Nucleoside triphosphate-dependent endonuclease for DNA fragmentation, a composition and a kit comprising the same

Abstract

The present disclosure relates to the technical field of DNA fragmentation for technologies such as next generation sequencing (NGS) or any other technologies where fragmentation of DNA to a certain uniform size is required. In particular, the present disclosure relates to a variant of a Nucleoside Triphosphate (NTP)-dependent restriction enzyme (McrBC variants selected from McrBL68YC, McrBL68FC, and combinations of certain ratios thereof) for nucleic acid fragmentation for methods that require fragmentation of DNA.

Description

NUCLEOSIDE TRIPHOSPHATE-DEPENDENT ENDONUCLEASE FOR DNA FRAGMENTATION, A COMPOSITION AND A KIT COMPRISING THE SAMEFIELD OF THE INVENTION

[0001] The present disclosure relates to the technical field of DNA sequencing technology in next generation sequencing (NGS). In particular, the present disclosure relates to a variant of a Nucleoside Triphosphate (NTP)-dependent restriction enzyme for nucleic acid fragmentation for methods that require fragmentation of nucleic acid to a size of 100 to 300 bp or longer.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention or that any publication specifically or implicitly referenced is prior art.

[0003] The first step of any DNA sequencing technology in next generation sequencing (NGS) is fragmenting the DNA substrate to a desired length. Two of the most common methods to generate nucleic acid fragments for library preparation includes mechanical methods and enzymatic digestion. Mechanical methods include acoustic shearing using ultrasonic sound waves (Bashkirov et al., 2019 U. S. Patent no. US10329598B2), hydrodynamic shearing and nebulization, while enzyme -based methods include enzymatic digestion using non-specific endonuclease such as DNasel or Fragmentase (two enzyme mix) (Hsieh et al., 2014 U. S. Patent no. US8703462B2) and transposase tagmentation reaction (Streemers et al., 2021 US2022 / 0213470A1). Although both physical and enzymatic methods are usually found to be effective, there are certain limitations tied to the existing technologies of DNA fragmentation.

[0004] Fragmentation using sonication not only requires high-cost instrumentation but also comes with challenges of high throughput scaling and sample loss. Hydrodynamic shearing requires large DNA inputs while the throughput is low. Nebulization is also used for shearing DNA fragments but it is only suitable for small number of samples. Moreover, sequencing biases are reported with different sonication conditions that are strongly reflected as bias in NGS reads (Ribarska et al., 2022). This bias produced by sonication of DNA fragments is a result of enhanced ultrasonic cleavage of dCpG dinucleotides (Grokhovsky et aZ.2011). They also result in oxidative damage resulting in mutation of the sequence (Costelloet al., 2013). Fragmentation methods based on hydrodynamic shearing are also found to produce similar bias (Poptsova et al., 2014). Moreover, mechanical shearing requires dedicated high-cost machinery, extensive optimization steps and continuous maintenance of temperature conditions for example during sonication. The fragment sizes are also usually larger than the most preferred length of ~ 300 bp for NGS sequencing. The method requires large quantity of sample.

[0005] Enzyme-based fragmentation emerged as a good alternative to mechanical shearing. Enzymatic digestion mostly employs non-specific endonuclease such as DNasel or NEBNext dsDNA Fragmentase and transposase tagmentation reaction or CRISPR-Cas. Fragmentase is reported to produce 100-800bp fragment size depending on the reaction time, with desired size fragments (100-300 bp for NGS) taking up at least 30 to 45 mins of time. Moreover, the fragment size analysis by Bioanalyzer 2100 shows a broadly distributed peak reaching up to 500-700bp even at 30mins. Additionally, it is a cocktail of two enzymes supplied in a suitable reaction buffer requiring careful handling during storage and transport (Hsieh et al., 2014 U. S. Patent no. US8703462B2). Finally, Fragmentase has also been known to produce unnecessary indels (Tanaka et al., 2020). Transposon based fragmentation methods employs transposases from the DDE transposase family that contains a conserved triad of acidic residues i.e., DDE motif. DDE transposases are not known to turnover under reaction conditions meaning after catalyzing a transposition event, the enzyme stays bound to the target DNA and hence cannot catalyze the second transposition event. Therefore, with transposases the effective amount of catalytically active transposome complex (transposase + transposon oligo) determines the extent of DNA fragmentation, depletion of which results in complete halt of generating DNA fragments of expected length. Moreover, post DNA fragmentation, transposomes create uniform overhanging sequences at both ends of the DNA fragments. These long complementary ends of fragmented DNA form stable secondary structures (stem-loops) which impedes primer annealing and therefore inhibits efficiency of downstream PCR amplification (Ukanis et al., 2002 European Patent no. EP310269 IB 1). Eventhough the transposase-based fragmentation is fast but the known sequence bias reduces its utility to certain range of samples. Also, the Tn5 transposase-based fragmentation requires large amount of protein for fragmentation reactions. The Tn5 transposase-oligonucleotide complex formation consumes ~9 times excess enzyme compared to oligonucleotides resulting in higher overall cost. Moreover, transposase based fragmentation requires a column cleanup and also not ideal for low GC genomes because of PCR based processing which limits its utility for highthroughput sampling (Lamble et al., 2013). Our invention enables the user to enzymatically fragment any DNA sample to a desired length efficiently since it allows multi turnover catalysis and the fragments generated do not create long overhangs thereby facilitating smooth downstream processing for WGS. Other proposed candidates of DNA fragmentation include Casl endonuclease which is known to generate substantially uniform length fragments. However, a 21 hours incubation is required to produce DNA fragments of size around 100-200 bp (Wiedenheft et al., 2018 U. S. Patent no. US10087431B2)). Pseudorandom fragmentation methods have also been a frequent proposal in the field of DNA fragmentation (Hindson et al., 2020 U. S. Patent publication no. US2020 / 0291472A1)). The entire fragmentation process requires several pairs of restriction enzymes (sometimes even 13 different pairs) to be used sequentially for generating fragments that might, in addition, require processing by sonication.

[0006] Therefore, there is a need in the art of an endonuclease for a single pot nucleic acid fragmentation method that produces optimal size nucleic acid fragments for methods that require fragmentation of nucleic acid to a size of 100 bp to 300 bp or longer fragments in time shorter than or equal to 15 mins and suitable for library preparation kits resulting in good sequencing information.OBJECTS OF THE INVENTION

[0007] Objects of the present disclosure is to generate ready-to-tag nucleic acid fragments of optimum length along with the cost effectiveness

[0008] It is an object of the present disclosure is to provide a nuclease for fragmenting the nucleic acid to a desired length.

[0009] It is an object of the present disclosure is to provide a variant of a Nucleoside Triphosphate (NTP)-dependent endonuclease for methods that require nucleic acid fragmentation to a size of 100 to 300 bp.

[0010] It is another object of the present disclosure is to provide a composition comprising a variant of a Nucleoside Triphosphate (NTP)-dependent endonuclease for methods that require nucleic acid fragmentation to a size of 100 to 300 bp.

