molecules comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0040]

[0021] The method of any one of

[0011] -

[0020] , wherein at least 80% of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I, such as a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0041]

[0022] The method of any one of

[0011] -

[0021] , wherein at least 90%, at least 95% or at least 99%, of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I, such as a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0042]

[0023] The method of any one of

[0011] -

[0022] , wherein the ssDNA molecule is subjected to liquid chromatography with tandem mass spectrometry (LC-MS / MS) in step (b).

[0024] The method of any one of

[0011] -

[0023] , wherein step (b) comprises acquiring a measured mass distribution for the cleavage products of step (a) by mass spectrometry, and comparing the measured mass distribution to the predicted mass distribution of the cleavage products.

[0043]

[0025] The method of any one of

[0011] -

[0024] , wherein step (b) comprises acquiring a measured mass for at least one cleavage product of step (a) by mass spectrometry, and comparing the measured mass to the predicted mass of the at least one cleavage product, whereby optionally at least a part of the nucleic acid sequence of the ssDNA molecule is verified.

[0044]

[0026] A use of a restriction enzyme for sequence-specifically cleaving a single-stranded DNA (ssDNA) molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0045]

[0027] A kit for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule, comprising:

[0046] (a) a first ssDNA molecule; and

[0047] (b) a restriction enzyme capable of cleaving the first ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0048]

[0028] The kit of

[0027] , wherein the first ssDNA molecule comprises a recognition motif for the restriction enzyme.

[0049]

[0029] The kit of

[0027] or

[0028] , wherein the restriction enzyme has a sequence selected from SEQ ID NOs: 1-6.

[0050]

[0030] The kit of any one of

[0027] -

[0029] , comprising two or more different restriction enzymes.

[0051]

[0031] The kit of

[0030] , wherein the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0052]

[0032] The kit of

[0030] or

[0031] , wherein the two or more different restriction enzymes are provided as a mixture.

[0053]

[0033] The kit of any one of

[0030] -

[0032] , wherein the first ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0054]

[0034] The kit of any one of

[0027] -

[0033] , further comprising (c) a second ssDNA molecule different from the first ssDNA molecule.

[0055]

[0035] The kit of

[0034] , wherein the second ssDNA molecule comprises a recognition motif for a restriction enzyme, optionally selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0056]

[0036] The kit of

[0034] or

[0035] , wherein the first ssDNA molecule and the second ssDNA molecule each comprise a recognition motif for a restriction enzyme, wherein the recognition motifs are different from one another.

[0057]

[0037] The kit of any one of

[0034] -

[0036] , wherein the first ssDNA molecule and the second ssDNA molecule each comprise only one recognition motif for a restriction enzyme.

[0058]

[0038] The kit of any one of

[0034] -

[0037] , wherein the second ssDNA molecule does not comprise a recognition motif for a restriction enzyme that is comprised in the first ssDNA molecule.

[0059]

[0039] A composition for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule comprising:

[0060] (a) a first ssDNA molecule; and

[0061] (b) a restriction enzyme capable of cleaving the first ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0062]

[0040] The composition of

[0039] , wherein the first ssDNA molecule comprises a recognition motif for the restriction enzyme.

[0063]

[0041] The composition of

[0039] or

[0040] , wherein the restriction enzyme has a sequence selected from SEQ IDNOs: 1-6.

[0064]

[0042] The composition of any one of

[0039] -

[0041] , comprising two or more different restriction enzymes.

[0065]

[0043] The composition of

[0042] , wherein the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0066]

[0044] The composition of

[0042] or

[0043] , wherein the first ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0067]

[0045] The composition of any one of

[0039] -

[0044] , further comprising (c) a second ssDNA molecule different from the first ssDNA molecule.

[0068]

[0046] The composition of

[0045] , wherein the second ssDNA molecule comprises a recognition motif for a restriction enzyme, optionally selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0069] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 : Diode array detector (DAD) chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Haelll.

[0070] FIG. 2: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Mspl.

[0071] FIG. 3 : DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Mbol.

[0072] FIG. 4: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Alul.

[0073] FIG. 5 : DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Xmil.

[0074] FIG. 6: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by EcoRI.

[0075] FIG. 7 : DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Rsal.

[0076] FIG. 8: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Bshl236I.

[0077] FIG. 9: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Csp6I.

[0078] FIG. 10: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by TaqI.

[0079] FIG. 11: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Mval. FIG. 12: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Tail.

[0080] FIG. 13: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by FspBI.

[0081] FIG. 14: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Eco47I.

[0082] FIG. 15: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Tasl.

[0083] FIG. 16: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Hint!.

[0084] FIG. 17: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by CviKI-1.

[0085] FIG. 18: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Fatl.

[0086] FIG. 19: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Nt.CVIPII.

[0087] FIG. 20: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Ssil.

[0088] FIG. 21: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Hin6I.

[0089] FIG. 22: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Beni.

[0090] FIG. 23: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule by Hhal.

[0091] FIG. 24: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 7) by Haelll and Bshl236I.

[0092] FIG. 25: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 15) by MspI and Tail. FIG. 26: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 10) by EcoRI and Csp6I.

[0093] FIG. 27: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 12) by Rsal and Csp6I.

[0094] FIG. 28: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 14) by Alul and TaqI.

[0095] FIG. 29: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 8) by Mspl and Mval.

[0096] FIG. 30: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 13) by Xmil and EcoRI.

[0097] FIG. 31: DAD chromatogram (256 nm) of results obtained from digestion of target ssDNA molecule (SEQ ID NO: 9) by Mbol, Alul, Xmil and Mval.

[0098] FIG. 32: DAD chromatogram (256 nm) of results obtained from digestion of two target ssDNA molecules (SEQ ID NOs: 10 and 12) by Csp6I.

[0099] FIG. 33: DAD chromatogram (256 nm) of results obtained from digestion of two target ssDNA molecules (SEQ ID NOs: 8 and 10) by Mval.

[0100] FIG. 34: DAD chromatogram (256 nm) of results obtained from digestion of two target ssDNA molecules (SEQ ID NOs: 7 and 8) by Hae III and Mspl.

[0101] FIG. 35: DAD chromatogram (256 nm) of results obtained from digestion of two target ssDNA molecules (SEQ ID NOs: 10 and 13) by EcoRI and Xmil.

[0102] DETAILED DESCRIPTION OF THE INVENTION

[0103] The disclosure will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0104] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton (Singleton et al., Dictionary of microbiology and molecular biology, 2nd ed., 1994, John Wiley and Sons, New York), Hale (Hale and Marham, The Harper Collins dictionary of biology, 1991, Harper Perennial, NY) and Walker (Walker and Cox, The Language of Biotechnology: A Dictionary of Terms. 1988, American Chemical Society, Washington, D.C. ISBN-0-8412-1499-1) provide one of skill with a general dictionary of many of the terms used in this invention. Practitioners are particularly directed to Sambrook (Sambrook et al., Molecular cloning: A laboratory manual, 1989, Cold Spring Harbor Laboratory Press), and Ausubel (Ausubel et al., Current protocols in molecular biology, 1993, John Wiley & Sons, Inc.), for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0105] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0106] Reference throughout this specification to "one embodiment" or "an embodiment" and variations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0107] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0108] The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0109] I. ssDNA cleavage

[0110] The disclosure provides a method for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule, comprising incubating a sample comprising the ssDNA molecule with a restriction enzyme capable of cleaving the ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. Typically, the ssDNA molecule comprises a recognition motif for the restriction enzyme.

[0111] A summary of the enzymes with recognition motifs, cutting behavior as known in the context of dsDNA cleavage, and origin is shown in Table 1: Table 1

[0112] # Enzyme Recognition Cutting of dsDNA Organism Gene motif

[0113] 1 Tail ACGT 5' ACGT / 3’ Thermus aquaticus Cel- taiIR 3’ / TGCA 5' 331

[0114] 2 Bshl2361 CGCG 5' CG / CG 3’ Bacillus sphaericus bshl236IR 3’ GC / GC 5' RFL6

[0115] 3 Csp61 GTAC 5' G / TAC 3’ Corynebacterium sp. csp6IR 3’ CAT / G 5'

[0116] 4 Mval CCAGG; 5' CC / WGG 3’ Micrococcus varians mvaIR CCTGG 3’ GG / WCC 5' RFL19

[0117] 5 CviKI-1 AGCT; 5' RG / CY 3’ CA-1A, Chlorella virus CviKI GGCC; 3’ YC / GR 5'

[0118] AGCC;

[0119] GGCT

[0120] 6 Fatl CATG 5' / CATG 3’ Flavobacterium aquatile Fatl 3’ GTAC / 5' NL3

[0121] 7 Nt.CviPII CCA; CCG; 5' / CCD 3’ Chlorella virus NY s- 1 Nt.CviPII CCT 3’ GGH / 5' nicking endonuclea se gene 8 TaqI TCGA 5' T / CGA 3’ Thermus Aquaticus YT- taqIR 3’ AGC / T 5' 1

[0122] 9 Mbol GATC 5' / GATC 3’ Moraxella bovis mboIR 3’ CTAG / 5'

[0123] 10 Xmil GTAGAC; 5' GT / MKAC 3’ Xanthomonas xmilR GTATAC; 3’ CAKM / TG 5' maltophilia Jo 21- 021 GTCGAC;

[0124] GTCTAC

[0125] 11 Tasl AATT 5' / AATT 3’ Thermus aquaticus Vn4- tasIR 3’ TTAA / 5' 211

[0126] 12 FspBI CTAG 5' C / TAG 3’ Flavobacterium sp. fspBIR 3’ GAT / C 5' RFL1

[0127] 13 Rsal GTAC 5' GT / AC 3’ Rhodopseudomonas rsaIR 3’ CA / TG 5' sphaeroides

[0128]

[0129] # Enzyme Recognition Cutting of dsDNA Organism Gene motif

[0130] 14 Hin6I GCGC 5' G / CGC 3’ Haemophilus influenzae

[0131] 3’ CGC / G 5' RFL6

[0132] 15 Eco47I GGWCC 5' G / GWCC 3’ Escherichia coli RFL47 eco47IR 3’ CCWG / G 5'

[0133] 16 Ssil CCGC 5' C / CGC 3’ Staphylococcus sciuri

[0134] 3’ GGC / G 5' RFL1

[0135] 17 Beni CCSGG 5' CC / SGG 3’ Bacillus centrosporus

[0136] 3’ GGS / CC 5' RFL1

[0137]

[0138] D = A, G or T; H = A, C or T; K= G or T; M = A or C; R = G or A; S = C or G; W = A or T; Y = C or T

[0139] In some embodiments, the restriction enzyme has an amino acid sequence selected from SEQ ID NOs: 1-6. The amino acid sequences of SEQ ID NOs: 1-6 are given in Table 2:

[0140] Table 2

[0141] Enzyme Sequence SEQ ID NO

[0142] Mval MSEYLNLLKEAIQNVVDGGWHETKRKGNTGIGKTFEDLLEKEE 1

[0143] DNLDAPDFHDIEIKTHETAAKSLLTLFTKSPTNPRGANTMLRNRY GKKDEYGNNILHQTVSGNRKTNSNSYNYDFKIDIDWESQVVRLE VFDKQDIMIDNSVYWSFDSLQNQLDKKLKYIAVISAESKIENEKK YYKYNSANLFTDLTVQSLCRGIENGDIKVDIRIGAYHSGKKKGK THDHGTAFRINMEKLLEYGEVKVIV

[0144] Nt.CviPII MYIYMSTPQAKTKYYEQRFVNDFYKELERNKVSLPVTIVLKDNL 2

[0145] GIKQVIQNGSGVRVLRDKANAKSPSKIKSEELGRHVTSKADIALF TEEKNGTKVDVAWISPQSHKDFLGKKITPAQYFDASSDVMFKTK IGQPKEIKELKNKMISLSVPLTATKYCWPKYKSGTSLRIWDDVQS TILMNMAIFGVEFGKAYCRNNANILMVGDPLIEVKDDKTIILTTK ENGFSLANGFAEYIPSKDKPIFFTKPTSGKKTVVDGKTIEGVSVWI IYRSYAGSKNRKIDDVLKNKIELISSSCSVKKKDNFVSIMQSKKIT SPPKSKKITSPPKSKKITSPSKSKKITNFFMKK

[0146] TaqI MASTQAQKALETFERFLASLDLESYQQKYRPIKTVEQDLPRELNP 3

[0147] LPDLYEHYWKALEDNPSFLGFEEFFDHWWEKRLRPLDEFIRKYF WGCSYAFVRLGLEARLYRTAVSIWTQFHFCYRWNASCELPLEA

[0148]

[0149] Enzyme Sequence SEQ ID NO APELDAQGIDALIHTSGSSTGIQIKKETYRSEAKSENRFLRKQRGT ALIEIPYTLQTPEELEEKPTGKSERRNLPSMGQGCTPFGPSRKRIR HFSGKLCEKH

[0150] Mbol MKLAFDDFLNSMSETNTTLDYFTDFDKVKKNVAQIEIHLNQLNY 4

[0151] LLGKDDLKQAVYDLYAECPNAFSILEILIAVRKKEQKKSLDEKG QVVTLNSYFQSADKIIDFLNNTGLADVFRDKNIKNLVDYVFGIEV GLDTNARKNRGGDNMSKAVQLLFDNADIYYKKEVRNTIFTDIES LGADVKQFDFVIKTKRKTYVIETNYYNSGGSKLNEVARAYTDV APKINQYSQYEFVWITDGQGWKTAKNKLQEAYTHIPSVYNLYTL HGFIEQLNSEGVIKDW

[0152] Rsal MERRFQLRWDEEELARAFKVTTKDVREYLTDGRRVSFIIERRLM 5

[0153] WENPGWKLAPSEGAGYDLLDPEGGMWEVRSITRQGVYFNPSNQ VGSGRKFNEDGFQLKMSGIKGFILSDIVGFPLVDVYVVPVENVLR WHQARALGANAKVSREKFLRDMVRDIRH

[0154] Hin6I MNLVELGSKTAKDGFKNEKDIADRFENWKENSEAQDWLVTMG 6

[0155] HNLDEIKSVKAVVLSGYKSDINVQVLVFYKDALDIHNIQVKLVS NKRGFNQIDKHWLAHYQEMWKFDDNLLRILRHFTGELPPYHSN TKDKRRMFMTEFSQEEQNIVLNWLEKNRVLVLTDILRGRGDFAA EWVLVAQKVSNNARWILRNINEVLQHYGSGDISLSPRGSINFGR VTIQRKGGDNGRETANMLQFKIDPTELFDI

[0156]

[0157] In some embodiments, the incubation is conducted in an aqueous composition comprising a buffering agent. Examples of suitable buffering agents include, for example, Tris, HEPES, or MOPS. In some embodiments, the buffering agent is Tris, such as TrisCi or TrisAcetate, for example at a concentration of 5 mM to 30 mM, such as 10 or 20 mM.

[0158] In some embodiments, the incubation is conducted in an aqueous composition comprising a magnesium salt. Examples of a magnesium salt include MgCE or magnesium acetate. Suitable concentrations for a magnesium salt include 2 mM to 15 mM, such as 5 or 10 mM. In some embodiments, the magnesium salt is magnesium acetate when the buffering agent is TrisAcetate.

[0159] In some embodiments, the incubation is conducted in an aqueous composition further comprising a salt in addition to a magnesium salt. In some embodiments, the salt is a potassium salt and / or a sodium salt. Examples of a potassium salt include KC1 or potassium acetate. Examples of a sodium salt include NaCl. In some embodiments, the aqueous composition comprises MgCh and one of NaCl or KC1. In some embodiments, the aqueous composition comprises magnesium acetate and potassium acetate. Suitable concentrations for a potassium or sodium salt include 40 mM to 150 mM, such as 50, 66 or 100 mM.

[0160] In some embodiments, the incubation is conducted in an aqueous composition comprising an albumin, such as BSA. Suitable concentrations for the albumin include 0.05 to 0.2 mg / ml, such as 0.1 mg / ml.

[0161] In some embodiments, the incubation is conducted in an aqueous composition of 10 mM TrisCi, 10 mM MgCh, 100 mM KC1 and 0.1 mg / ml BSA. In some embodiments, the incubation is conducted in an aqueous composition of 10 mM TrisCi, 10 mM MgCh, 50 mM NaCl and 0.1 mg / ml BSA. In some embodiments, the incubation is conducted in an aqueous composition of 10 mM TrisCi, 5 mM MgCh, 100 mM NaCl and 0.1 mg / ml BSA. In some embodiments, the incubation is conducted in an aqueous composition of 10 mM TrisCi, 10 mM MgCh, 100 mM KC1 and 0.1 mg / ml BSA. In some embodiments, the incubation is conducted in an aqueous composition of 20 mM TrisAcetate, 50 mM potassium acetate, 10 mM magnesium acetate, and 0.1 mg / ml BSA.

