Salt active nuclease compositions and methods

Abstract

The present disclosure relates, according to some embodiments, to systems, apparatus, compositions, methods, and workflows that include salt active nucleases (e.g., salt active nuclease variants) with desirable properties including, for example, salt tolerance.

Description

SALT ACTIVE NUCLEASE COMPOSITIONS AND METHODSCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Application No. 63 / 639,305 filedApril 26, 2024 and U.S. Provisional Application No. 63 / 640,498 filed April 30, 2024. Thecontents of all of the above are hereby incorporated in their entirety by reference.SEQUENCE LISTING STATEMENTThis disclosure includes a Sequence Listing submitted electronically in .xml formatunder the file name “NEB-501.xml" created on April 26, 2024, and having a size of 15.4 KB.This Sequence Listing is incorporated herein in its entirety by this reference.BACKGROUNDMany purification methods involve one or more steps that include high salt conditions.High salt conditions may be desirable, for example, to stabilize one or more molecules ofinterest, to control its interactions (e.g., drive its association or dissociation) with one or morecomponents present in the composition in which it exists, and / or to support desirable conditionsfor cell lysis. However, high salt conditions may limit or prevent activity of enzymes used toremove contaminants such as polynucleotides during a purification of a protein, metabolite,virus, cell or other material of interest.SUMMARYAccordingly, needs have arisen for improved nucleases that are active in the presenceof salts. The present disclosure provides salt active nucleases (including salt active nucleasevariants) and related methods, kits, and compositions. In some embodiments, a salt activenuclease variant may have an amino acid sequence that is ≥90% (e.g., ≥92%, ≥94%, ≥95%,≥96%, ≥98%, ≥99%) identical to a reference salt active nuclease (e.g., one or more of SEQ IDNOS:1-4), having at its position corresponding to position 52 of SEQ ID NO:1 an amino acidother than Q (glutamine) (e.g., may be S52 (serine52)), and having catalytic activity as anendonuclease and / or an endoribonuclease at a total salt concentration of 250 mM to 1 M. Insome embodiments, a salt active nuclease variant may have an amino acid sequence that isidentical to SEQ ID NO:2 or identical to SEQ ID NO:3. A salt active nuclease variant identicalto SEQ ID NO:3 may have, for example, a signal peptide, a purification tag, or a linker at X1and / or X215. For example, a X1 may be a signal peptide, a purification tag, or a linker andX215 may be any amino acid or absent, or (b) X1 may be any amino acid or absent and X215may be a purification tag or a linker or X215. In a salt active nuclease variant having less than100% identity to a reference sequence, each non-identical position may constitute a substitutionrelative to the reference. Salt active nuclease variants having less than 100% identity (e.g.,with each non-identical position constituting a substitution) to a reference sequence maycomprise conservative (e.g., exclusively conservative) substitutions relative to the referenceand / or non-conservative (e.g., zero, one, two, three, four, figure, six, or more non-conservativesubstitutions). According to some embodiments, a salt active nuclease variant may comprisean amino acid sequence having at least 98% identical to SEQ ID NO:2 and wherein X47-X51and X53-X56 are each, independently, any amino acid. According to some embodiments, asalt active nuclease variant may comprise an amino acid sequence having at least 98% identicalto SEQ ID NO:2 and wherein X47, X49-X51, and X53-X55 each, independently, may be C,D, E, H, K, N, P, Q, R, S, T, or Y, and X48 and X56 are each, independently, A, L, I, M, W,F, C, G, or P.A salt active nuclease variant, according to some embodiments, may have ≥30% (e.g.,≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%) of its peak catalytic activity in the presence of atotal salt concentration of 250 mM to 1 M (e.g., 250 mM to 750 mM, 300 mM to 600 mM), thesalt consisting essentially of NaCl, KCl, or NaCl and KCl. In some embodiments, a salt activenuclease variant may have ≥30% (e.g., ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%) of its peakcatalytic activity in the presence of a total salt concentration of 50 mM to 150 mM (e.g., 75mM to 125 mM), the salt consisting essentially of MgCl2, Mg(NO3)2, or MgCl2 and Mg(NO3)2.A salt active nuclease variant may have, in some embodiments, ≥60% (e.g., ≥65%,≥70%, ≥75%, ≥80%) of its peak catalytic activity at pH ≥ 8. According to some embodiments,a salt active nuclease may have ≥60% (e.g., ≥65%, ≥70%, ≥75%, ≥80%) of its peak catalyticactivity at 40°C - 55°C. A salt active nuclease may be an immobilized salt active nucleasevariant comprising the salt active nuclease variant and a solid support (e.g., a magnetic bead,an agarose bead, a polystyrene bead, a polyacrylamide bead or a chitin bead.).The present disclosure also provides compositions including salt active nucleases. Forexample, compositions may include a salt active nuclease variant and one or more salts (e.g., atotal of 50 mM – 150 mM of MgCl2, Mg(NO3)2, or MgCl2 and Mg(NO3)2; a total of 250 mM1 M of NaCl, KCl, or NaCl and KCl). In some embodiments, a composition may comprisea buffering agent and / or optionally one or more other materials of interest (e.g., proteins,polysaccharides, cells, phage particles, viral particles, liposomes, and / or micelles). Salt activenucleases may be included in fusions. For example, a fusion protein may comprise a singlepolypeptide chain, the single peptide chain comprising (a) a salt active nuclease variant and (b)a SSO7d DNA binding peptide, a transcription factor, an antibody, protein A, a maltose bindingdomain, a histidine tag, a chitin binding domain, an alpha mating factor, an Oʻ-alkylguanine-DNA alkyltransferase, and / or albumin.The present disclosure also provides methods including salt active nucleases. A methodmay comprise, for example, hydrolyzing DNA and RNA. In some