[0011] It is yet another object of the present disclosure is to provide a kit comprising a variant of a Nucleoside Triphosphate (NTP)-dependent endonuclease for methods that require nucleic acid fragmentation to a size of 100 to 300 bp.SUMMARY OF THE INVENTION

[0012] Aspects of the present disclosure relate to the technical field of DNA sequencing technology in next generation sequencing (NGS). In particular, the present disclosure relates to a variant of a Nucleoside Triphosphate (NTP)-dependent restriction enzyme for nucleic acid fragmentation for methods that require fragmentation of DNA.

[0013] In an aspect, the present disclosure generates optimal size nucleic acid fragments without creating unnecessary indel artifacts and the NGS reads is expected to reflect reads with minimal sequence bias.

[0014] In an aspect, the present disclosure provides a variant of a Nucleoside Triphosphate (NTP)-dependent endonuclease McrBC for nucleic acid fragmentation with special application in producing optimal size nucleic acid fragments during library preparation for whole genome sequencing (WGS) and other applications of NGS that require fragmentation of DNA to a size of 100 to 300 bp.

[0015] In an aspect of the present disclosure, the variants of McrBC comprise mutations at position L68, including substitutions such as L68Y (SEQ ID NO: 3), L68F (SEQ ID NO: 4), and combinations thereof, or protein homologues thereof. In alternative aspect, the substitution at position L68 may also be replaced with an amino acid having a longer side chain, including but not limited to phenylalanine (F), methionine (M), arginine (R), and tryptophan (W).

[0016] In an aspect of the present disclosure, the variant of the McrBC is having a peptide sequence having at least 50-100% similarity SEQ ID NO: 3 or having atleast 50-100% similarity to SEQ ID NO: 4, with the proviso that the variant of the McrBC is selected from McrBL68YC (Variant 1) and McrBL68FC (variant 2)..

[0017] In an aspect, the present disclosure provides a method of fragmenting a nucleic acid into fragments, comprising the steps of:(a) contacting the nucleic acid sample with a variant of McrBC selected from variant 1 or variant 2 or a combination thereof in presence of guanosine triphosphate (GTP), and a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent;(b) incubating the reaction mixture at about 37°C for about 15 minutes; and (c) heat-inactivating the reaction at about 65 °C for about 5 minutes to obtain DNA fragments having an average size of 100 to 300 base pairs or longer.

[0018] In another preferred aspect, the present disclosure provides a reagent composition for fragmenting or sizing a nucleic acid comprises:(i) a variant of McrBC selected from variant 1 or variant 2 or a combination thereof; (ii) a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent; and(iii) nucleoside triphosphate (NTP),wherein the McrBC variants are freshly purified, or flash frozen and stored at -80°C or at -20°C, or stored in liquid state at -20°C with a suitable percentage of glycerol ranging from 0%to 30%.

[0019] In yet another preferred aspect, the present disclosure provides a kit for fragmenting or sizing a nucleic acid comprises: (i) the reagent composition as disclosed herein; and (ii) user instructions specifying reaction conditions.

[0020] In another preferred aspect, the present disclosure provides a reagent composition for fragmenting or sizing a nucleic acid comprises:(i) a variant of McrBC selected from variant 1 or variant 2 or a combination thereof; (ii) a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent;(iii) nucleoside triphosphate (NTP); and(iv) trehalose or any other stabilizer of the variants of McrBC during lyophilization, wherein the lyophilized reagent composition comprises a preformed enzyme mixture containing components (i) to (iv), and is stored at room temperature or 4°C, which retains enzymatic activity upon reconstitution.

[0021] In yet another preferred aspect, the present disclosure provides a kit for fragmenting or sizing a nucleic acid comprises: (i) lyophilized reagent composition as disclosed herein and stored at either room temperature or 4°C; and (ii) user instructions specifying reconstitution of the lyophilized reagent composition with nuclease-free water prior to use, and reaction conditions.

[0022] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1 shows the 0.8% agarose gel image for the cleavage of mammalian genomic DNA (extracted from HEK cells) by variant 1. The fourth lane shows the complete fragmentation of the methylated human genomic DNA into lengths of 100-200 bp on addition of the cofactor GTP.

[0024] Figure 2 shows the 0.8% agarose gel image for the cleavage of bacterial genomic DNA (extracted from NEB turbo cells) by variant 1. Upon addition of the cofactor GTP, the unmethylated bacterial genomic DNA is completely fragmented into short fragments.

[0025] Figure 3. shows the 0.8% agarose gel image for the cleavage of bacterial genomic DNA (extracted from NEB turbo cells) by variant 1 and variant 2 separately. Upon addition of the cofactor GTP, the unmethylated bacterial genomic DNA is completely fragmented into short fragments by both variant 1 and variant 2.

[0026] Figure 4. shows the 0.8% agarose gel image for the cleavage of bacterial genomic DNA (extracted from NEB turbo cells) by variant 3 which is the cocktail of variant 1 and variant 2 in specific ratios. Upon addition of the cofactor GTP, the unmethylated bacterial genomic DNA is completely fragmented into short fragments by variant 3 for both the ratios used.

[0027] Figure 5. shows the Tapestation profile of the fragmented DNA by variant 3 (V1: V2 = 10 nM: 100 nM) for bacterial genomic DNA substrate.

[0028] Figure 6. shows the fragmentation of the bacterial (Escherichia coli) genomic DNA by variant 1 to a size of 100-300 bp in 15 mins.

[0029] Figure 7. shows the fragmentation of the bacterial (Escherichia coli) genomic DNA by the kit having lyophilized reagent composition to a size of 100-300 bp in 15 mins.

[0030] Figure 8. Shows the bioanalyzer profile of the E. coli fragmented gDNA by variant 1 to an average size of 159 bp and the fragment size ranging from 77 bp - 486 bp.

[0031] Figure 9. shows the Bioanalyzer profile of the QC passed libraries prepared from the E. coli fragmented gDNA (gel depicted in Fig. 7) showed a narrow distribution of size from 204-490 bp with an average fragment size of 293 bp.

[0032] Figure 10. shows the fragmentation of the methylated mammalian genomic DNA from HEK by the kit having lyophilized reagent composition in 15 mins.

[0033] Figure 11. shows the bioanalyzer profile of the QC passed libraries prepared from the fragmented HEK gDNA showed a narrow distribution of size from 200-493 bp with an average fragment size peak at 302 bp.

[0034] Figure 12. shows the fragmentation of the GC-rich Pseudomonas aeruginosa genome by variant 1 in 15 min is comparable with an hour’s time.

[0035] Figure 13. shows the fragmentation of the AT-rich Plasmodium falciparum genome by the kit having lyophilized reagent composition in 15 min.Figure 14. shows the comparable coverage profile of reads (for fragmented E. coli gDNA) at 640X and 45X with fragmentation by variant 1 for 15 mins time.DETAILED DESCRIPTION OF THE INVENTION

[0036] The following is a detailed description of embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0037] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0038] Reference throughout this specification to “one embodiment” or “an embodiment” 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.

[0039] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense, including “and / or” unless the content clearly dictates otherwise.

[0040] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digitsand by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0041] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it is individually recited herein.

[0042] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0043] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0044] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0045] Embodiments of the present disclosure relates to the technical field of DNA sequencing technology in next generation sequencing (NGS). In particular, the present disclosure relates to a variant of a Nucleoside Triphosphate (NTP)-dependent restriction enzyme for nucleic acid fragmentation for methods that require fragmentation of nucleic acid.