[0162] The incubation is typically carried out for a time period that allows the cleavage of the ssDNA molecule. In some embodiments, the incubation is carried out for at least 15 min, at least 30 min or at least 1 hour. In some embodiments, the incubation is carried out for 4 hours or less, 3 hours or less or 2 hours or less. In some embodiments, the incubation is carried out for 15 min to 4 hours, such as for 30 min to 4 hours or 1 hour to 4 hours, for example for 2 hours.

[0163] The incubation is typically carried out at a temperature that allows the cleavage of the ssDNA molecule. In some embodiments, the incubation is carried out at a temperature of 30 °C to 45 °C, such as 35 °C to 40 °C, for example 37 °C.

[0164] In some embodiments, the sample comprising the ssDNA molecule is incubated with two or more different restriction enzymes. In some embodiments, at least one of the two or more different restriction enzymes is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. Typically, the ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0165] In some embodiments, the sample comprises two or more different ssDNA molecules. In some embodiments, at least one of the two or more different ssDNA molecules comprises a recognition motif for the restriction enzyme. In some embodiments, each of the two or more different ssDNA molecules comprises a recognition motif for the restriction enzyme. In some embodiments, when the sample is incubated with two or more different restriction enzymes, at least one of the two or more different ssDNA molecules comprises a recognition motif for at least one of the two or more different restriction enzymes. In some embodiments, when the sample is incubated with two or more different restriction enzymes, each of the two or more different ssDNA molecules comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0166] In some embodiments, at least 80% of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, at least 90%, at least 95% or at least 99%, of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0167] In some embodiments, the method further comprises purifying the cleavage product after the incubation, for instance by liquid chromatography.

[0168] In some embodiments, the cleavage products are analyzed, for example for their size or mass. Size analysis can be conducted, e.g. by electrophoresis, such as gel electrophoresis, or by chromatography, such as liquid chromatography (LC), including high performance LC (HPLC). Mass analysis can be conducted, e.g. by mass spectrometry.

[0169] The disclosure also provides a use of a restriction enzyme for sequence-specifically cleaving a single-stranded DNA (ssDNA) molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0170] The disclosure also provides a kit for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule, comprising:

[0171] (a) a first ssDNA molecule; and

[0172] (b) a restriction enzyme capable of cleaving the first ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0173] In some embodiments, the first ssDNA molecule comprises a recognition motif for the restriction enzyme. In some embodiments, the first ssDNA molecule serves as a positive control molecule for cleavage by a restriction enzyme, for which it comprises a recognition motif. In some embodiments, the first ssDNA molecule serves as a negative control for restriction enzymes, for which it does not comprise a recognition motif.

[0174] In some embodiments, the restriction enzyme has a sequence selected from SEQ ID NOs: 1-6.

[0175] In some embodiments, the kit comprises two or more different restriction enzymes. In some embodiments, the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, the two or more different restriction enzymes are provided as a mixture. In some embodiments, the first ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0176] In some embodiments, the kit further comprises (c) a second ssDNA molecule different from the first ssDNA molecule. In some embodiments, the second ssDNA molecule comprises a recognition motif for a restriction enzyme, optionally selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0177] In some embodiments, the first ssDNA molecule and the second ssDNA molecule each comprise a recognition motif for a restriction enzyme, wherein the recognition motifs are different from one another. In some embodiments, the first ssDNA molecule and the second ssDNA molecule each comprise only one recognition motif for a restriction enzyme. In some embodiments, the second ssDNA molecule does not comprise a recognition motif for a restriction enzyme that is comprised in the first ssDNA molecule. In some embodiments, the first and the second ssDNA molecules serve as positive controls for two different restriction enzymes, for which they comprise a recognition motif. In some embodiments, the first and the second ssDNA molecules serve as negative controls for restriction enzymes, for which they do not comprise a recognition motif.

[0178] The disclosure also provides a reaction mix for sequence-specific cleavage of a ssDNA molecule. In some embodiments, the disclosure thus provides a composition for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule comprising:

[0179] (a) a first ssDNA molecule; and

[0180] (b) a restriction enzyme capable of cleaving the first ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. Typically, the first ssDNA molecule comprises a recognition motif for the restriction enzyme.

[0181] In some embodiments, the restriction enzyme has a sequence selected from SEQ ID NOs: 1-6. In some embodiments, the composition comprises two or more different restriction enzymes. In some embodiments, the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, the first ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0182] In some embodiments, the composition further comprises (c) a second ssDNA molecule different from the first ssDNA molecule. In some embodiments, the second ssDNA molecule comprises a recognition motif for a restriction enzyme, optionally selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0183] II. Mass spectrometry

[0184] The disclosure also provides a method for analyzing a single-stranded DNA (ssDNA) molecule, comprising the steps:

[0185] incubating a sample comprising the ssDNA molecule with a restriction enzyme capable of cleaving the ssDNA molecule, thereby sequence-specifically cleaving the ssDNA molecule; and

[0186] analyzing the cleavage products of step (a) by mass spectrometry.

[0187] In some embodiments, the restriction enzyme is a Type II restriction enzyme. In some embodiments, the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I. In some embodiments, the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKLl, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKL 1, Fatl, Nt.CVIPII, TaqI, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0188] Typically, the ssDNA molecule comprises a recognition motif for the restriction enzyme.

[0189] In some embodiments, the ssDNA molecule is at least 35 nucleotides (nt) in length, such as at least 50 nt. In some embodiments, the ssDNA molecule is no more than 2000 nt in length, such as no more than 1000 nt, no more than 500 nt, no more than 200 nt or no more than 100 nt. In some embodiments, the ssDNA is 35 nt to 2000 nt, 35 nt to 1000 nt, 35 nt to 500 nt, 35 nt to 200 nt, or 35 nt to 100 nt in length. In some embodiments, at least one cleavage product of step (a) is less than 35 nt in length, such as less than 25 nt, less than 15 nt or less than 10 nt.

[0190] The incubation in step (a) can be carried out as described herein. In some embodiments, the restriction enzyme has a sequence selected from SEQ ID NOs: 1-6.

[0191] In some embodiments, the sample comprising the ssDNA molecule is incubated with two or more different restriction enzymes. In some embodiments, the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3 Al and TthHB8I. In some embodiments, at least one of the two or more different restriction enzymes is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, the two or more different restriction enzymes are selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. Typically, the ssDNA molecule comprises a recognition motif for at least one of the two or more different restriction enzymes. In some embodiments, the ssDNA molecule comprises a recognition motif for each of the two or more different restriction enzymes.

[0192] In some embodiments, the sample comprises two or more different ssDNA molecules. In some embodiments, at least one of the two or more different ssDNA molecules comprises a recognition motif for the restriction enzyme, or, when the sample is incubated with two or more different restriction enzymes, at least one of the two or more different ssDNA molecules comprises a recognition motif for at least one of the two or more different restriction enzymes.

[0193] In some embodiments, the at least two ssDNA molecule are each at least 35 nucleotides (nt) in length, such as at least 50 nt. In some embodiments, the ssDNA molecule is no more than 2000 nt in length, such as no more than 1000 nt, no more than 500 nt, no more than 200 nt or no more than 100 nt. In some embodiments, the ssDNA is 35 nt to 2000 nt, 35 nt to 1000 nt, 35 nt to 500 nt, 35 nt to 200 nt, or 35 nt to 100 nt in length. In some embodiments, at least one cleavage product of step (a) is less than 35 nt in length.

[0194] In some embodiments, at least 80% of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I. In some embodiments, at least 80% of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni. In some embodiments, at least 90%, at least 95% or at least 99%, of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I. In some embodiments, at least 90%, at least 95% or at least 99%, of all ssDNA molecules comprised in the sample comprise a recognition motif for at least one restriction enzyme selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Mbol, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil and Beni.

[0195] The term “Mass Spectrometry” (“Mass Spec” or “MS”) relates to an analytical technology used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z". MS technology generally includes (1) ionizing compounds to form charged compounds; and (2) detecting the mass-to-charge ratio and calculating the molecular weight of the charged compounds. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units. The MS method may be performed either in "negative ion mode", wherein negative ions are generated and detected, or in "positive ion mode" wherein positive ions are generated and detected.

[0196] “Time-of-flight” (TOF) MS is a method wherein an ion’s mass-to-charge ratio is determined via a time of flight measurement. Ions are accelerated by an electric field of known strength. The velocity of the ion depends on the mass-to-charge ratio. The time for ion to reach a detector at a known distance is measured. Based on this measurement, the ion can be identified.