embodiments, a method forhydrolyzing DNA and RNA may comprise contacting (a) a composition comprising DNA andRNA, and (b) a salt active nuclease variant to form a reaction mixture comprising DNAhydrolysis products and RNA hydrolysis products. A reaction mix may have any desired pH(e.g., 6.5-12, 8-10) and / or any desired temperature (e.g., 2°C-60°C, 40°C-55°C). In someembodiments, a composition comprising DNA and RNA and / or a reaction mixture may furthercomprise (a) a total salt concentration of 250 mM to 1 M, the salt consisting essentially of (i)NaCl, (ii) KCl, or (iii) NaCl and KCl or (b) a total salt concentration of 50 mM to 150 mM, thesalt consisting essentially of (i) MgCl2, (ii) Mg(NO3)2, or (iii) MgCl2 and Mg(NO3)2. In someembodiments, the reaction mixture comprises ≤10% of the DNA that was in the compositioncomprising DNA and RNA (prior to contact with the nuclease) and / or ≤10% of the RNA thatwas in the composition comprising DNA and RNA (prior to contact with the nuclease). Amethod may further comprise detecting the amount of DNA and / or RNA is present in thecomposition comprising DNA and / or RNA and / or may further comprise detecting the amountof DNA and / or RNA is present in the reaction mixture. In some embodiments, a compositioncomprising DNA and RNA may further comprise one or more DNA-binding proteins (e.g.,histones). For example, a composition comprising DNA and RNA may further comprise oneor more nucleosomes. In some embodiments, a method may include isolating and / or purifyinga material of interest from the composition and / or the reaction mixture.BRIEF DESCRIPTION OF THE FIGURESThe file of this patent contains at least one drawing executed in color. Copies of thispatent with color drawing(s) will be provided by the Patent and Trademark Office uponrequest and payment of the necessary fee.FIGURES 1A, 1B, and 1C show nuclease activity of example salt active nucleasesvsEndA and vsEndA-Q52S on dsDNA (FIGURE 1A), ssDNA (FIGURE 1B) and RNA(FIGURE 1C) substrates at various NaCl concentrations.FIGURE 2 shows nuclease activity of an example salt active nuclease vsEndA-Q52Sin various NaCl concentrations compared to 2 commercially available endonucleases.FIGURE 3 shows nuclease activity of example salt active nucleases vsEndA andvsEndA-Q52S on dsDNA across a range of pHs.FIGURE 4 shows nuclease activity of example salt active nucleases vsEndA andvsEndA-Q52S on dsDNA across a range of temperatures.FIGURE 5 shows nuclease activity of example salt active nucleases vsEndA (left gel)and vsEndA-Q52S (right gel) on lambda DNA at pH 8.5 and 500 mM NaCl.FIGURE 6 shows nuclease activity of example salt active nucleases vsEndA (left gel)and vsEndA-Q52S (right gel) on a total RNA extract from HEK cells at pH 8.5 and 500 mMNaCl.FIGURE 7 shows nuclease activity of example salt active nucleases vsEndA (left gel)and vsEndA-Q52S (right gel) on a fluorescent RNA substrate at pH 8.5 and 500 mM NaCl.FIGURES 8A, 8B, and 8C show nuclease activity of example salt active nucleasesvsEndA and vsEndA-Q52S in the presence of KCl (FIGURE 8A), MgCl2 (FIGURE 8B), andMg(NO3)2 (FIGURE 8C).FIGURE 9 shows nuclease activity of an example salt active nuclease vsEndA-Q52Sand 2 commercially available endonucleases on calf thymus DNA at 500 mM NaCl pH 8.5 at25°C (upper gel) and 4°C (lower gel).FIGURES 10A and 10B shows an example embodiment of linear release offluorescence from dsDNA (FIGURE 10A) or ssDNA (FIGURE 10B) substrates relative tothe concentration of both enzymes.FIGURE 11 shows nuclease activity of an example salt active nuclease vsEndA-Q52Sand a commercially available endonuclease (Supplier B) on mononucleosomal DNA at 100mM, 250 mM, and 500 mM NaCl.BRIEF DESCRIPTION OF THE SEQUENCESSome embodiments of this disclosure relate to the following provided sequences ofexample polynucleotides and / or example polypeptides.SEQ ID NO:1 is an example salt active nuclease variant.APPSSFSKAKKEAVKIYLDYPTSFYCGCDITWKNKKKGIPELESCGYQVRKSEKRASRIEWEHVVPAWQFGHQRQCWQKGGRKNCTRNDKQFKSMEADLHNLVPAIGEVNGDRSNFRFSQWNGSKGAFYGQCAFKVDFKGRVAEPPAQSRGAIARTYLYMNNEYKFNLSKAQRQLMEAWNKQYPVSTWECTRDERIAKIQGNHNQFVYKACTKSEQ ID NO:2 is an example salt active nuclease variant, wherein X47, X49-X51, andX53-X55 independently may be any amino acid (e.g., one of C, D, E, H, K, N, P, Q, R, S, T,and Y), X52 is any amino acid other than Q (e.g., C, D, E, H, K, P, R, S, T or Y) and X48 andX56 may be any amino acid (e.g., one of A, L, I, M, W, F, C, G, and P). The underlinedamino acids may be spatially close to X52 and amenable to conservative (or non-conservative) substitution to improve cold activity, salt activity, salt tolerance, and / or saltindifference.APPSSFSKAKKEAVKIYLDYPTSFYCGCDITWKNKKKGIPELESCGYXXXXXXXXXSRIEWEHVVPAWQFGHQRQCWQKGGRKNCTRNDKQFKSMEADLHNLVPAIGEVNGDRSNFRFSQWNGSKGAFYGQCAFKVDFKGRVAEPPAQSRGAIARTYLYMNNEYKFNLSKAQRQLMEAWNKQYPVSTWECTRDERIAKIQGNHNQFVYKАСТКSEQ ID NO:3 is an example salt active nuclease variant, wherein X1 may be apurification tag, a linker, any amino acid or may be absent, X53 is any amino acid other thanQ (e.g., C, D, E, H, K, N, P, R, S, T or Y), and X215 may be a signal peptide, a purificationtag, a linker, any amino acid or may be absent.XAPPSSFSKAKKEAVKIYLDYPTSFYCGCDITWKNKKKGIPELESCGYQVRKXEKRASRIEWEHVVPAWQFGHQRQCWQKGGRKNCTRNDKQFKSMEADLHNLVPAIGEVNGDRSNFRFSQWNGSKGAFYGQCAFKVDFKGRVAEPPAQSRGAIARTYLYMNNEYKFNLSKAQRQLMEAWNKQYPVSTWECTRDERIAKIQGNHNQFVYKACTKXSEQ ID NO:4 is an example salt active nuclease variant, wherein a cleaved signalpeptide is underlined.MKLIRLVISLIAVSFTVNVMAAPPSSFSKAKKEAVKIYLDYPTSFYCGCDITWKNKKKGIPELESCGYQVRKSEKRASRIEWEHVVPAWQFGHQRQCWQKGGRKNCTRNDKQFKSMEADLHNLVPAIGEVNGDRSNFRFSQWNGSKGAFYGQCAFKVDFKGRVAEPPAQSRGAIARTYLYMNNEYKFNLSKAQRQLMEAWNKQYPVSTWECTRDERIAKIQGNHNQFVYKACTKSEQ ID NO:5 is an example salt active nuclease (vsEndA; Q2XSL7), wherein a(cleavable) 21-amino acid signal peptide is underlined.MKLIRLVISLIAVSFTVNVMAAPPSSFSKAKKEAVKIYLDYPTSFYCGCDITWKNKK[PELESCGYQVRKQEKRASRIEWEHVVPAWQFGHQRQCWQKGGRKNCTRNDKQFKSMEADLHNLVPAIGEVNGDRSNFRFSQWNGSKGAFYGQCAFKVDFKGRVAEPPAQSRGAIARTYLYMNNEYKFNLSKAQRQLMEAWNKQYPVSTWECTRDERIAKIQGNHNQFVYKACTKSEQ ID NO:6 is an example salt active nuclease (VvnI)APPSSFSAAKQQAVKIYQDHPISFYCGCDIEWQG_KKGIPNLETCGYQVRKQOTRASRIEWEHVVPAWQFGHHRQCWQKGGRKNCSKNDQQFRLMEADLHNLTPAIGEVNGDRSNFNFSQWNGVDGVSYGRCEMQVNFKQRKVMPQTELRGSIARTYLYMSQEYGFQLSKQQQQLMQAWNKSYPVDEWECTRDDRIAKIQGNHNPFVQQSCOTOSEQ ID NO:7 is an example hairpin dsDNA substrate comprising 5' FAM labeledand 3' black hole quencher ("BHQ1") labeled ends. When the probe is heated to 95°C andthen gradually cooled, it folds into a hairpin structure in which the quencher quenches thefluorophore. Cleavage of the DNA hairpin by a nuclease (e.g., a SAN) releases FAM,resulting in an increase in fluorescence that can be measured at 517 nm using aspectrophotometer.56-FAM-TCTAAGCCGTGTACATTTTTTGTACACGGCTTAGA-BHQ1-3'SEQ ID NO:8 is an example linear ssDNA substrate comprising 5' FAM and 3'BHQ1 labeled ends.56-FAM-TGAAGTAATCTGTTA-BHQ1-3'SEQ ID NO:9 is an example linear RNA substrate optionally comprising a FAM-labeled 5' end.56-FAM-AAGGAGAAGAGAAGAGGAAGAAAACUAACACAGGAGAGAGAAGGASEQ ID NO:10 is an example DNA sequence encoding an example salt activenuclease.GCACCACCAAGCAGTTTTTCTAAAGCTAAGAAAGAGGCTGTGAAAATTTACCTGGACТАСССТАСTAGTTTTTACTGTGGTTGTGATATTACATGGAAAAATAAAAAGAAAGGAATTCCGGAGTTAGAGTCATGTGGGTACCAGGTCCGTAAAAGCGAAAAGCGTGCGAGCCGTATCGAGTGGGAACACGTCGTACCTGCATGGCAATTTGGACATCAGCGTCAATGCTGGCAGAAAGGGGGACGCAAAAATTGCACTCGCAACGATAAACAATTCAAAAGCATGGAGGCAGATTTGCATAACCTTGTACCTGCCATCGGCGAGGTGAATGGAGATCGCTCTAACTTCCGCTTTTCGCAGTGGAATGGGTCAAAGGGGGCGTTCTATGGGCAATGCGCCTTCAAGGTAGATTTCAAAGGTCGCGTTGCGGAGCCACCAGCCCAGTCCCGTGGGGCCATTGCCCGCACTTATTTATACATGAATAACGAGTATAAGTTTAATTTGAGCAAAGCCCAGCGTCAACTTATGGAAGCCTGGAATAAACAATATCCGGTGAGCACCTGGGAGTGCACTCGTGATGAGCGTATTGCCAAAATCCAGGGGAATCATAACCAATTCGTCTATAAGGCGTGCACTAAGTGADETAILED DESCRIPTIONThe present disclosure relates, in some embodiments, to systems, apparatus,compositions, and / or methods for cleaving polynucleotides in the presence of salt (e.g., ≥250mM salt). For example, the present disclosure provides systems, apparatus, compositions,methods, and workflows that include salt active nucleases with desirable properties including,for example, salt tolerance. A composition may comprise, in some embodiments, a salt activenuclease. For example, a composition may include a catalytically active salt active nucleasecomprising an amino acid sequence having ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥98%,or ≥99% identity to SEQ ID NO:1 and ≥50 mM, ≥100 mM, ≥150 mM, ≥200 mM, ≥250 mM,≥300 mM, ≥350 mM, ≥400 mM, ≥450 mM, ≥500 mM, ≥600 mM, ≥700 mM, ≥800 mM, or≥900 mM salt (e.g., NaCl).General ConsiderationsAspects of the present disclosure can be understood in light of the provideddescriptions, figures, sequences, embodiments, section headings, and examples, none of whichshould be construed as limiting the entire scope of the present disclosure in any way.Accordingly, the innovations set forth herein should be construed in view of the full breadthand spirit of the disclosure.Each of the individual embodiments described and illustrated herein has discretecomponents and features which can be readily separated from or combined with thecomponents and / or features of any of the other several embodiments without departing fromthe scope or spirit of the present teachings. Lists of example species within a particular genusmay vary in length at different places throughout the disclosure. Species lists shortened forconvenience shall not be construed to exclude example species listed elsewhere in thespecification. Any recited method can be carried out in the order of events recited or in anyother order which is logically possible.Unless otherwise expressly stated to be required herein, each component, feature, andmethod step disclosed herein is optional and the disclosure contemplates embodiments inwhich each optional element may be expressly excluded. As such, this statement is intendedto serve as antecedent basis for use of such exclusive terminology as "solely," "only" and thelike in connection with the recitation of claim elements or use of a “negative" limitation. It isfurther intended to serve as antecedent basis for use of such elective terminology as"optionally" and the like in connection with the recitation of one or more claim elements.Unless otherwise defined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which this disclosurebelongs. Still, certain terms are defined herein with respect to embodiments of the disclosureand for the sake of clarity and ease of reference.Sources of commonly understood terms and symbols may include: standard treatisesand texts such as Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, NewYork, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975);Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York,1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (OxfordUniversity Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A PracticalApproach (IRL Press, Oxford, 1984); Singleton, et al., Dictionary of Microbiology andMolecular biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, theHarper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) and the like.As used herein and in the appended claims, the singular forms “a” and “an” includeplural referents unless the context clearly dictates otherwise. For example, the term "a protein"refers to one or more proteins, i.e., a single protein and multiple proteins.Numeric ranges are inclusive of the numbers defining the range. All numbers shouldbe understood to encompass the midpoint of the integer above and below the integer i.e., thenumber 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55 etc. When samplenumerical values are provided, each alone may represent an intermediate value in a range ofvalues and together may represent the extremes of a range unless specified. Percent rangeswith only one end point (e.g., ≥ 90% or ≤ 10%) optionally include a second endpoint at themaximum or minimum percentage (e.g., ≥ 90% includes a range of 90%-100% and ≤ 10%includes a range of 0%-10%). Ranges (including percent ranges) with only one end point (e.g.,≥ 