[0046] In an embodiment, the present disclosure is ease of use, scalable workflow and robustness to generate ready-to-tag nucleic acid fragments of optimum length along with the cost effectiveness.

[0047] In an embodiment, the present disclosure generates optimal size fragments without creating unnecessary indel artifacts and the NGS reads is expected to reflect reads with minimal sequence bias.

[0048] In another embodiment, the present disclosure is applicable to any field that requires fragmenting nucleic acid as part of their technical routine will greatly benefit from this invention selected from but not limited to including processing DNA substrates from metagenomic samples, removing any DNA contamination from RNA samples, generation of chromatin fragments during chromatin immunoprecipitation (ChIP), and the like.

[0049] McrBC is a restriction enzyme with a tetradecameric complex of McrB and McrC subunits. The recognition site for this enzyme is a methylated cytosine on a dinucleotide RmC, where R is purine. McrB has an N-terminal DNA binding domain and a C-terminal domain which has GTPase activity, while McrC harbors the endonuclease domain.

[0050] Methylation of cytosine at the 5th position is the central epigenetic modification of eukaryotic genomic DNA. Nearly 60%-80% of the 28 million CpG dinucleotides are methylated in the human genome. Since recognition site of McrBC is only a two nucleotide sequence (RmC) and a methylated genome would contain several such sites, it implies that upon treatment with McrBC such a methylated genomic DNA would be cleaved into multiple small fragments and would appear as smear on an agarose gel corresponding to the average size fragments generated. McrBC is already used to study methylation patterns in human DNA.

[0051] In an embodiment of the present disclosure, the size of fractionated nucleic acid fragments ranges from about 1 to 100000 base pairs (bp). For example 10-10000 bp, 100-1000 bp, 100-900 bp, 100-800 bp, 100-700 bp, 100-600 bp, 100-500 bp, and the like. Preferably 100-400 bp, and more preferably 100-300 bp.

[0052] In an embodiment, the present disclosure is applicable to all kinds of nucleic acid samples ranging from and above 100 bp PCR generated products to all kinds of genomic deoxyribonucleic acid (DNA) selected from but not limited to genomic DNA, plasmid DNA, chromatin DNA, cell free DNA of multiple origin.

[0053] In an embodiment, the present disclosure is applicable to double stranded (ds) DNA.

[0054] In an embodiment of the present disclosure, the genomic DNA is a plant genomic DNA, animal genomic DNA, mammalian genomic DNA, cellular DNA, unicellular or multicellular microbial genomic DNA, bacteriophage genomic DNA, any other form of extrachromosomal DNA, synthetic DNA and the like. By the term “genome”, it is intended to refer the entire genome or said substantial subset of the genome.

[0055] In an embodiment, the present disclosure provides a variant of a Nucleoside Triphosphate (NTP) -dependent endonuclease McrBC for nucleic acid fragmentation withspecial application in producing optimal size DNA fragments during library preparation for whole genome sequencing (WGS) and other applications of NGS that require fragmentation of nucleic acid to a size of 100 to 300 bp or more.

[0056] In an embodiment of the present disclosure, the variants of McrBC comprise mutations at position L68, including substitutions such as L68Y (SEQ ID NO: 3), L68F (SEQ ID NO: 4), and combinations thereof, or protein homologues thereof. In alternative embodiment, the substitution at position L68 may also be replaced with an amino acid having a longer side chain, including but not limited to phenylalanine (F), methionine (M), arginine (R), and tryptophan (W).

[0057] In an embodiment of the present disclosure, the variant of the McrBC is produced by a method disclosed in Nirwan et al., Nucleic Acids Res 2019.

[0058] In an embodiment of the present disclosure, the variant of the McrBC is having a peptide sequence having atleast 50-100% similarity SEQ ID NO: 3 or having atleast 50-100% similarity to SEQ ID NO: 4, with the proviso that the variant of the McrBC is selected from a peptide having mutations at positions McrBL68YC (Variant 1) or McrBL68FC (variant 2).

[0059] In an embodiment of the present disclosure, the variant 1 of the McrB is encoded by a nucleic acid sequence having SEQ ID NO: 1.Nucleic acid sequence of McrBL68YC (Variant 1) (SEQ ID NO: 1) ATGGAATCTATTCAACCCTGGATTGAAAAATTTATTAAGCAAGCACAGCAACAACGTTCGC AATCCACTAAAGATTATCCAACGTCTTACCGTAACCTGCGAGTAAAATTGAGTTTCGGTTA TGGTAATTTTACGTCTATTCCCTGGTTTGCATTTCTTGGAGAAGGTCAGGAAGCTTCTAAC GGTATATATCCCGTTATTT TTATTATAAAGATTTTGATGAGTTGGTTTTGGCTTATGGTA TAAGCGACACGAATGAACCACATGCCCAATGGCAGTTCTCTTCAGACATACCTAAAACAAT CGCAGAGTATTTTCAGGCAACTTCGGGTGTATATCCTAAAAAATACGGACAGTCCTATTAC GCCTGTTCCCAAAAAGTCTCACAGGGTATTGATTACACCCGATTTGCCTCTATGCTGGACA ACATAATCAACGACTATAAATTAATATTTAATTCTGGCAAGAGTGTTATTCCACCTATGTC AA AC T G AT G AT AC T G T C T G GAAGAT G C G T T AAAT GATTTGTTTATCCCT G A C C AC ATAGAGACGATACTCAAACGATTAACCATCAAAAAAAATATTATCCTCCAGGGGCCGCCCG GCGTTGGAAAAACCTTTGTTGCACGCCGTCTGGCTTACTTGCTGACAGGAGAAAAGGCTCC GCAACGCGTCAATATGGTTCAGTTCCATCAATCTTATAGCTATGAGGATTTTATACAGGGC TATCGTCCGAATGGCGTCGGCTTCCGACGTAAAGACGGCATATTTTACAATTTTTGTCAGC AAGCTAAAGAGCAGCCAGAGAAAAAGTATATTTTTATTATAGATGAAATCAATCGTGCCAA TCTCAGTAAAGTATTTGGCGAAGTGATGATGTTAATGGAACATGATAAACGAGGTGAAAAC TGGTCTGTTCCCCTAACCTACTCCGAAAACGATGAAGAACGATTCTATGTCCCGGAGAATG TTTATATCATCGGTTTAATGAATACTGCCGATCGCTCTCTGGCCGTTGTTGACTATGCCCT CGCAGACG TTTTCT TC T G ATTGAGCC GG TTGATAC CCACAGTTCCGG T TTTTTACTGAATAAAAAAGCAGAACCTTCATTTGTTGAGTCTTTATGCCAAAAAATGAACG AGTTGAACCAGGAAATCAGCAAAGAGGCCACTATCCTTGGGAAAGGATTCCGCATTGGGCA TAGTTACTTCTGCTGTGGGTTGGAAGATGGCACCTCTCCGGATACGCAATGGCTTAATGAA ATTGTGATGACGGATATCGCCCCTTTACTCGAAGAATATTTCTTTGATGACCCCTAT AAC AAC AGAAAT GGAC C ACAAAT TAT TAGGGGAC T CAT AGCorresponding polypeptide sequence of McrBL68YC (Variant 1) (SEQ ID NO: 3): MESIQPWIEKFIKQAQQQRSQSTKDYPTSYRNLRVKLSFGYGNFTSIPWFAELGEGQEASN GIYPVIYYYKDFDELVLAYGISDTNEPHAQWQFSSDIPKTIAEYFQATSGVYPKKYGQSYY ACSQKVSQLGIDYTRFASMLDNIINDYKLIENSGKSVIPPMSKTESYCLEDALNDLFIPET TIETILKRLTIKKNIILQGPPGVGKTFVARRLAYLLTGEKAPQRVNMVQFHQSYSYEDFIQ GYRPNGVGFRRKDGIFYNFCQQAKEQPEKKYIFIIDEINRANLSKVFGEVMMLMEHDKRGE NWSVPLTYSENDEERFYVPENVYIIGLMNTADRSLAWDYALRRRFSFIDIEPGFDTPQFR NFLLNKKAEPSFVESLCQKMNELNQEISKEATTLGKGFRTGHSYFCCGLEDGTSPDTQWLN EIVMTDIAPLLEEYFFDDPYKQQKWTNKLLGDS*