[0197] Most sample workflows in MS further include sample preparation and / or enrichment steps, wherein e.g. the analyte(s) of interest are separated from the matrix, e.g. sample constituents different from the analyte, using e.g. gas or liquid chromatography. Typically, for the mass spectrometry measurement, the following three steps are performed:

[0198] (1.) a sample comprising an analyte of interest is ionized, usually by adduct formation with cations, often by protonation to cations. Ionization sources include but are not limited to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

[0199] (2.) the ions are sorted and separated according to their mass and charge. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) may be used as ion filter.

[0200] (3.) the separated ions are then detected, e.g. in multiple reaction mode (MRM), and the results are displayed on a chart. The term "electrospray ionization" or "ESI," refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. A solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[0201] The term "atmospheric pressure chemical ionization" or "APCI," refers to mass spectrometry methods that are similar to ESI; however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. The ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N2 gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar entity.

[0202] "Multiple reaction mode" or "MRM" is a detection mode for a MS instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0203] “Tandem mass spectrometry” or “MS / MS” involves multiple steps of mass spectrometry selection, wherein fragmentation of an analyte occurs in between the stages. In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MSI). Ions of a particular mass-to-charge ratio (precursor ions or parent ion) are selected and fragment ions (or daughter ions) are created by collision-induced dissociation, ionmolecule reaction, or photodissociation. The resulting ions are then separated and detected in a second stage of mass spectrometry (MS2).

[0204] Since a mass spectrometer separates and detects ions of slightly different masses, it easily distinguishes different isotopes of a given element. Mass spectrometry is thus, an important method for the accurate mass determination and characterization of analytes, including but not limited to nucleic acids.

[0205] Mass spectrometric determination may be combined with additional analytical methods including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or ion mobility-based separation techniques.

[0206] In some embodiments, the ssDNA molecule is subjected to liquid chromatography with tandem mass spectrometry (LC-MS / MS) in step (b). Mass spectrometry, including LC-MS / MS, is commonly known in the art, see for example Limbach, 1996, Mass Spectrom. Rev., 15: 297-336. In brief, molecules comprised in a composition are separated by size by a preceding LC step, and then subjected to tandem MS.

[0207] When the intended nucleic acid sequence of the ssDNA molecule is known, a fragmentation pattern, and thereby also a mass distribution, can be predicted for the cleavage products of step (a). Analyzing the ssDNA molecule may thus comprise determining whether a measured fragmentation pattern of the cleavage products of step (a) corresponds to their predicted fragmentation pattern. For example, this may be determined based on the results from mass spectrometry in step (b). In other words, the analysis by mass spectrometry in step (b) may provide a measured mass distribution for the cleavage products of step (a) which can be compared to their predicted mass distribution.

[0208] When the intended nucleic acid sequence of the ssDNA molecule is known, comparing the measured mass distribution to a predicted mass distribution may allow to verify whether the nucleic acid sequence of the analyzed ssDNA molecule corresponds to the intended nucleic acid sequence. In some embodiments, analyzing the ssDNA molecule may comprise verifying at least a part of the nucleic acid sequence of the ssDNA molecule. For example, this may be determined based on the results from mass spectrometry in step (b). In other words, the analysis by mass spectrometry in step (b) may provide a measured mass for a given cleavage product of step (a) which can be compared to its predicted mass. If the measured and the predicted mass match, the cleavage product may be said to have the intended nucleic acid sequence, thereby verifying its sequence. By cleaving with different restriction enzymes and measuring the mass of the various resulting cleavage products, the degree of certainty of sequence verification may be improved.

[0209] III. Examples

[0210] Example 1: Sequence Specific Hydrolysis of ssDNA

[0211] Target ssDNA oligonucleotides for each enzyme to be tested were designed to avoid intermolecular and intramolecular base-pairing to identify bona fide ssDNA cleavage. To this end, OligoAnalyzer (https: / / eu.idtdna.com / calc / analyzer) or UNAFold Web server (http: / / www.unafold.org / mfold / applications / dna-folding-form.php) were used. A summary of the enzymes tested and the sequence of the target ssDNA oligonucleotide is provided in Table 3. All enzymes used are available commercially, but can also be prepared by standard techniques for protein purification (see, for example, Petrusyte and Janulaitis, Eur.J .Biochem. 1982, 121:377-381). Fatl, CviKI-1, Nt.CVIPII, HpyF3I and Bsp 1431 were purchased from New England Biolabs, the remaining restriction enzymes used were purchased from Thermo Scientific. Table 3

[0212] # Enzyme Target ssDNA oligonucleotide SEQ ID NO of target ssDNA 1 Haelll ATATGGCCATATCGCGATAT 7

[0213] 2 MspI TTAGCATGATACCTGGATGGACCTACCAGGTCCGGT 8

[0214] ATTT

[0215] 3 Mbol ATGATAGCTAATCGGTCCAAGATCAAATTGCAGCGT 9

[0216] CCTGGCCGTCTACAAGTACCATGG

[0217] 4 Alul ATGATAGCTAATCGGTCCAAGATCAAATTGCAGCGT 9

[0218] CCTGGCCGTCTACAAGTACCATGG

[0219] 5 Xmil ATGATAGCTAATCGGTCCAAGATCAAATTGCAGCGT 9

[0220] CCTGGCCGTCTACAAGTACCATGG

[0221] 6 HpyF3I CGATGAATTCATGTACTTAG 10

[0222] 7 Bspl43I GGACTAAGCTGATCTGAGCTGATTT 11

[0223] 8 EcoRI CGATGAATTCATGTACTTAG 10

[0224] 9 Rsal ACCTAAGTACTTACGCGAGTCTTAATCGGT 12

[0225] 10 Bshl236I ATATGGCCATATCGCGATAT 7

[0226] 11 Trull ACCTAAGTACTTACGCGAGTCTTAATCGGT 12

[0227] 12 Csp6I ACCTAAGTACTTACGCGAGTCTTAATCGGT 12

[0228] 13 TaqI AAGTACAGTAGGTACGTAATGCGTCGACAATGAATT 13

[0229] CTTGGTTAATAGCA

[0230] 14 Taal GATACCGTATCGATTCGACAGTAGCTGCTTACGAT 14

[0231] 15 Satl GATACCGTATCGATTCGACAGTAGCTGCTTACGAT 14

[0232] 16 Mval ATGATAGCTAATCGGTCCAAGATCAAATTGCAGCGT 9

[0233] CCTGGCCGTCTACAAGTACCATGG

[0234] 17 Hinlll CGATGAATTCATGTACTTAG 10

[0235]

[0236] # Enzyme Target ssDNA oligonucleotide SEQ ID NO of target ssDNA 18 Cfrl3I TTAGCATGATACCTGGATGGACCTACCAGGTCCGGT 8

[0237] ATTT

[0238] 19 Tail CTGAGCCCGGCAGATCCGGAGCTGTAGACGTCGAC 15

[0239] GTCGACGTCTACCTGA

[0240] 20 FspBI TATTGCCTACTAGAATTGCGCCGCTTAGCTAATTGC 16

[0241] GCAA

[0242] 21 Eco47I TTAGCATGATACCTGGATGGACCTACCAGGTCCGGT 8

[0243] ATTT

[0244] 22 TasI CGATGAATTCATGTACTTAG 10

[0245] 23 Ppu21I AAGTACAGTAGGTACGTAATGCGTCGACAATGAATT 13

[0246] CTTGGTTAATAGCA

[0247] 24 HincII AAGTACAGTAGGTACGTAATGCGTCGACAATGAATT 13

[0248] CTTGGTTAATAGCA

[0249] 25 Ecol05I AAGTACAGTAGGTACGTAATGCGTCGACAATGAATT 13

[0250] CTTGGTTAATAGCA

[0251] 26 HpyCH4V ATGATAGCTAATCGGTCCAAGATCAAATTGCAGCGT 9

[0252] CCTGGCCGTCTACAAGTACCATGG

[0253] 27 Hinfl GATACCGTATCGATTCGACAGTAGCTGCTTACGAT 14

[0254] 28 CviKI-1 ATATGGCCATATCGCGATAT 7

[0255] 29 Fatl ATGATAGCTAATCGGTCCAAGATCAAATTGCAGCGT 9

[0256] CCTGGCCGTCTACAAGTACCATGG

[0257] 30 Nt. CVIPII ATATGGCCATATATATATATCGCGATATATATATAT 17

[0258] GGCCATATCGCGATAATA

[0259] 31 Hin6I CCTACTACCGGGGCGCCGCTTAGCTAATTGCGCAAC 18

[0260] CCGGTATATGGTA

[0261] 32 Beni CCTACTACCGGGGCGCCGCTTAGCTAATTGCGCAAC 18

[0262] CCGGTATATGGTA

[0263]

[0264] # Enzyme Target ssDNA oligonucleotide SEQ ID NO of target ssDNA 33 SslI CCTACTACCGGGGCGCCGCTTAGCTAATTGCGCAAC 18

[0265] CCGGTATATGGTA

[0266] 34 Hhal ATATGGCCATATCGCGATAT 7

[0267]

[0268] For sequence specific hydrolysis of ssDNA, 2.5 pg of target ssDNA oligonucleotides was incubated with 3 pL of the respective restriction enzyme to be tested and 3 pL of lOx reaction buffer, provided by the vendor, followed by the addition of ultrapure water to a final volume of 30 pL. The reaction mixture was incubated 2h at 37 °C. After hydrolysis the reaction mixture is transferred into an HPLC glass vial and measured directly. If multiple digestions with different enzymes are performed, the protocol is scaled accordingly.