90 or ≤ 10) optionally include a second endpoint 10% higher or 10% lower than the providedendpoint (e.g., ≥ 90 includes a range of 90-99 and ≤ 10 includes a range of 1-10). Concentrationpercentages are w / v percentages unless otherwise indicated.In the context of the present disclosure, "buffer" and "buffering agent" refer to achemical entity or composition that itself resists and, when present in a solution, allows suchsolution to resist changes in pH when such solution is contacted with a chemical entity orcomposition having a higher or lower pH (e.g., an acid or alkali). Examples of suitable non-naturally occurring buffering agents that may be used in disclosed compositions, kits, andmethods include HEPES, MES, MOPS, TAPS, tricine, and Tris. Additional examples ofsuitable buffering agents that may be used in disclosed compositions, kits, and methods includeACES, ADA, BES, Bicine, CAPS, carbonic acid / bicarbonic acid, CHES, citric acid, DIPSO,EPPS, histidine, MOPSO, phosphoric acid, PIPES, POPSO, TAPS, TAPSO, andtriethanolamine.As used herein, "catalytically active" refers to the property of a molecule (e.g., aproteinaceous molecule or macromolecule) to function as a catalyst of one or more chemicalreactions relative to one or more substrates and products. A catalytically active salt activenuclease or salt active nuclease variant, for example, hydrolyzes one or morepolydeoxyribonuceic acids to yield (however briefly) products comprising at least one 5'-phosphorylated oligonucleotide. Catalytic activity of salt active nuclease and / or salt activenuclease variants may be assessed using existing techniques applied to one or more modelsubstrates and / or one or more substrates of interest. For example, effective assays for catalyticactivity of salt active nuclease may include size fractionation of products (e.g., on gels or othermatrices), radioactive assays, and fluorometric assays (e.g., #234056, # abab252898, AbCamPLC, Cambridge, U.K.; PicoGreen, Nucl. Acids Res. 2003 31(18):e111)). Catalytic activitymay be assessed with respect to loss of original DNA (e.g., percent of original DNAremaining), concentration of DNA (e.g., above 10 nts, above 8 nts, above 6 nts, above, above3 nts in length, 4 nts, or above 2 nts in length), and / or metrics that serve as a proxy thereof.Peak catalytic activity or peak activity is the highest observed catalytic activity inreactions representing the full range of total salt concentrations, but otherwise maintained underthe same conditions (e.g., buffer, temperature, reaction time, type and quantity of substrate,quantity of enzyme, pH). For example, to assess peak catalytic activity empirically, a salttolerant nuclease variant may be divided into a plurality of aliquots. Each aliquot may becombined with a buffering agent, a test substrate (e.g., SEQ ID NOS:7-9), and differingconcentrations of salt (e.g., 0 mM, 250 mM, 500 mM, 750 mM, and 1 M), incubated for aselected time at a selected temperature, and assayed for nuclease activity by any methodprovided herein or otherwise available. Peak activity for the salt tolerant nuclease variantassayed would be the highest observed activity among the tested concentrations (e.g., 0 mM,250 mM, 500 mM, 750 mM, and 1 M). The activity at the other salt concentrations may beexpressed as a percentage of the peak activity.In the context of the present disclosure, "container" refers to a human-made container.A container may comprise one or more walls (e.g., defining an interior volume) and optionallyone or more openings. Containers comprising one or more openings may further comprise oneor more closures (e.g., removable closures) for some or all such openings. A closure optionallymay comprise an aperture or a septum, for example, to provide fluid communication with avolume of the container and a connected or inserted tube or syringe. Examples of containersinclude boxes, cartons, bottles, tubes (e.g., test tubes, microcentrifuge tubes), plates (e.g., 96-well, 384-well plates), vials, pipette tips, and ampules. Containers and / or closures maycomprise any desired material including paper, plastics, glass, silicone, composites, metals,alloys, or combinations thereof. Containers and / or closures may comprise materials that arecompostable, recyclable, and / or sustainable.In the context of the present disclosure and with respect to an amino acid residue or anucleotide base position, "corresponding to" refers to positions that lie across from one anotherwhen sequences are aligned, e.g., by the BLAST algorithm. An amino acid position in afunctional or structural motif in one endonuclease may correspond to a position within afunctionally equivalent functional or structural motif in another endonuclease.With respect to polynucleic acid, "digest," as used herein, refers to hydrolyzing orotherwise reducing the size of such polynucleic acid. Unless qualified, digesting a polynucleicacid (e.g., DNA or RNA) includes all degrees of hydrolyzing or otherwise reducing the size ofsuch polynucleic acid, partially up to and including fully. Sites of hydrolysis may be regardedas independent of the nucleotide sequence (“non-specific”), even if some sequence bias isobserved under some conditions.In the context of the present disclosure, "fusion" refers to two or more polypeptides,subunits, or proteins covalently joined to one another (e.g., by a peptide bond). For example,a protein fusion may refer to a non-naturally occurring polypeptide comprising a protein ofinterest covalently joined to a second polypeptide. Examples of a second polypeptide includea reporter protein (e.g., a green fluorescent protein), a purification tag (e.g., a 6xHis or 8xHistag), and expression tag, a polynucleotide binding protein, an enzyme, a conjugation tag (e.g.,a SNAP® tag), and a peptide linker (e.g., a flexible linker, an inflexible linker, a cleavablelinker). Unless otherwise disclosed, the protein of interest may be nearer to the N-terminal endor nearer to the C-terminal end than the second polypeptide to which it is joined. A fusionprotein may have one or more