[0060] In an embodiment of the present disclosure, the variant 2 of the McrBC is encoded by a nucleic acid sequence having SEQ ID NO: 2.Nucleic acid sequence of McrBL68FC (Variant 2) (SEQ ID NO: 2) ATGGAATCTATTCAACCCTGGATTGAAAAATTTATTAAGCAAGCACAGCAACAACGTTCGC AATCCACTAAAGATTATCCAACGTCTTACCGTAACCTGCGAGTAAAATTGAGTTTCGGTTA TGGTAATTTTACGTCTATTCCCTGGTTTGCATTTCTTGGAGAAGGTCAGGAAGCTTCTAAC GGTATATATCCCGTTATTTTCTATTATAAAGATTTTGATGAGTTGGTTTTGGCTTATGGTA TAAGCGACACGAATGAACCACATGCCCAATGGCAGTTCTCTTCAGACATACCTAAAACAAT CGCAGAGTATTTTCAGGCAACTTCGGGTGTATATCCTAAAAAATACGGACAGTCCTATTAC GCCTGTTCCCAAAAAGTCTCACAGGGTATTGATTACACCCGATTTGCCTCTATGCTGGACA ACATAATCAACGACTATAAATTAATATTTAATTCTGGCAAGAGTGTTATTCCACCTATGTC AAAAACTGAATCATACTGTCTGGAAGATGCGTTAAATGATTTGTTTATCCCTGAAACCACA ATAGAGACGATACTCAAACGATTAACCATCAAAAAAAATATTATCCTCCAGGGGCCGCCCG GCGTTGGAAAAACCTTTGTTGCACGCCGTCTGGCTTACTTGCTGACAGGAGAAAAGGCTCC GCAACGCGTCAATATGGTTCAGTTCCATCAATCTTATAGCTATGAGGATTTTATACAGGGC TATCGTCCGAATGGCGTCGGCTTCCGACGTAAAGACGGCATATTTTACAATTTTTGTCAGC AAGCTAAAGAGCAGCCAGAGAAAAAGTATATTTTTATTATAGATGAAATCAATCGTGCCAA TCTCAGTAAAGTATTTGGCGAAGTGATGATGTTAATGGAACATGATAAACGAGGTGAAAAC TGGTCTGTTCCCCTAACCTACTCCGAAAACGATGAAGAACGATTCTATGTCCCGGAGAATG TTTATATCATCGGTTTAATGAATACTGCCGATCGCTCTCTGGCCGTTGTTGACTATGCCCT ACGCAGACGATTTTCTTTCATAGATATTGAGCCAGGTTTTGATACACCACAGTTCCGGAAT TTTTTACTGAATAAAAAAGCAGAACCTTCATTTGTTGAGTCTTTATGCCAAAAAATGAACG AGTTGAACCAGGAAATCAGCAAAGAGGCCACTATCCTTGGGAAAGGATTCCGCATTGGGCA TAGTTACTTCTGCTGTGGGTTGGAAGATGGCACCTCTCCGGATACGCAATGGCTTAATGAA ATTGTGATGACGGATATCGCCCCTTTACTCGAAGAATATTTCTTTGATGACCCCTATAAAC AAC AGAAAT GG C CAAC AAT T AT T GGGG AC T CAT AGCorresponding polypeptide sequence of McrBL68FC (Variant 2) (SEQ ID NO: 4): MESIQPWIEKFIKQAQQQRSQSTKDYPTSYRNLRVKLSFGYGNFTSIPWFAFLGEGQEASN GIYPVIFYYKDFDELVLAYGISDTNEPHAQWQFSSDIPKTIAEYFQATSGVYPKKYGQSYY ACSQKVSQGIDYTRFASMLDNIINDYKLIFNSGKSVIPPMSKTESYCLEDALNDLFIPETT IETILKRLTIKKNIILQGPPGVGKTFVARRLAYLLTGEKAPQRVNMVQFHQSYSYEDFIQG YRPNGVGFRRKDGIFYNFCQQAKEQPEKKYIFIIDEINRANLSKVFGEVMMLMEHDKRGEN WSVPLTYSENDEERFYVPENVYIIGLMNTADRSLAWDYALRRRFSFIDIEPGFDTPQFRNFLLNKKAEPSFVESLCQKMNELNQEISKEATTLGKGFRTGHSYFCCGLEDGTSPDTQWLNE IVMTDIAPLLEEYFFDDPYKQQKWTNKLLGDS*

[0061] In an embodiment of the present disclosure, the variant 3 is the combination cocktail of variant 1 and variant 2 in certain ratios, the sequence of which are mentionedabove. In some embodiments, the ratio of variant 1:variant 2 is 1:10 to 10:1 for example 10 nM:100 nM.

[0062] In an embodiment of the present disclosure, the variant 1 or variant 2 of the McrBC results in efficient cleavage of both methylated and non-methylated DNA.

[0063] In another preferred embodiment, the present disclosure provides a reagent composition for fragmenting or sizing a nucleic acid comprises:(i) a variant of McrBC selected from variant 1 or variant 2 or a combination thereof; (ii) a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent; and(iii) nuceloside triphosphate (NTP),wherein the McrBC is freshly purified, or flash frozen and stored at -80°C or at - 20°C, or stored in liquid state at -20°C with a suitable percentage of glycerol ranging from 0 to 30%.

[0064] In another preferred embodiment, the present disclosure provides a reagent composition for fragmenting or sizing a nucleic acid comprises:(i) a variant of McrBC selected from variant 1 or variant 2 or a combination thereof; (ii) a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent;(iii) nucleoside triphosphate (NTP); and(iv) trehalose or any other stabilizer of the protein during lyophilization, wherein the lyophilized reagent composition comprises a preformed enzyme mixture containing components (i) to (iv), and is stored at room temperature or 4°C, which retains enzymatic activity upon reconstitution.