[0269] The target oligonucleotides used for each enzyme tested, and results obtained are shown in Fig. 1-22. Exemplary results for an enzyme not capable of cleaving the ssDNA target molecule (Hhal) is shown in Fig. 23. A summary of the identified activities on the target ssDNA oligonucleotides is given in Table 4.

[0270] Table 4

[0271] Activity on ssDNA

[0272] Capable of cleaving Not capable of cleaving

[0273] Haelll HpyF3I

[0274] MspI Bspl43I

[0275] Mbol Trull

[0276] Alni Taal

[0277] Xmil Satl

[0278] EcoRI Hint 11

[0279] Rsal Cfrl3I

[0280] Bshl236I Ppu21I

[0281] Csp6I Hindi

[0282]

[0283] Activity on ssDNA

[0284] Capable of cleaving Not capable of cleaving

[0285] TaqI Ecol05I

[0286] Mval HpyCH4V

[0287] Tail Hhal

[0288] FspBI

[0289] Eco47I

[0290] TasI

[0291] Hinfl

[0292] CviKI-1

[0293] Fatl

[0294] Nt.CVIPII

[0295] Hin6I

[0296] Beni

[0297] Ssil

[0298]

[0299] The results demonstrate that of the tested restriction enzymes some (cf. left column of Table 4) but not all (cf. right column of Table 4) were capable of cleaving the target ssDNA oligonucleotides used. The test included the enzymes Haelll, MspI, Alul, EcoRI, and Hint! that had been previously reported by Nishigaki et al. (supra) to cleave ssDNA. The above results for these enzymes are in agreement with the prior findings. The remaining enzymes in the left column of Table 4 are newly identified herein as being capable of cleaving ssDNA substrates.

[0300] It was further tested if ssDNA molecules could be cleaved by multiple enzymes by incubating a combinations of two or more enzymes with a target ssDNA oligonucleotides. The results are shown in Fig. 24-31.

[0301] The specificity of ssDNA cleavage could be further confirmed by incubating a single enzyme or combinations of enzymes with one or two target ssDNA oligonucleotides, wherein the tested enzyme target only one or both oligonucleotides. The results are shown in Fig. 32-35. The results demonstrate the possibility of performing sequence specific cleavage on one or more target ssDNA molecules at the same time, using one or more enzymes in a single reaction.

[0302] Example 2: Sequence verification of ssDNA with LC-MS / MS

[0303] For sequence verification of oligonucleotides with mass spectrometry, a Waters Premier UPLC equipped with a diodearray detector (DAD) combined with an Waters Xevo G2-XS QToF 4k and electrospray ionization (ESI-MS) was used. Operating parameters: negative ion mode, sensitivity mode, capillary voltage 2.5 kV, sampling cone 130 V, source offset 100 V, desolvation temperature 400 °C, source temperature 100 °C, desolvation gas flow 800 1 / h, cone gas flow 10 1 / h. The instrument was operated in MSE mode. Here a low collision energy of 6 V was selected and ramped for the high energy. It is advised to use a ramp of 20-25 V for <30-mers and a ramp of 20-40 V for >30-mers. For separation an Acquity Premier UPLC BEH C18 1,7pm 2,1x150mm at 60 °C and a flow rate of 0.4 ml / min were used in combination with a binary mobile phase of 10 mM dibutylamine and 50 mM hexafluoroisopropanol as aqueous buffer A and an organic buffer B of 10 mM dibutylamine and 50 mM hexafluoroisopropanol in 50 / 50 H2O and pure acetonitrile (LC-MS grade, purity > 99.95). The gradient started at 15% solvent B for 0.5 min, followed by an increase to 100 % solvent B till 5 min. From 5 to 5.5 min, solvent B was maintained at 100 %, before returning to 15 % solvent B in 0.1 min and a 0.4 min re-equilibration period.

[0304] Results for Haelll are shown in Table 5.

[0305] Table 5

[0306] Incubation time: 120 min

[0307] Predicted Monoisotopic Number of Peaks Intensity Fragment

[0308] mass, Da mass [M-H] Da nucl.

[0309] 1 1830.355 1829.3767 7.46E+06 ATATGG 6

[0310] 2 4220.7456 4299.7261 2.29E+07 CCATATCGCGATAT 14

[0311]

[0312] Monoisotopic mass corresponds to the measured mass. Predicted mass is the mass predicted based on the intended nucleic sequence of a given fragment. The number of nucl. indicates the number of nucleotides of the measured fragment. Of note, generally, potential mass differences of 79 Da between the predicted and measured mass can be explained by the absence or presence of a phosphate group. Potential mass differences of 22 Da between the predicted and measured mass can be explained by the formation of sodium adducts. Results for digestions with single restriction enzymes are given in Tables 6-26. Results for MspI are shown in Table 6.

[0313] Table 6

[0314] Incubation time: 120 min

[0315] Predicted mass, Monoisotopic mass Number Peaks Intensity Fragment

[0316] Da [M-H] Da of nucl.

[0317] 1 2414.4351 2413.4297 1.51E+07 CGGTATTT 8

[0318] TTAGCATGATACCTGGAT

[0319] 2 9819.6562 9818.7598 1.27E+08 32 GGACCTACCAGGTC

[0320] 3 12296.0471 12295.1367 2.26E+07 Intact mass 40

[0321]

[0322] Results for Mbol are shown in Table 7.

[0323] Table 7

[0324] Incubation time: 120 min

[0325] Predicted Monoisotopic Number Peaks Intensity Fragment

[0326] mass, Da mass [M-H] Da of nucl.

[0327] 1 6122.0681 6121.0962 1.40E+07 ATGATAGCTAATCGGTCCAA 20

[0328] GATCAAATTGCAGCGTCCTG

[0329] 2 12275.0596 12354.1270 7.54E+06 40 GCCGTCTACAAGTACCATGG

[0330] 3 18459.0835 18458.1758 1.97E+07 Intact mass 60

[0331]

[0332] Results for Alul are shown in Table 8.

[0333] Table 8

[0334] Incubation time: 120 min

[0335] Predicted Monoisotopic Number Peaks Intensity Fragment

[0336] mass, Da mass [M-H] Da ofnucl.

[0337] 1 2143.4124 2142.4150 1.74E+07 ATGATAG 7

[0338] CTAATCGGTCCAAGATCAAATT

[0339] 2 16253.7153 16332.3975 4.89E+06 GCAGCGTCCTGGCCGTCTACAA 53

[0340] GTACCATGG

[0341]

[0342] Results for Xmil are shown in Table 9.

[0343] Table 9

[0344] Incubation time: 120 min

[0345] Predicted Monoisotopic Number Peaks Intensity Fragment

[0346] mass, Da mass [M-H] Da ofnucl.

[0347] 1 4558.8096 4637.8276 5.82+07 CTACAAGTACCATGG 15

[0348] ATGATAGCTAATCGGTCCAAG

[0349] 2 13838.3181 13837.4043 1.86E+07 ATCAAATTGCAGCGTCCTGGCC 45

[0350] G

[0351]

[0352] Results for EcoRI are shown in Table 10.

[0353] Table 10

[0354] Incubation time: 120 min

[0355] Predicted Monoisotopic mass Number Peaks Intensity Fragment

[0356] mass, Da [M-H] Da ofnucl.