heterologous domains added to the N-terminus, C-terminus, andor the middle portion of the protein. A fusion may comprise a non-naturally occurringcombined polypeptide chain comprising two proteins or two protein domains joined directly toeach other by a peptide bond or joined through a peptide linker. If two parts of a fusion proteinare "heterologous", they are not part of the same protein in its natural state. In someembodiments, a fusion may comprise a variant salt active nuclease covalently joined to asecond polypeptide. In some embodiments, a variant salt active nuclease may include a fusionto an exogenous DNA binding domain, examples of which are provided in Table 1 of U.S.Patent No. 11,259,184. Examples of fusion proteins include a salt active nuclease variant fusedto an SSO7d DNA binding peptide (see for example, US Patent 6,627,424), a transcriptionfactor (see for example, US patent 10,041,051), an antibody, protein A (e.g., SpA), a bindingdomain suitable for immobilization such as maltose binding domain (MBP), a histidine tag("His-tag"), chitin binding domain, alpha mating factor or an O-alkylguanine-DNAalkyltransferase (e.g., SNAP-Tag®, New England Biolabs, Ipswich, MA (see for example USpatents 7,939,284 and 7,888,090)), and / or albumin. The binding peptide may be used toimprove solubility or yield of the salt active nuclease variant during the production of theprotein reagent. Other examples of fusion proteins include fusions of a salt active nuclease anda heterologous targeting sequence, a linker, an epitope tag, a detectable fusion partner, such asa fluorescent protein, ẞ-galactosidase, luciferase and / or functionally similar peptides.Components of a fusion protein may be joined by one more peptide bonds, disulfide linkages,and / or other covalent bonds.In the context of the present disclosure, “immobilized" refers to covalent attachment ofan enzyme to a solid support with or without a linker. Examples of solid supports include beads(e.g., magnetic, agarose, polystyrene, polyacrylamide, chitin). Beads may include one or moresurface modifications (e.g., O6-benzyleguanine, polyethylene glycol) that facilitate covalentattachment and / or activity of an enzyme of interest. For example, a support may comprise aligand and an enzyme may have a receptor for such ligand or an enzyme may comprise a ligandand a support may comprise a receptor for such ligand. Receptor-ligand binding may becovalent or non-covalent. Non-covalent attachment (e.g., avidin:biotin, chitin:CBP) may beuseful in some embodiments, for example, where the level of dissociation of the binding partneris deemed tolerable. A linker may be disposed between a support and an enzyme. For example,linker disposed between a support and an enzyme may have a first covalent bond to the supportand a second covalent bond to the enzyme. An immobilized enzyme comprising a ligand-receptor attachment may have a linker disposed between the support and the ligand-receptorattachment, a linker disposed between the enzyme and the ligand-receptor attachment, or both.An immobilized enzyme comprising a linker may also comprise an optional covalent bonddirectly between the enzyme and the support. A linker may be of any desired length and haveany desired range of motion. A peptide linker may comprise one or more repeats (e.g., 1-10repeats) of glycine-serine.In the context of the present disclosure, “modified nucleotide" refers to nucleotideshaving a modification on the sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose,and hexose); and / or in the phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages); and / or in the nucleotide base (e.g., as described in US 8,383,340;WO 2013 / 151666; US 9,428,535 B2; US 2016 / 0032316). Examples of modified nucleotidesinclude pseudouridine and N1-methyl-pseudouridine.In the context of the present disclosure, “non-naturally occurring" refers to a molecule(e.g., a polynucleotide, polypeptide, carbohydrate, or lipid) or composition that does not existin nature. Such a molecule or composition may differ from naturally occurring molecules orcompositions in one or more respects. For example, a polymer (e.g., a polynucleotide,polypeptide, or carbohydrate) may differ in the kind and arrangement of the component parts(e.g., nucleotide sequence, amino acid sequence, or sugar molecules). A polymer may differfrom a naturally occurring polymer with respect to the molecule(s) to which it is linked. Forexample, a "non-naturally occurring" polypeptide (e.g., protein) may differ from naturallyoccurring polypeptides in its secondary, tertiary, or quaternary structure, by having (or lacking)a chemical bond (e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfidebond, an ester bond, and ether bond, and others) to a lipid, a carbohydrate, a second polypeptide(e.g., a fusion protein), or any other molecule. Similarly, a "non-naturally occurring"polynucleotide or nucleic acid may comprise (or lack) one or more other modifications (e.g.,an added label or other moiety) to the 5'- end, the 3' end, and / or between the 5'- and 3'-ends(e.g., methylation) of the nucleic acid. A "non-naturally occurring" molecule or compositionmay differ from naturally occurring compositions in one or more of the following respects: (a)having components that are not combined in nature, (b) having components in ratios and / orconcentrations not found in nature, (c) lacking one or more components otherwise found innaturally occurring molecules or compositions (e.g., a cell-free composition, a chromosome-free composition, a histone-free composition, a polymerase-free composition, a cell membrane-free composition), (d) having a form not found in nature (e.g., dried, freeze dried, lyophilized,crystalline, aqueous, immobilized), and (e) having one or more additional components beyondthose found in nature (e.g., a buffering agent, a detergent, a dye, a solvent or a preservative).With reference to an amino acid, "position" refers to the place such amino acid occupiesin the primary sequence of a peptide or polypeptide numbered from its amino terminus to itscarboxy terminus. A position in one primary sequence may correspond to a position in a secondprimary sequence, for example, where the two positions are opposite one another when the twoprimary sequences are aligned using an alignment algorithm (e.g., BLAST (Journal ofMolecular Biology. 