[0065] In another embodiment of the present disclosure, the buffer is selected from a Trisbased, phosphate-based, or HEPES-based buffering agent suitable for maintaining the enzymatic activity of the McrBC variant during DNA fragmentation. In some embodiments, the monovalent salt in the buffer is selected from the group consisting of potassium chloride (KCl), sodium chloride (NaCl), lithium chloride (LiCl), ammonium chloride (NH₄Cl), and functional equivalents thereof. In some embodiments, the divalent salt in the buffer is selected from the group consisting of magnesium chloride (MgCl₂), manganese chloride (MnCl₂), calcium chloride (CaCl₂), zinc chloride (ZnCl₂), and functional equivalents thereof. In some embodiments, the reducing agentin the buffer is selected from the group consisting of dithiothreitol (DTT), [3-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), glutathione, cysteine, or functional equivalents thereof. In a preferred embodiment, the buffer comprises Tris-Cl, potassium chloride (KC1), magnesium chloride (MgCl₂), and dithiothreitol (DTT). In some embodiments, the buffer provides a final IX working concentration and maintains a pH of approximately 7.5-8.5, preferably about pH 8.0.

[0066] In certain embodiments, the buffer is present at about 0.01% to 2% w / v, the monovalent salt is present at about 0.01% to 5% w / v, the divalent salt is present at about 0.001% to 2% w / v, and the reducing agent is present at about 0.0001% to 1% w / v. In a more specific embodiment, the buffer contains: Tris-Cl at approximately 0.12% w / v (corresponding to 10 mM), KC1 at approximately 0.37% w / v (corresponding to 50 mM), MgCl₂ at approximately 0.095% w / v (corresponding to 10 mM), and DTT at approximately 0.015% w / v (corresponding to 1 mM), wherein the buffer provides a final IX working concentration and maintains a pH of approximately 7.5-8.5, preferably about pH 8.0.

[0067] In another embodiment of the present disclosure, the amount of the target nucleic acid sample in the reagent composition ranges from 1 ng to 1 pg. Preferably, 1 ng to 1 pg and most preferably 1 ng to 500 ng.

[0068] In another embodiment of the present disclosure, the concentration of the variant 1 or variant 2 or variant 3 of the McrBC in the reagent composition ranges from 0.1 nM to 10 pM. Preferably, 1 nM to 1 pM and most preferably 0.1 pM to 1 pM.

[0069] In another embodiment of the present disclosure, the reagent composition further comprises one or more reagents for tagging the fragmented nucleic acid with adaptors, reagents that allows end repair and dA-tailing would enable subsequent adaptor ligation all in the same vial.

[0070] In yet another embodiment, the present disclosure provides a kit for fragmenting or sizing the nucleic acid comprises: (i) the reagent composition as disclosed herein; and (ii) user instructions specifying reaction conditions.

[0071] In yet another preferred aspect, the present disclosure provides a kit for fragmenting or sizing the nucleic acid comprises: (i) lyophilized reagent composition as disclosed herein and stored at either room temperature or 4°C; and (ii) user instructions specifying reconstitution of the lyophilized reagent composition with nuclease-free water prior to use, and reaction conditions.

[0072] In another embodiment of the present disclosure, the amount of the target nucleic acid sample in the kit ranges from 1 ng to 1 pg. Preferably, 1 ng to 1 pg and most preferably 1 ng to 500 ng.

[0073] In another embodiment of the present disclosure, the concentration of the variant 1 or variant 2 or variant 3 of the McrBC in the kit ranges from 0.1 nM to 10 pM. Preferably, 1 nM to 1 pM and most preferably 0.1 pM to 1 pM.

[0074] In yet another embodiment, the present disclosure provides a method for fragmentation of nucleic acids into fragments using the variant 1 and / or variant 2 by the protocol mentioned in the cited paper Nirwan et al., Nucleic Acids Res., 2019.

[0075] In a preferred embodiment, the present invention provides a method for fragmentation of a nucleic acid comprising the steps of:(a) contacting the nucleic acid sample with a variant of McrBC selected from variant 1 or variant 2 or a combination thereof in presence of guanosine triphosphate (GTP), and in presence of nucleoside triphosphate (NTP), and a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent;(b) incubating the reaction mixture at about 37°C for about 15 minutes; and (c) heat-inactivating the reaction at about 65 °C for about 5 minutes to obtain DNA fragments having an average size of 100 to 300 base pairs or longer.

[0076] In another preferred embodiment, the present invention provides a method for fragmentation of a nucleic acid sample to a size of 100 to 300 bp comprising the steps of:(a) contacting a nucleic acid sample with the reagent composition or the kit as disclosed herein in presence of guanosine triphosphate (GTP);(b) incubating the reaction mixture at about 37°C for about 1 hour; and(c) heat-inactivating the reaction at about 65 °C for about 20 minutes,wherein the method produces DNA fragments having an average size of 100 to 300 base pairs.

[0077] In yet another embodiment of the present disclosure, the method produces ready-to-tag desirable sized nucleic acid fragments without the unnecessary indels artifacts or sequence information loss.

[0078] In yet another embodiment of the present disclosure, the kit further comprises one or more reagents for tagging the fragmented nucleic acid with adaptors, reagents that allows end repair and dA-tailing would enable subsequent adaptor ligation all in the same vial.

[0079] In yet another embodiment of the present disclosure, the kit further comprises at least one fourth primer pair which hybridises to a DNA sequence of the library having a length from 100 bp to 300 bp, wherein the first, second, third and fourth primer pairs give rise, when amplified by PCR, to PCR products having different size from one another.

[0080] In further embodiment, the present disclosure may be applied to any field of technology / study / analysis / sector where uniform fragmentation of a nucleic acid substrate of any origin (bacterial, human, insects, plants, bacteriophages, cell free DNA etc.) of any size or length (ranging from 30 bp to genomic DNA), of any conformation (plasmid; supercoiled, nicked, linear, circular) is required producing desirable uniform length fragments in the range of 100-300 bp or longer within 15 minutes.Examples

[0081] Material and method:Reaction buffer reagents: Tris powder, KC1 and MgCE powder from Sigma company and DTT powder from Promega company.Genomic DNA was isolated using the Qiagen genomic DNA extraction kit.The enzyme was purified using the protocol mentioned in the cited paper Nirwan et al., Nucleic Acids Res 2019.

[0082] Example 1: Cleavage of mammalian genomic DNA (extracted from HEK cells) by variant 1 (McrBL68YC)

[0083] 200 ng of mammalian genomic DNA extracted from HEK (human embryonic kidney) cells (by Qiagen genomic DNA extraction kit) was subjected to DNA cleavage by variant 1 at a concentration of 100 nM in the presence of the nucleotide GTP (at 2 mM concentration) in a total volume of 10 pL reaction. The reaction was kept at 37°C for Ihour after which it was subjected to heat inactivation at 65°C for 20mins. The entire 10 pL reaction volume was loaded onto a 0.8% agarose gel (containing ethidium bromide for DNA visualization under UV) after adding 2pL of DNA loading dye. After a 45 mins to Ihr of gel electrophoresis, the cleaved fragments were visualized using BioRad GelDocGo Imager. The cleaved fragments can be seen as a smear on gel around the DNA band size of 100-200bp in the fourth lane. Lane 1 is the DNA ladder used to estimate the size of the fragments. Lane 2 denotes the untreated HEK genomic DNA substrate used for the assay. Lane 3 contains the genomic DNA substrate along with the enzyme of interest i.e., variant 1 but without the nucleotide GTP. Lane 4 is the demonstration of the fragmentation of the mammalian genomic DNA into fragment size of 100-200 bp by variant 1 in the presence of GTP at 2 mM. Thebuffer composition used in the reaction includes the following: 10X TMDK buffer (Tris Mg DTT KCl): 100 mM Tris-Cl; pH 8.0, 500 mM KCl, 100 mM MgCl2and 10 mM DTT. 1µL of the 10X buffer was added to the whole 10µL reaction to obtain IX final concentration of the components. Cleavage of HEK genomic DNA by variant 1 is a demonstration of its nuclease activity for methylated DNA substrate (Figure 1).