[0357] 1 1501.2985 1501.3125 5.40+06 CGATG 5

[0358]

[0359] Incubation time: 120 min

[0360] Predicted Monoisotopic mass Number Peaks Intensity Fragment

[0361] mass, Da [M-H] Da ofnucl.

[0362] 2 4642.8042 4562.8442 1.24E+07 AATTCATGTACTTAG 15

[0363]

[0364] Results for Rsal are shown in Table 11.

[0365] Table 11

[0366] Incubation time: 120 min

[0367] Predicted mass, Monoisotopic Number Peaks Intensity Fragment

[0368] Da mass [M-H] Da of nucl.

[0369] 1 2391.4617 2391.4998 5.82E+07 ACCTAAGT 8

[0370] 2 6801.2163 6801.2163 8.61E+07 ACTTACGCGAGTCTTAATCGGT 22

[0371]

[0372] Results for Bshl236I are shown in Table 12.

[0373] Table 12

[0374] Incubation time: 120 min

[0375] Predicted mass, Monoisotopic Number of Peaks Intensity Fragment

[0376] Da mass [M-H] Da nucl.

[0377] 1 1869.3018 1869.3287 8.87E+06 CGATAT 6

[0378] 2 4259.7681 4281.7529 9.76E+06 ATATGGCCATATCG 14

[0379]

[0380] Results for Csp6I are shown in Table 13.

[0381] Table 13

[0382] Incubation time: 120 min

[0383] Predicted mass, Monoisotopic Number Peaks Intensity Fragment

[0384] Da mass [M-H] Da ofnucl.

[0385] 1 2087.4048 2087.4268 3.19E+07 ACCTAAG 7

[0386] 2 7105.2593 7105.2710 1.99E+08 TACTTACGCGAGTCTTAATCGGT 23

[0387]

[0388] Results for TaqI are shown in Table 14.

[0389] Table 14

[0390] Incubation time: 120 min

[0391] Monoisotopic

[0392] Predicted Number Peaks mass [M-H] Intensity Fragment

[0393] mass, Da ofnucl.

[0394] Da

[0395] 1 7437.282 7436.3491 1.16E+07 AAGTACAGTAGGTACGTAATGCGT 24

[0396] 2 7989.3723 8068.4292 1.98E+07 CGACAATGAATTCTTGGTTAATAGCA 26

[0397] 3 15488.6101 15487.6563 3.46E+07 Intact mass 50

[0398]

[0399] Results for Mval are shown in Table 15.

[0400] Table 15

[0401] Incubation time: 120 min

[0402] Predicted Monoisotopic Number Peaks Intensity Fragment

[0403] mass, Da mass [M-H] Da of nucl.

[0404] 1 6732.1501 6811.1421 6.04E+07 TGGCCGTCTACAAGTACCATGG 22 ATGATAGCTAATCGGTCCA

[0405] 2 11664.9776 11662.9238 8.86E+06 38

[0406] AGATCAAATTGCAGCGTCC

[0407]

[0408] Incubation time: 120 min

[0409] Predicted Monoisotopic Number Peaks Intensity Fragment

[0410] mass, Da mass [M-H] Da of nucl.

[0411] 3 18459.0835 18458.4219 6.40E+05 Intact mass 60

[0412]

[0413] Results for Tail are shown in Table 16.

[0414] Table 16

[0415] Incubation time: 120 min

[0416] Predicted mass, Monoisotopic mass Number Peaks Intensity Fragment

[0417] Da [M-H] Da of nucl.

[0418] 1 1791.3441 1870.3042 7.63E+06 CGACGT 6

[0419] 2 2368.441 2367.4543 6.96E+06 CTACCTGA 8

[0420] 3 3644.644 3723.6218 1.93E+07 CGACGTCGACGT 12

[0421] 4 4221.7409 4300.7222 4.32E+07 CGACGTCTACCTGA 14

[0422] 5 6075.0408 6154.0415 7.70E+06 CGACGTCGACGTCTACCTGA 20 CTGAGCCCGGCAGATCCG

[0423] 6 9557.607 9556.6777 3.55E+07 31

[0424] GAGCTGTAGACGT CTGAGCCCGGCAGATCCG

[0425] 7 11410.9072 11408.9248 4.45E+07 37 GAGCTGTAGACGTCGACGT

[0426] 8 15694.6039 15693.7227 5.12E+07 Intact mass 51

[0427]

[0428] Results for FspBI are shown in Table 17.

[0429] Table 17

[0430] Incubation time: 120 min

[0431] Monoisotopic

[0432] Predicted Number Peaks mass [M-H] Intensity Fragment

[0433] mass, Da of nucl.

[0434] Da

[0435] 1 2976.5324 2975.5283 6.59E+06 TATTGCCTAC 10 TAGAATTGCGCCGCT

[0436] 2 9201.5578 9280.4932 5.72E+05 30 TAGCTAATTGCGCAA

[0437] 3 12240.046 12239.1162 6.42E+05 Intact mass 40

[0438]

[0439] Results for Eco47I are shown in Table 18.

[0440] Table 18

[0441] Incubation time: 120 min

[0442] Monoisotopic

[0443] Predicted Number Peaks mass [M-H] Intensity Fragment

[0444] mass, Da of nucl.

[0445] Da

[0446] 2994.5505,

[0447] 1 2995.5511 1.11E+06 GACCTACCAG(+p) 10

[0448] 3074.5034

[0449] 3335.5901,

[0450] 2 3336.5792 3.39E+06 GTCCGGTATTT(+p) 11

[0451] 3415.5547

[0452] 3336.5792 3415.5417, 1.81E+06 GTCCGGTATTT(+p),

[0453] 3 19 5840.0052 5839.0195 3.67E+06 TTAGCATGATACCTGGATG

[0454] 4 8897.5121 8896.5469 1.11E+07 TTAGCATGATACCTGGATGGACCTACCAG 29

[0455]

[0456] Results for TasI are shown in Table 19.

[0457] Table 19

[0458] Incubation time: 120 min

[0459] Predicted mass, Monoisotopic Number of Peaks Intensity Fragment

[0460] Da mass [M-H] Da nucl.

[0461] 1 1502.298 1501.2888 3.06E+06 CGATG 5

[0462] 2 4563.8022 4562.7979 2.03E+05 AATTCATGTACTTAG 15

[0463] 3 6128.056 6127.0957 7.78E+06 CGATGAATTCATGTACTTAG 20

[0464]

[0465] Results for Hinfl are shown in Table 20.

[0466] Table 20

[0467] Incubation time: 120 min

[0468] Predicted mass, Monoisotopic Number Peaks Intensity Fragment

[0469] Da mass [M-H] Da of nucl.

[0470] 1 3643.6487 3642.6194 8.01E+06 GATACCGTATCG 12

[0471] 2 7035.2005 7114.1182 1.57E+07 ATTCGACAGTAGCTGCTTACGAT 23

[0472] 3 10740.805 10739.7646 5.67E+07 Intact mass 35

[0473]

[0474] Results for CviKI-1 are shown in Table 21.

[0475] Table 21

[0476] Incubation time: 120 min

[0477] Predicted mass, Monoisotopic Number of Peaks Intensity Fragment

[0478] Da mass [M-H] Da nucl.

[0479] 1 1830.355 1829.8702 1.15E+07 ATATGG 6

[0480]

[0481] Incubation time: 120 min

[0482] Predicted mass, Monoisotopic Number of Peaks Intensity Fragment

[0483] Da mass [M-H] Da nucl.

[0484] 2 4220.7456 4299.9141 9.34E+05 CCATATCGCGATAT 14

[0485]

[0486] Results for Fatl are shown in Table 22.

[0487] Table 22

[0488] Incubation time: 120 min

[0489] Monoisotopi

[0490] Predicted Number Peaks c mass [M- Intensity Fragment

[0491] mass, Da of nucl.

[0492] H] Da

[0493] 1 1502.298 1501.2897 4.39E+06 CATGG 5 ATGATAGCTAATCGGTCCAAGATC

[0494] 2 16894.8297 16893.8516 5.76E+06 AAATTGCAGCGTCCTGGCCGTCTA 28

[0495] CAAGTAC

[0496]

[0497] Results for Nt.CVIPII are shown in Table 23.

[0498] Table 23

[0499] Incubation time: 120 min

[0500] Predicted mass, Monoisotopic mass Number Peaks Intensity Fragment

[0501] Da [M-H] Da of nucl.