215 (3): 403–410) using default parameters (e.g., expect threshold 0.05,word size 3, max matches in a query range 0, matrix BLOSUM62, Gap existence 11 extension1, and conditional compositional score matrix adjustment) or custom parameters). An aminoacid position in one sequence may correspond to a position within a functionally equivalentmotif or structural motif that can be identified within one or more other sequence(s) in adatabase by alignment of the motifs.As used herein, "salt" refers to a material comprising an organic or inorganic cation ofa base (e.g., Na+, Ca2+, K+, Mn+, Mg2+) and an organic or inorganic anion of an acid (e.g., Cl-,CO32-, NO3, SO42-, CH3COO). Examples of salt include NaCl, KCl, CaCl2, MgCl2,Mg(NO3)2, MnSO4, K2SO4, and NaHCO3. In the context of salt tolerance and salt indifference,a salt may be a monovalent salt (e.g., NaCl), a divalent salt (e.g., MgCl2, CaCl2), an organicsalt (NaCH3COO), and / or an inorganic salt.As used herein, “salt indifferent" refers to a property or capacity to display activity bothin the absence of salt and across a range of concentrations of one or more salts. A saltindifferent salt active nuclease variant, for example, may display DNA-binding activity and / orcatalytic activity in the absence or presence of one or more salts (e.g., from 0 M to 1 M salt)that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, or at least 60% of such variant's peak activity. For example, a salt activenuclease variant may have at least 20% of its peak catalytic activity under conditions spanninga range of total salt from 0 M to 1 M. Salt indifferent enzymes may be distinguished from saltsensitive enzymes, which have little or no activity (e.g., less than 20% of peak catalytic activity)in the absence of salt (salt-requiring enzymes) or have little or no activity (e.g., less than 20%of peak catalytic activity) in the presence of salt (salt-labile enzymes).As used herein, “salt tolerant” refers to a property or capacity to display activity in thepresence of one or more salts. A salt tolerant salt active nuclease variant, for example, maydisplay DNA-binding activity and / or catalytic activity in the presence of one or more salts.Binding activity may be evaluated in suitable biochemical terms, for example, binding affinity(Kd). Similarly, catalytic activity may be evaluated in suitable biochemical terms, for example,Michaelis constant (Km) and / or maximal reaction velocity (Vmax). Catalytic activity and / orDNA binding of a salt tolerant salt active nuclease variant may be less sensitive to the presenceof salt than a reference enzyme (e.g., a corresponding wild-type enzyme). A salt tolerant saltactive nuclease variant, for example, may bind a (single stranded or double stranded) nucleicacid (DNA and / or RNA) and / or hydrolyze the nucleic acid to yield products comprising at leastone 5'-phosphorylated nucleic acid strand and / or at least one 3'-hydroxylated nucleic acidstrand in the presence of at least 150 mM, at least 250 mM, at least 500 mM, or at least 900mM salt. Catalytic activity of a salt tolerant salt active nuclease variant in the presence of 200mM salt may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 98% of the activity of the same salt tolerant salt active nuclease variant in theabsence of salt when assayed by any method disclosed herein. A salt tolerant salt activenuclease variant may display at least 20%, at least 22%, at least 24%, at least 26%, at least28%, at least 30%, at least 35%, at least 40%, or at least 50% of its peak catalytic activity inthe presence of up to 1 M salt.A salt active nuclease variant (e.g., in the presence of 100 mM, 200 mM, 300 mM, 400mM, 500 mM salt) may be less processive than a corresponding reference (e.g., wild-type)enzyme under the same conditions with or without a measurable difference in overall catalyticactivity. For example, a salt active nuclease variant may bind and release substrate moleculesmore frequently than a corresponding reference (e.g., wild-type) enzyme under the sameconditions. Short substrates may obscure this property with both variant and referenceenzymes appearing to cut the substrate equally well. One approach to resolving this differenceincludes contacting each enzyme with a mixture of two or more substrates, wherein a firstsubstrate is short (e.g., ≤100 nts) and comprises a detection system and a second substrate islong (e.g., ≥500 nts) without a detection system. The long substrate may be included in molarexcess over the short substrate. Without limiting any embodiment to any particular mechanismof action, a less processive endoribonuclease is expected to switch substrates more frequentlyand digest (e.g., at least once) more substrate molecules in the mixture compared to a moreprocessive enzyme.In the context of the present disclosure, "salt active nuclease" refers to any enzyme thatdigests one or more salt active nuclease substrates (e.g., single- and double-stranded DNA andRNA, including, in each case, linear and circular forms) in the presence of salt (e.g., total salt≥50 mM, ≥100 mM, ≥150 mM, ≥200 mM, ≥250 mM, ≥300 mM, ≥350 mM, ≥400 mM, ≥450mM, ≥500 mM, ≥600 mM, ≥700 mM, ≥800 mM, ≥900 mM, or ≥ 1 M salt) and optionally inthe absence of salt.In the context of the present disclosure, “salt active nuclease substrate" refers to amolecule having at least one bond that is cleavable by a salt active nuclease and / or a salt activenuclease variant. Examples of salt active nuclease substrates include single-stranded DNA,double-stranded DNA, single-stranded RNA, double-stranded RNA, and RNA:DNA duplexes.Examples of salt active nuclease substrates include linear, circular, and branchedpolynucleotides. Salt active nuclease substrates may be associated (e.g., non-covalently bound)with one or