[0084] Example 2: Cleavage of bacterial genomic DNA (extracted from NEB turbo cells) by variant 1 (McrBL68YC)

[0085] 200 ng of bacterial genomic DNA extracted from Escherichia coli (strain NEBturbo;non-methylated genome) cells was subjected to DNA cleavage by variant 1 at a concentration of 100 nM in the presence of the nucleotide GTP (at 2 mM concentration) in a total volume of 10 pL reaction. The reaction was kept at 37°C for 1 hour after which it was subjected to heat inactivation at 65°C for 20mins. The entire 10 pL reaction volume was loaded onto a 0.8% agarose gel (containing ethidium bromide for DNA visualization under UV) after adding 2 pL of DNA loading dye. After a 45 mins to Ihr of gel electrophoresis, the cleaved fragments were visualized using BioRad GelDocGo Imager. The cleaved fragments can be seen as a bunched smear on gel in the third lane. Lane 1 denotes the untreated bacterial genomic DNA substrate used for the assay. Lane 2 contains the genomic DNA substrate along with variant 1 but without the nucleotide GTP. Lane 3 is the demonstration of the fragmentation of the bacterial genomic DNA into fragment size of approximate 100-200 bp by variant 1 in the presence of GTP at 2 mM. The buffer composition used in the reaction includes the following: 10X TMDK buffer (Tris Mg DTT KCl): 100 mM Tris-Cl; pH 8.0, 500 mM KCl, 100 mM MgCl2and 10 mM DTT. 1 µL of the 10X buffer was added to the whole 10 µL reaction to obtain 1X final concentration of the components. Cleavage of E. coli NEBturbo genomic DNA by variant 1 is a demonstration of its nuclease activity for unmethylated DNA substrate (Figure 2).

[0086] Therefore, variant 1 can be employed in fragmentation of both methylated and nonmethylated DNA substrates with similar nuclease efficiencies and fragment size.

[0087] Example 3: Cleavage of bacterial genomic DNA (extracted from NEB turbo cells) by variant 1 (McrBL68YC) and variant 2 (McrBL68FC) separately

[0088] 200ng of bacterial genomic DNA extracted from Escherichia coli (strain NEBturbo;non-methylated genome) cells was subjected to DNA cleavage by variant 1 and variant 2 in two separate reactions at a concentration of 100 nM each in the presence of the nucleotide GTP (at 2 mM concentration) in a total volume of 10 pL reaction each. Thereaction was kept at 37°C for 1 hour after which it was subjected to heat inactivation at 65°C for 20 mins. The entire 10 pL reaction volume was loaded onto a 0.8% agarose gel (containing ethidium bromide for DNA visualization under UV) after adding 2 pL of DNA loading dye. After a 45 mins to 1 hr of gel electrophoresis, the cleaved fragments were visualized using BioRad GelDocGo Imager. Lane 1 denotes the untreated bacterial genomic DNA substrate used for the assay. Lane 2 contains the genomic DNA substrate along with variant 2 and the nucleotide GTP. Lane 2 demostrates the fragmentation of genomic DNA by variant 2. Lane 3 contains the genomic DNA substrate variant 1 alongwith the nucleotide GTP. Lane 3 demostrates the fragmentation of the genomic DNA by variant 1. The buffer composition used in the reaction includes the following: 10X TMDK buffer (Tris Mg DTT KCl): 100mM Tris-Cl; pH 8.0, 500mM KCl, 100 mM MgCl2and 10mM DTT. 1µL of the 10X buffer was added to the whole 10µL reaction to obtain 1X final concentration of the components. Cleavage of E.coli NEBturbo genomic DNA by variant 1 and variant 2 is a demonstration for comparing their nuclease activities for unmethylated DNA substrate (Figure 3).

[0089] Example 4: Cleavage of bacterial genomic DNA (extracted from NEB turbo cells) by variant 3 (a combination of McrBL68YC and McrBL68FC)

[0090] 200ng of bacterial genomic DNA extracted from Escherichia coli (strain NEBturbo;non-methylated genome) cells was subjected to DNA cleavage by variant 3, combining variant 1 and variant 2 in two different ratios. The first combination uses variant 1 and variant 2 in the ratio of 1:20 i.e., 5 nM of variant 1 and 100 nM of variant 2. The second combination uses variant 1 and variant 2 in the ratio of 1:10 i.e., 10 nM of variant 1 and 100 nM of variant 2. Both reactions of variant 3 occurred in the presence GTP at concentration of 2 mM in a total volume of 10 pL reaction. The reaction was kept at 37°C for 1 hour after which it was subjected to heat inactivation at 65°C for 20 mins. The entire 10 pL reaction volume was loaded onto a 0.8% agarose gel (containing ethidium bromide for DNA visualization under UV) after adding 2 pL of DNA loading dye. After a 45 mins to 1 hr of gel electrophoresis, the cleaved fragments were visualized using BioRad GelDocGo Imager. Lane 1 denotes the untreated bacterial genomic DNA substrate used for the assay. Lane 2 demostrates the fragmentation of genomic DNA by 1: 20 ratio of variant 1 and variant 2 in the presence of GTP. Lane 3 demostrates the fragmentation of genomic DNA by 1: 10 ratio of variant 1 and variant 2 in the presence of GTP. This gel is a demonstration of the diverse range of DNA fragments that can be generated by different combinations of variant 1 andvariant 2 i.e., variant 3 can be used to generate diverse kinds of desired DNA fragments from any DNA substrate.

[0091] The buffer composition used in the reaction includes the following: 10X TMDK buffer (Tris Mg DTT KCl): 100mM Tris-Cl; pH 8.0, 500 mM KCl, 100 mM MgCl2and 10 mM DTT. 1µL of the 10X buffer was added to the whole 10µL reaction to obtain 1X final concentration of the components. Cleavage of E. coli NEBturbo genomic DNA by variant 3 is a demonstration for a range of DNA fragments size (most preferably >150bp - Ikb) that can be generated (Figure 4).

[0092] Example 5: Tapestation profile of the fragmented DNA by variant 3 (a combination of McrBL68YC and McrBL68FC)

[0093] 200 ng of bacterial genomic DNA extracted from Escherichia coli (strain NEBturbo;non-methylated genome) cells was subjected to DNA cleavage by variant 3, combination of variant 1 and variant 2 in the ratio of 1:10 i.e., 10 nM of variant 1 and 100 nM of variant 2. Three identical reactions of 10 pL volume was set in the presence GTP at concentration of 2 mM. The reaction was kept at 37°C for 1 hour after which it was subjected to heat inactivation at 65°C for 20 mins. At the end of incubation, the three 10 pL volumes were pooled and the DNA sample were quantified on Qubit Flurometer and the fragmented sample was loaded on a high sensitivity DNA screen tape to determine the fragment size. The concentration of fragmented DNA was 7 ng / pL. Figure 5 depicts the tapestation profile of the fragment DNA illustrating that the average size of the fragments turned out to be 282 bp. The table indicates the quality control remarks for the fragmented DNA by variant 3. In essence the QC parameters and the tapestation profile indicates not only the sensitivity of this method for concentration of fragmented DNA as low as 7ng but also suggests the desirable fragment length suitable for most sequencing techniques (Figure 5).