[0502] 1 1830.355 1829.3499 3.09E+07 ATATGG 6

[0503] 2 4846.8604 4845.8691 1.45E+07 CCATATCGCGATAATA 16 CCATATATATATATCG

[0504] 3 9815.675 9894.6826 1.06E+08 32

[0505] CGATATATATATATGG

[0506]

[0507] Results for Ssil are shown in Table 24.

[0508] Table 24

[0509] Incubation time: 120 min

[0510] Predicted Monoisotopic Number Peaks Intensity Fragment

[0511] mass, Da mass M-H [Da] ofnucl.

[0512] 1 4840.8346 4839.8550 2.30E+07 CCTACTACCGGGGCGC 16 CGCTTAGCTAATTGCGCAAC

[0513] 2 10123.7019 10202.7002 3.04E+07 33 CCGGTATATGGTA

[0514] 3 15026.4923 15025.5527 1.20E+08 Intact mass 49

[0515]

[0516] Results for Hin6I are shown in Table 25.

[0517] Table 25

[0518] Incubation time: 120 min

[0519] Predicted Monoisotopic mass Number Peaks Intensity Fragment

[0520] mass, Da (Maxent 3) (Da) of nucl.

[0521] 1 3933.6935 3932.698 2.94E+07 CCTACTACCGGGG 13 2 5158.8892 5237.7861 1.59E+07 CGCCGCTTAGCTAATTG 17 3 5810.0109 5888.9395 1.64E+07 CGCAACCCGGTATATGGTA 19

[0522]

[0523] Results for Beni are shown in Table 26.

[0524] Table 26

[0525] Incubation time: 120 min

[0526] Predicted Monoisotopic mass Number Peaks Intensity Fragment

[0527] mass, Da (Maxent 3) (Da) of nucl.

[0528] 1 2617.4834 2616.4709 3.41E+06 CCTACTACC 9 2 3698.6576 3777.5967 6.04E+06 CGGTATATGGTA 12 GGGGCGCCGCTTAGCTAATT

[0529] 3 8586.4525 8665.4053 4.46E+05 28

[0530] GCGCAACC

[0531]

[0532] Incubation time: 120 min

[0533] Predicted Monoisotopic mass Number Peaks Intensity Fragment

[0534] mass, Da (Maxent 3) (Da) of nucl.

[0535] CCTACTACCGGGGCGCCGC

[0536] 4 11265.8916 11264.7646 9.04E+05 37

[0537] TTAGCTAATTGCGCAACC

[0538] 5 15033.78 15034.0010 2.36E+06 Intact mass (av. mass)

[0539]

[0540] Results for digestions with two or more restriction enzymes are given in Tables 27-34.

[0541] Results for HaeIII+Bshl236I are shown in Table 27.

[0542] Table 27

[0543] Incubation time: 120min

[0544] Predicted mass, Monoisotopic mass Number of Peaks Intensity Fragment

[0545] Da [M-H] Da nucl.

[0546] 1 1830.355 1829.3563 2.73E+07 ATATGG 6

[0547] 2 1790.3488 1869.3293 1.26E+07 CGATAT 6

[0548] 3 2368.441 2367.4573 2.05E+07 CCATATCG 8

[0549] 4 4260.7518 4259.7817 1.35E+06 ATATGGCCATATCG 14

[0550]

[0551] Results for Mspl+Tail are shown in Table 28.

[0552] Table 28

[0553] Incubation time: 120min

[0554] Predicted mass, Monoisotopic Number Peaks Intensity Fragment

[0555] Da mass [M-H] Da of nucl.

[0556] 1 1791.3441 1870.3170 2.43E+05 CTGAGC or CGACGT 6

[0557] 2 2368.441 2447.4084 1.79E+06 CTACCTGA 8

[0558]

[0559] Incubation time: 120min

[0560] Predicted mass, Monoisotopic Number Peaks Intensity Fragment

[0561] Da mass [M-H] Da of nucl.

[0562] 3 4864.8459 4863.8750 4.37E+07 CTGAGCCCGGCAGATC 16

[0563] 4 6484.1055 6563.1899 1.53E+07 CGGAGCTGTAGACGTCGACGT 21

[0564] CTGAGCCCGGCAGATC CGGAGCTGTAGACGT

[0565] 5 9557.6073 9556.7754 4.37E+06 or 31 CCGGCAGATCCGGAG CTGTAGACGTCGACGT CTGAGCCCGGCAGATCCGG

[0566] 10853.0225, 1.21E+07

[0567] AGCTGTAGACGTCGACGT

[0568] 6 11410.907 or 37

[0569] 11416.5137 8.04E+06 CCGGCAGATCCGGAGCTGT AGACGTCGACGTCGACGT

[0570] 7 15694.6039 15702.5137 2.23E+07 Intact mass 51

[0571]

[0572] Results for EcoRI+Csp6I are shown in Table 29.

[0573] Table 29

[0574] Incubation time: 120min

[0575] Predicted mass, Monoisotopic Number of Peaks Intensity Fragment

[0576] Da mass [M-H] Da nucl.

[0577] 1 1502.298 1501.2961 3.60E+07 CGATG 5

[0578] 2 2094.3945 2173.3838 4.64E+06 TACTTAG 7

[0579] 3 2407.4519 2486.4170 1.20E+06 AATTCATG 8

[0580] 4 4563.8022 4562.8379 4.56E+06 AATTCATGTACTTAG 15

[0581]

[0582] Results for RsaI+Csp6I are shown in Table 30.

[0583] Table 30

[0584] Incubation time: 120 min

[0585] Predicted mass, Monoisotopic Number of Peaks Intensity Fragment

[0586] Da mass [M-H] Da nucl.

[0587] 1 2088.4066 2087.3982 1.50E+07 ACCTAAG 7

[0588] 2 2392.4523 2391.4617 6.20E+06 ACCTAAGT 8 TACTTACGCGAGTCT

[0589] 3 7026.1888 7105.2319 9.74E+07 23

[0590] TAATCGGT

[0591]

[0592] Results for AluI+TaqI are shown in Table 31.

[0593] Table 31

[0594] Incubation time: 120min

[0595] Predicted mass, Monoisotopic mass Number of Peaks Intensity Fragment

[0596] Da [M-H] Da nucl.

[0597] 1 1477.2914 1556.2594 1.77E+06 CGATT+p 5

[0598] 2825.4990, 2.78E+07, CGACAGTAG+p or

[0599] 2 3025.5503 10

[0600] 3024.5613 3.16E+06 GATACCGTAT

[0601] 3 3305.5847 3384.5564 8.12E+06 CTGCTTACGAT+p 11

[0602] 4364.7690, CGATTCGACAGTAG+

[0603] 4 4564.7975 2.78E+07 p or 15

[0604] 4563.8301 GATACCGTATCGATT GATACCGTATCGATT

[0605] 5 7373.2645 7372.3223 5.93E+07 24

[0606] CGACAGTAG

[0607]

[0608] Results for Mspl+Mval are shown in Table 32.

[0609] Table 32

[0610] Incubation time: 120min

[0611] Predicted mass, Monoisotopic Number of Peaks Intensity Fragment

[0612] Da mass [M-H] Da nucl.

[0613] 1 1502.298 1501.2932 3.38E+06 AGGTC 5

[0614] 2 2414.4351 2493.4092 3.73E+07 CGGTATTT+p 8

[0615] 3 3931.6995 3930.7217 3.87E+06 TTAGCATGATACC 13

[0616] 4057.6892, AGGTCCGGTATTT or 13 4 4261.7471 3.29E+07

[0617] 4340.7544 TGGATGGACCTACC 14 TGGATGGACCTACCA

[0618] 5 5826.0009 5905.0313 2.03E+07 19 GGTC TTAGCATGATACCTG

[0619] 6 8255.4024 8254.5088 4.62E+07 27 GATGGACCTACC TTAGCATGATACCTG

[0620] 8254.5352, GATGGACCTACC or

[0621] 7 8302.3918 1.53E+07 27

[0622] 8381.4385 TGGATGGACCTACCA GGTCCGGTATTT

[0623] 8 12296.0471 12295.2676 1.07E+07 Intact mass 40

[0624]

[0625] Results for Xmil+EcoRI are shown in Table 33.

[0626] Table 33

[0627] Incubation time: 120min

[0628] Predicted Monoisotopic Number of Peaks Intensity Fragment

[0629] mass, Da mass [M-H] Da nucl.