more other materials (e.g., biological materials such as proteins, lipids, and / orcarbohydrates, non-biological materials such as magnetic beads or other supports).In the context of the present disclosure, “salt active nuclease variant" refers to any non-naturally occurring salt active nuclease that digests one or more salt active nuclease substrates(e.g., single- and double-stranded DNA and RNA) in the presence of salt (e.g., total salt ≥150mM, ≥200 mM, ≥250 mM, ≥300 mM, ≥350 mM, ≥400 mM, ≥450 mM, ≥500 mM, ≥600 mM,≥700 mM, ≥800 mM, ≥900 mM, or ≥ 1 M salt) and optionally in the absence of salt. A saltactive nuclease variant, in some embodiments, may have an amino acid sequence sharing anydesired degree of sequence identity with positions 22-234 of SEQ ID NO:5 up to (butexcluding) 100% identity. According to some embodiments, a salt active nuclease variant maycomprise an amino acid sequence having ≥85%, ≥88%, ≥90%, ≥92%, ≥93%, ≥95%, ≥96%,≥97%, ≥98% ≥99% identity to one or more of SEQ ID NOS:1-5 and have catalytic activity(e.g., ssDNA nuclease activity, dsDNA nuclease activity, ssRNA nuclease activity, ssRNAnuclease activity, and / or duplex DNA:RNA nuclease activity). For example, salt activenuclease variant may comprise an amino acid sequence having a serine (e.g., a Q->Ssubstitution or another conservative substitution) at a position corresponding to position 52 ofSEQ ID NO:1 (a "Q52S" substitution) and / or corresponding to position 73 of SEQ ID NO:5 (a"Q73S" substitution). In some embodiments, a salt active nuclease variant may comprise anamino acid sequence having at least 96%, at least 97%, at least 98% or at least 99% identity toone or more of SEQ ID NOS:1-4, wherein the amino acid at the position corresponding toposition 52 of any of SEQ ID NOS:1-4 is not Q (e.g., is instead C, D, E, H, K, N, P, R, S, T orY). In some embodiments, a salt active nuclease variant may comprise an amino acid sequencehaving at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:3,wherein the amino acid at the position corresponding to position 53 of any of SEQ ID NO:3 isnot Q (e.g., is instead C, D, E, H, K, N, P, R, S, T or Y). In some embodiments, a salt activenuclease variant may comprise an amino acid sequence having ≥85%, ≥88%, ≥90%, ≥92%,≥93%, ≥95%, ≥96%, ≥97%, ≥98% ≥99% identity to positions 22-234 of SEQ ID NO:4,wherein the amino acid at the position corresponding to position 73 of SEQ ID NO:4 is not Q(e.g., is instead C, D, E, H, K, N, P, R, S, T or Y). A salt tolerant nuclease variant, accordingto some embodiments, may comprise an amino acid sequence having ≥85%, ≥88%, ≥90%,≥92%, ≥93%, ≥95%, ≥96%, ≥97%, ≥98% ≥99% identity to any of SEQ ID NOS:1-4, whereineach substitution (e.g., relative to amino acids 22-234 of SEQ ID NO:5) is a conservativesubstitution or wherein all substitutions are conservative substitutions except one (which isnon-conservative) or wherein all substitutions are conservative substitutions except two orwherein all substitutions are conservative substitutions except three or wherein all substitutionsare conservative substitutions except four or wherein all substitutions are conservativesubstitutions except five.A salt tolerant nuclease variant may be salt tolerant and / or salt indifferent. Catalyticactivity of a salt active nuclease variant may persist across a range of salt concentrations,temperatures and / or pH. For example, a salt active nuclease variant may display catalyticactivity under such a range of conditions and / or following removal from exposure to conditionswithin such a range. A salt active nuclease variant may have catalytic activity at and / orfollowing exposure to temperatures in ranges X to Y, where X is any of 1°C, 2°C, 4°C, 10°C,15°C, 20°C, 25°C, 30°C and Y is any of 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C,65°C and X < Y. For example, a salt active nuclease variant may have catalytic activity atand / or following exposure to temperatures in ranges 1°C–65°C, 2°C-60°C, 10°C-60°C, 20°C-55°C, 35°C-55°C. According to some embodiments, a salt active nuclease variant has higher(e.g., ≥25%, ≥35%, ≥50%, ≥75% higher) activity at 250 mM salt and 50°C than acorresponding wild type enzyme. A salt active nuclease variant may have catalytic activity atand / or following exposure to a pH in ranges X⁺ to Y†, where X is any of 6.5, 7, 7.5, 8, 8.5 andYt is any of 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 and X*<Y†. For example, a salt active nucleasevariant may have catalytic activity at and / or following exposure to pH in ranges 6.5–12, 7–11.5, 7.5-11, 7.5-10.5, 8-10, 8.5-9.5.A salt active nuclease variant may comprise one or more amino acids in addition to aSEQ ID NOS:1-5. For example, a salt active nuclease variant may comprise (e.g., at its aminoterminal end or carboxy terminal end) 1-25 amino acids. Such additional amino acids mayenable, facilitate and / or enhance translation, expression, cellular sorting, inactivation (e.g., byincluding a protease recognition and / or cleavage site), and / or purification. Such additionalamino acids may constitute a linker, for example, to a support (e.g., a magnetic bead) or anotherprotein.A salt active nuclease variant may have one or more other desirable properties (beyondretaining activity in high salt compositions) including, for example, readily binding cationexchange media attributable to its high pI (e.g., 9 ≤ pI ≤ 10), reduced binding affinity to actin,mucolytic activity, and / or phosphodiesterase / hydrolytic activity.In the context of the present disclosure, “substitution" refers to an amino acid residueat a position in a comparator amino acid sequence that differs with respect to a correspondingposition of a reference amino acid sequence, where the comparator and reference sequencesare at least 60% identical to each other or at least 70% identical to each other or at least 80%identical to each other. A reference sequence and comparator sequence may have the samelength or similar lengths (e.g., differing by ≤ 12%, ≤ 5%, < 1%). A substitute amino acidresidue at a position, in addition to