[0094] Example 6: Fragmentation of various types of DNA samples by the lyophilized reagent composition containing McrBL68YC (Variant 1, lyophilized preformed reaction mix)Reaction buffer composition Standard enzyme reaction Lyophilized preformed reaction (for lOpL reaction) (for 200 ng of DNA) mix (for 200 ng of DNA) Tris-Cl (pH 8.0) 10 mM 10 mMKC1 50 mM 50 mMMgCl210 mM 10 mMDTT 1 mM 1 mMGTP 4 mM 2 mMvariant 1 100 nM 200 nMTrehalose (w / v) - 5%Table 1 shows the table summarizing the reaction conditions and buffer compositions used for setting up the DNA fragmentation reaction by variant 1 including both the standard enzyme reaction and the reaction for the lyophilized variant 1 preformed reaction mix. The tabular summary includes enzyme and substrate concentrations, incubation time, temperature and buffer formulations (pH, final salt concentrations etc.) used in each experimental condition.Further, the reaction components (1-7) were lyophilized for 24 hours to obtain lyophilized preformed reaction mix, and the lyophilized mixture was reconstituted by adding 1 mg / mL BSA, the required amount of DNA, and an additional 2 mM of GTP to achieve a total GTP concentration of 4 mM. MilliQ water was then added to make up the total reaction volume to 10 pL. Reconstitution required the use of ice-cold MilliQ water, which was added to the walls of the vial, followed by a short spin to obtain a homogeneous reaction mixture.A 10 pL standard enzyme reaction setup was prepared in which 200 ng of E. coli genomic DNA was fragmented by variant 1 (100 nM) in the presence of GTP (4 mM) for 15 minutes, and the entire reaction volume was subsequently loaded onto a 0.8% agarose gel to visualize the size of the fragmented DNA. The DNA fragment sizes were estimated using a 1 kb DNA ladder as a reference. As shown in Figure 6, the E. coli genomic DNA was efficiently fragmented by variant 1 (100 nM) only in the presence of GTP (4 mM), producing DNA fragments within the size range of approximately 100-300 bp.A 10 pL reaction was prepared in which 200 ng of E. coli genomic DNA was fragmented by the lyophilized preformed variant 1 (200 nM) reaction mix in the presence of GTP (4 mM) for 15 minutes, and 3 pL of the reaction mixture was loaded on a 0.8% agarose gel to visualize the fragmented DNA. As shown in Figure 7, the E. coli genomic DNA was efficiently fragmented within the size range of 100-300 bp.A 10 pL reaction containing 200 ng of E. coli genomic DNA was fragmented using the lyophilized preformed variant 1 reaction mix (as mentioned in Table 1) in the presence of GTP (4 mM) for 15 minutes, and the fragmented DNA was analyzed using a Bioanalyzer. As shown in Figure 8, the Bioanalyzer profile displayed the average size of the DNA fragmentsto be 159 bp with fragment size ranging from 77 bp to 486 bp. Furthermore, figure 9 depicts the bioanalyzer profile of the QC-passed, size-selected DNA libraries distributed within a narrow range of 204-490 bp, with an average fragment size of 293 bp. These libraries were subsequently used for whole-genome sequencing (WGS) analysis.A 10 pL reaction containing 200 ng of HEK (mammalian) genomic DNA was fragmented using the lyophilized preformed variant 1 reaction mix (200 nM) in the presence of GTP (4 mM) for 15 minutes. A 3 pL portion of the reaction mixture was loaded on a 0.8% agarose gel to visualize the fragmented DNA, which was observed to fall within the 100-300 bp range. As shown in Figure 10, the remaining 7 pL of the reaction was sent for fragment size confirmation using a Bioanalyzer.A 10 pL reaction containing 200 ng of HEK (mammalian) genomic DNA was fragmented using the lyophilized preformed variant 1 reaction mix (200 nM) in the presence of GTP (4 mM) for 15 minutes, and the fragmented DNA was analyzed using a Bioanalyzer. As shown in Figure 11, the Bioanalyzer profile displayed QC-passed, size-selected DNA libraries distributed within a narrow range of 200-493 bp, with an average fragment size of 302 bp. The results demonstrated the efficiency of variant 1 in fragmenting methylated mammalian genomic DNA within 15 minutes.A standard 10 pL enzyme reaction was prepared in which 200 ng of Pseudomonas aeruginosa genomic DNA was fragmented by variant 1 (100 nM) in the presence of GTP (4 mM) for 15 minutes, and the entire reaction volume was loaded onto a 0.8% agarose gel to visualize the fragmented DNA. As shown in Figure 12, the genomic DNA was efficiently fragmented within the 100-300 bp range. A comparative analysis between the 15-minute and 60-minute reactions showed similar fragmentation efficiency, indicating that variant 1 achieved maximum activity within minimal bench time. The results demonstrated the capability of variant 1 to efficiently fragment GC-rich genomes (>65%) to the desired size range.A 10 pL reaction containing 200 ng of Plasmodium falciparum genomic DNA was fragmented using the lyophilized preformed variant 1 (200 nM) reaction mix in the presence of GTP (4 mM) for 15 minutes, and a 3 pL portion of the reaction mixture was loaded onto a 0.8% agarose gel to visualize the fragmented DNA. As shown in Figure 13, the DNA fragments were observed within the 100-300 bp range, demonstrating the efficiency of variant 1 in fragmenting AT-rich genomes (~70% AT) effectively within a short reaction time.Table 2. Reaction parameters for variant 1 applicable in general and Whole genome sequencing (WGS) analysis of the sequencing parameters for the representative bacterial genome (E. coli) fragmented by variant 1 (preformed lyophilized master mix) in 15 mins.Reaction and WGS parametersPreformed Lyophilized reaction mixassessed1. DNA input amount As low as 10 ng to mgApplicable to any kind of DNA (PCR, Plasmid, 2. DNA originGenomic DNA of any origin and composition) <15 mins (bench time)3. Time of incubation at 37°C Heat inactivation at 65°C / 5 mins (for further processing)4. Intermediate DNA purificationNot requiredstep post fragmentation5. Average DNA fragment size 159 bp ( 77- 486 bp) (for E. coli gDNA)6. Percentage genome coverage 99.72% (for E. coli gDNA)7. Mean mapping quality 40.7 (for E. coli gDNA)8. GC% 52.98% (for E. coli gDNA)Table 2 shows the general reaction parameters applicable for fragmentation of any DNA sample with quantity as low as 10 ng and irrespective of its AT or GC richness and methylation status. Fragments generated by variant 1 for any genome are compatible with any library preparation kit without the need of any intermediate DNA purification step, libraries prepared from only one representative genomic DNA (E. coli) was chosen for WGS analysis for assessing sequencing parameters. The genome coverage analysis upon WGS of libraries prepared from variant 1 generated fragments showed >99% coverage (Table 2) which indicates that variant 1 fragmentation not only produces short fragments of uniform size but also allows almost full coverage of the genome with such insert lengths. Moreover, a high mean mapping quality score of 40.7 is a great indication of very high confidence in read alignment. The entire sequencing analysis has been done with a starting DNA input of just ~6ng / pL suggesting the invention’s feasibility for even scarce sample availability in exceptional cases. Moreover, a very close observed to expected GC content (52.98%: 51%) proved that the enzymatic digestion did not create any sequencing bias as is frequently observed with other enzyme-based techniques.As shown in Figure 14, the genome coverage profdes of sequencing reads at 45 x mean depth and at 640 x demonstrated comparable distribution patterns. The similarity in coverage profdes indicated that the DNA fragments generated by variant 1 were capable of producing high-quality sequencing data with reliable genome representation, even at a lower read depth of 2 million reads. This result highlights the efficiency and robustness of variant 1-generated DNA fragments for high-confidence sequencing applications.ADVANTAGES OF THE PRESENT DISCLOSURE