[0630] 1 2417.4589 2496.4421 3.91E+06 CGACAATG 8

[0631] 2 5509.9576 5588.9985 3.26E+06 AATTCTTGGTTAATAGCA 18

[0632]

[0633] Incubation time: 120min

[0634] Predicted Monoisotopic Number of Peaks Intensity Fragment

[0635] mass, Da mass [M-H] Da nucl.

[0636] AAGTACAGTAGGTACGTA

[0637] 7436.3730 4.21E+07 24 3 7437.282 ATGCGT; or

[0638] 5588.9836 1.09E+07 18

[0639] AATTCTTGGTTAATAGCA

[0640]

[0641] Results for Mbol+Alul+Xmil+Mval are shown in Table 34.

[0642] Table 34

[0643] Incubation time: 120min

[0644] Predicted mass, Monoisotopic Number Peaks Intensity Fragment

[0645] Da mass [M-H] Da of nucl.

[0646] 1 2143.41 2142.4160 1.58E+07 ATGATAG 7

[0647] 2 2111.38 2190.3574 3.72E+05 TGGCCGT+p 7

[0648] 3 3916.69 3995.6863 8.22E+05 CTAATCGGTCCAA+p 13

[0649] 4 4558.80 4637.8262 4.11E+07 CTACAAGTACCATGG+p 15 TGGCCGTCTACAAGTACCATGG

[0650] 5 6732.15 6811.2349 1.20E+07 22 +P CTAATCGGTCCAAGATCAAATT

[0651] 6 9459.60 9538.6748 7.01E+06 31 GCAGCGTCC+p

[0652] Av. mass

[0653] 7 11664.97 11718.7354 1.13E+07 CTAATCGGTCCAAGATCAAATT 38

[0654] GCAGCGTCCTGGCCGT+p

[0655]

[0656] Results for digestions of two or more target ssDNA oligonucleotides with one or more restriction enzymes are given in Tables 35-38.

[0657] Results for digestion of SEQ ID NOs 10 and 12 with Csp6I are shown in Table 35. This demonstrates a scenario in which one enzyme cleaves two different ssDNA molecules. Table 35

[0658] Incubation time 120min

[0659] Predicted Monoisotopic Number Peaks Intensity Fragment

[0660] mass, Da mass [M-H] Da of nucl.

[0661] 1 2088.4066 2087.3982 2.90E+07 ACCTAAG 7

[0662] 2 2094.3945 2173.3376 1.19E+07 TACTTAG+p 7

[0663] 3 3971.7057 3970.6487 1.58E+07 CGATGAATTCATG 13

[0664] 4 6128.056 6127.0205 7.42E+07 Intact SEQ ID NO: 10 20

[0665] TACTTACGCGAGTCTTAA

[0666] 5 7026.1888 7105.1313 6.02E+07 23

[0667] TCGGT+p

[0668]

[0669] Results for digestion of SEQ ID NOs 8 and 10 with Mval are shown in Table 36. This demonstrates a scenario in which one enzyme cleaves only one of two different ssDNA molecules.

[0670] Table 36

[0671] Incubation time: 120 min

[0672] Predicted Monoisotopic Number Peaks Intensity Fragment

[0673] mass, Da mass [M-H] Da of nucl.

[0674] 1 3931.6995 3930.6584 2.87E+07 TTAGCATGATACC 13

[0675] 2 3978.6889 4057.6174 2.75E+07 AGGTCCGGTATTT+p 13

[0676] 14

[0677] 3 4261.7471 4340.6680 1.05E+07 TGGATGGACCTACC+p

[0678] 4 6128.056 6127.0200 8.27E+07 Intact SEQ ID NO: 10 20

[0679] TTAGCATGATACCTGGATG

[0680] 5 8254.283 8254.2832 1.62E+05 27

[0681] GACCTACC

[0682]

[0683] Results for digestion of SEQ ID NOs 7 and 8 with Haelll+MspI are shown in Table 37. This demonstrates a scenario of cleavage by two enzymes, wherein each enzyme cleaves only one of two different ssDNA molecules.

[0684] Table 37

[0685] Incubation time

[0686] Monoisotopic

[0687] Predicted Number Peaks mass [M-H] Intensity Fragment

[0688] mass, Da of nucl.

[0689] Da

[0690] 1 1830.355 1829.3496 4.43E+07 ATATGG 6

[0691] 2 2414.4351 2493.3906 3.19E+06 CGGTATTT+p 8

[0692] 3 4220.7456 4299.6943 8.14E+07 CCATATCGCGATAT+p 14

[0693] Av. Mass

[0694] 4 9819.6562 9823.4854 4.61E+05 TTAGCATGATACCTGGATG 32

[0695] GACCTACCAGGTC

[0696]

[0697] Results for digestion of SEQ ID NOs: 10 and 13 with EcoRI+Xmil are shown in Table 38. This demonstrates a scenario of cleavage by two enzymes, wherein one enzymes cleaves only one of two different ssDNA molecules and the other enzyme cleaves both of two different ssDNA molecules.

[0698] Table 38

[0699] Incubation time: 120 min

[0700] Predicted Monoisotopic Number Peaks Intensity Fragment

[0701] mass, Da mass [M-H] Da of nucl.

[0702] 1 1502.298 1501.2877 2.52E+07 CGATG 5

[0703] 2 2417.4589 2496.4060 2.97E+07 CGACAATG+p 8

[0704] 3 4563.8022 4642.7524 5.84E+07 AATTCATGTACTTAG+p 15

[0705] 4 5509.9576 5588.8970 1.93E+07 AATTCTTGGTTAATAGCA+p 18

[0706]

[0707] Incubation time: 120 min

[0708] Predicted Monoisotopic Number Peaks Intensity Fragment

[0709] mass, Da mass [M-H] Da of nucl.

[0710] 5588.9199

[0711] 6.99E+06 AATTCTTGGTTAATAGCA+p or

[0712] 5 7437.282 7440.6191 (av. /

[0713] 4.49E+06 AAGTACAGTAGGTACGTAATGCGT

[0714] Mass)

[0715]

Claims

PATENT CLAIMS1. A method for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule, comprising incubating a sample comprising the ssDNA molecule with a restriction enzyme capable of cleaving the ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni and Mbol.

2. The method of claim 1, wherein the sample comprising the ssDNA molecule is incubated with two or more different restriction enzymes.

3. The method of claim 1 or 2, wherein the sample comprises two or more different ssDNA molecules.

4. A method for analyzing a single-stranded DNA (ssDNA) molecule, comprising the steps:(a) incubating a sample comprising the ssDNA molecule with a restriction enzyme capable of cleaving the ssDNA molecule, thereby sequence-specifically cleaving the ssDNA molecule; and(b) analyzing the cleavage products of step (a) by mass spectrometry.

5. The method of claim 4, wherein the restriction enzyme is a Type II restriction enzyme, such as a restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni, Mbol, AccI, AccII, Avail, Alul, Bbel, Ddel, EcoRI, Haell, Haelll, HapII, Hpall, Hhal, Hinfl, MspI, Sau3AI and TthHB8I.

6. The method of claim 4 or 5, wherein the restriction enzyme is a Type II restriction enzyme, such as a restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-l, Fatl, Nt.CVIPII, TaqI, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni and Mbol.

7. The method of any one of claims 4-6, wherein the sample comprising the ssDNA molecule is incubated with two or more different restriction enzymes.

8. The method of any one of claims 4-7, wherein the sample comprises two or more different ssDNA molecules.

9. The method of any one of claims 4-8, wherein the ssDNA molecule is subjected to liquid chromatography with tandem mass spectrometry (LC-MS / MS) in step (b).

10. A kit for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule, comprising:(a) a first ssDNA molecule; and(b) a restriction enzyme capable of cleaving the first ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni and Mbol.

11. The kit of claim 10, comprising two or more different restriction enzymes.

12. The kit of claim 10 or 11, further comprising(c) a second ssDNA molecule different from the first ssDNA molecule.

13. The kit of claim 12, wherein the first ssDNA molecule and the second ssDNA molecule each comprise a recognition motif for a restriction enzyme, wherein the recognition motifs are different from one another.

14. A composition for sequence-specific cleavage of a single-stranded DNA (ssDNA) molecule comprising:(a) a first ssDNA molecule; and(b) a restriction enzyme capable of cleaving the first ssDNA molecule, wherein the restriction enzyme is selected from Tail, Bshl236I, Csp6I, Mval, CviKI-1, Fatl, Nt.CVIPII, TaqI, Xmil, TasI, FspBI, Rsal, Hin6I, Eco47I, Ssil, Beni and Mbol.

15. The composition of claim 14, comprising two or more different restriction enzymes.

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