[0095] The present invention can be applied to any field of technology / study / analysis / sector where uniform fragmentation of a DNA substrate of any origin (bacterial, human, insects, plants, bacteriophages, cell free DNA etc.) of any size or length (ranging from lOObp to genomic DNA), of any conformation (plasmid; supercoiled, nicked, linear, circular) is required producing desirable uniform length fragments in the range of 100-300 or more bp in less than or equal to 15 minutes. This application can be extended to all methods that require nucleic acid fragmentation including processing DNA substrates from metagenomic samples, removing any DNA contamination from RNA samples, generation of chromatin fragments during chromatin immunoprecipitation (ChIP) applicable to practically all kinds of DNA samples including genomic DNA, plasmid DNA, chromatin DNA, cell free DNA of multiple origin.References:1. Bashkirov, I. (2019). Methods and apparatuses for nucleic acid shearing by sonication (U. S Patent No. US 10, 329, 598 B2). United States Patent. https: / / patents.google.com / patent / US9127306B2 / en.2. Guan (2021). Method for fragmenting dna by nick translation. (U. S Patent No. US 11, 021, 701 B2) United States Patent. https: / / patents.google.com / patent / US11021701B2 / en.3. Steemers (2022). Methods and compositions for nucleic sequencing. (U. S Patent No.2022 / 0213470 Al) United States Patent.https: / / patents.google.com / patent / US20220213470Al / en?oq=US+2022%2f0213470+Al.4. Ribarska et al. BMC Genomics (2022). Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing, https: / / doi.org / 10.1186 / sl2864-022-08316-y5. Poptsova et al.(2014). Non-random DNA fragmentation in next-generation sequencing. Scientific Reports, 4, 1-6. https: / / doi.org / 10.1038 / srep045326. Belyaev. (2015) Methods and compositions for dna fragmentation and tagging by transposases.(U. S Patent No. 9, 005, 935 B2) United States Patent. https: / / patents.google.com / patent / US9005935B2 / en?oq=US+9%2c005%2c935+B2.7. Wiedenheft et al.2018. Methods of generating nucleic acid fragments. (U. S Patent No. 10, 087, 431 B2) United States Patent. https: / / patents.google.com / patent / US10087431B2 / en 8. Hindson et al. (2020). Methods and systems for processing polynucleotides. (U. S Patent No. 2020 / 0291472 Al). United States Patent. https: / / patents.google.com / patent / US20200291472Al / en.9. Nirwan et al., Nature Communications 2019. Structure -based mechanism for activation of the AAA+ GTPase McrB by the endonuclease McrC. https: / / doi.org / 10.1038 / s41467-019- 11084-1.

Claims

We Claim:

1. A McrBC vanant for generating nucleic acid fragments having an average size in the range of 100 to 300 base pairs or longer from a nucleic acid sample, wherein the McrBC variant comprises a polypeptide sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

2. The McrBC variant as claimed in claim 1, wherein the McrBC variant comprises a mutation at position L68Y (McrBL68YC variant), or a mutation at position L68F (McrBL68FC variant) relative to wild-type McrBC.

3. The McrBC variant as claimed in claim 2, wherein the McrBL68YC variant is encoded by a nucleic acid sequence having SEQ ID NO: 1.

4. The McrBC variant as claimed in claim 2, wherein the McrBL68FC variant is encoded by a nucleic acid sequence having SEQ ID NO: 2.

5. The McrBC variant as claimed in any one of claims 1-4, wherein the McrBC variant is a combination of the McrBL68YC and McrBL68FC variants in a molar ratio of 1: 10 to 10:1, respectively.

6. The McrBC variant as claimed in claim 1, wherein the McrBC variants exhibit nuclease activity at a pH of 7.0 to 8.0 and at a temperature of 25°C to 40°C.

7. A method of fragmenting a nucleic acid sample into fragments, comprises the steps of:(a) contacting the nucleic acid sample with a McrBC variant as claimed in claim 1 in presence of nucleoside triphosphate (NTP), and a buffer;(b) incubating the reaction mixture at about 37°C for less than or equal to 15 minutes; and(c) heat-inactivating the reaction at about 65 °C for about 5 minutes to obtain DNA fragments having an average size of 100 to 300 base pairs or longer.

8. The method as claimed in claim 7, wherein the nucleic acid sample is a methylated or a non-methylated form of deoxyribo nucleic acid (DNA) selected from genomic DNA, plasmid DNA, chromatin DNA, extrachromosomal DNA, and cell free DNA.

9. The method as claimed in claim 8, wherein the buffer comprises a monovalent salt, a divalent metal ion source, and a reducing agent.

10. The method as claimed in claim 8, wherein the nucleic acid sample is present in an amount of 1 ng to 500 ng.

11. A reagent composition for fragmenting or sizing a nucleic acid sample, comprising: (i) a McrBC variant as claimed in any one of claims 1 to XX, and combinations thereof;(ii) a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent; and(iii) nucleoside triphosphate (NTP),Wherein the McrBC is freshly purified, or flash frozen and stored at -80°C or at - 20°C, or stored in liquid state at -20°C with 0 to 30% glycerol.

12. A kit for fragmenting or sizing a nucleic acid sample, comprising:(i) the reagent composition as claimed in claim 11; and(ii) user instructions specifying reaction conditions.

13. A lyophilized reagent composition for fragmenting or sizing a nucleic acid sample, comprising:(i) a McrBC variant as claimed in any one of claims 1 to XX, and combinations thereof;(ii) a buffer comprising a monovalent salt, a divalent metal ion source, and a reducing agent;(iii) nucleoside triphosphate (GTP); and(iv) trehalose or any other stabilizer of protein during lyophilization,wherein the lyophilized reagent composition comprises a preformed enzyme mixture containing components (i) to (iv), and is stored at room temperature or 4°C, which retains enzymatic activity upon reconstitution.

14. A kit for fragmenting or sizing a nucleic acid sample, comprising:(i) the lyophilized reagent composition as claimed in claim 13;(ii) nuclease-free water for reconstitution; and(iii) user instructions specifying reconstitution of the lyophilized reagent composition with nuclease-free water prior to use, and reaction conditions.

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