differing from the corresponding position of a referenceamino acid sequence, may differ from the amino acid at the corresponding position of allnaturally-occurring sequences that are at least 60% identical to each other or at least 70%identical to each other or at least 80% identical to the reference sequence. Optionally, asubstitute amino acid may have different properties than the amino acid in the correspondingposition of the reference sequence. Optionally, a substitute amino acid may have similarproperties to the amino acid in the corresponding position of the reference sequence (a"conservative” substitution). For example, a non-polar amino acid (e.g., A, V, L, I, M, W, andF (and optionally C, G, and P) may substitute for another non-polar amino acid; a polar aminoacid (e.g., N, Q, S, T, and Y) may substitute for another polar amino acid (e.g., C, D, E, H, K,N, P, Q, R, S, T, and Y); a positively charged amino acid (e.g., H, K, and R) may substitute foranother positively charged amino acid; and a negatively charged amino acid (e.g., D and E)may substitute for another negatively charged amino acid. A substitute amino acid may be anatural amino acid (e.g., replacing another natural amino acid or a non-natural amino acid). Asubstitute amino acid may be a non-natural amino acid (e.g., replacing a natural amino acid oranother non-natural amino acid).All publications, patents, and patent applications mentioned in this specification areincorporated herein in their entirety by reference to the same extent as if each individualpublication, patent, or patent application was specifically and individually indicated to beincorporated by reference. Reagents referenced in this disclosure may be made using availablematerials and techniques, obtained from the indicated source, and / or obtained from NewEngland Biolabs, Inc. (Ipswich, MA).CompositionsThe present disclosure relates, in some embodiments, to compositions for reducingthe amount (e.g., molar amount) of a polynucleotide in a sample and / or samples having oneor more polynucleotides. Compositions may comprise, according to some embodiments, anyof the salt active nuclease variants disclosed herein, a salt active nuclease substrate (e.g.,SSRNA, dsRNA, ssDNA, and / or dsDNA), and, optionally, one or more salts. Salt activenuclease variant compositions may comprise, in some embodiments, any of the salt activenuclease variants disclosed herein (e.g., variants having an amino acid sequence sharing anydesired degree of sequence identity with positions 22-234 of SEQ ID NO:5 up to (butexcluding) 100% identity), a salt active nuclease substrate, and one or more of a salt (e.g.,NaCl, MgCl2), a protein (e.g., albumin, topoisomerase, polymerase), DNA, RNA, a bufferingagent, a cell (e.g., intact or digested), a biological fluid or secretion (e.g., mucus, pus), and / or(non-naturally occurring) combinations thereof. Compositions may comprise one or moresalts (e.g., any of the salts disclosed here alone or in any combination) at a total saltconcentration of, for example, ≥150 mM, ≥200 mM, ≥250 mM, ≥300 mM, ≥350 mM, ≥400mM, ≥450 mM, ≥500 mM, ≥600 mM, ≥700 mM, ≥800 mM, ≥900 mM, or ≥ 1 M. Salt activenuclease variants included in compositions may comprise an amino acid sequence having≥85%, ≥88%, ≥90%, ≥92%, ≥93%, ≥95%, ≥96%, ≥97%, ≥98% ≥99% identity to one or moreof SEQ ID NOS: 1-5 and have catalytic activity (e.g., ssDNA, dsDNA, ssRNA, ssRNA,and / or duplex DNA:RNA nuclease activity). In each case, salt present may be a single saltspecies or a mixture of salts. In each case, salt present may comprise monovalent and / ordivalent salts. A salt active nuclease variant composition may comprise one or more ionic,non-ionic, and / or zwitterionic detergents (e.g., octoxinol, polysorbate 20), crowding agents,sugars, starches, cellulose, lipids, and oils.Salt active nuclease variants and compositions thereof may have any desirable degreeof purity including, for example, cell paste, crude extract, partially purified, and / or purifiedpreparations. Salt active nuclease variants and compositions thereof may have any desirableform including, for example, a liquid, a gel, a film, a powder, a cake, and / or any dried orlyophilized form. A salt active nuclease variant composition may comprise one or morestabilizers including, for example, an aptamer, a monosaccharide, a disaccharide, atrisaccharide, a tetrasaccharide, starch, cellulose, dextrin, and dextran.In some embodiments, a salt active nuclease variant may be encoded by a nucleic acidsequence having ≥85%, ≥88%, ≥90%, ≥92%, ≥93%, ≥95%, ≥96%, ≥97%, ≥98% ≥99%identity to a sequence (e.g., a codon optimized sequence) encoding the amino acid sequenceof any of SEQ ID NOS: 1-5. For example, a salt active nuclease variant may be encoded by anucleic acid sequence having ≥85%, ≥88%, ≥90%, ≥92%, ≥93%, ≥95%, ≥96%, ≥97%, ≥98%≥99% identity to SEQ ID NO:10. A nucleic acid encoding a salt active nuclease variant maybe included in an expression cassette, expression vector, or other expressible form suitablefor in vitro or in vivo expression (e.g., in E. coli or other bacteria or P. pastoris or otheryeast).KitsThe present disclosure further relates to kits including salt active nuclease variants.For example, a kit may include a salt active nuclease variant, other enzymes (e.g.,polymerases, enzymes other than polymerases, or both), buffering agents, or combinationsthereof. Enzymes may be included in a storage buffer. Any suitable storage buffer may beused, for example, buffers comprising one or more of a cryoprotectant (e.g., a polyol such asglycerol, an antifreeze protein), a salt, a detergent, a reducing agent, a sugar, a chelator, andan antimicrobial agent and having a pH tolerated by the enzyme to be stored, for example,between pH 6 and 9. A composition or kit may include a reaction buffer which may be inconcentrated form, and the buffer may contain additives (e.g. glycerol), salt (e.g. NaCl, KCl),reducing agent, EDTA or detergents, among others. Detergents include nonionic detergents(e.g., t-octylphenoxypolyethoxyethanol), anionic detergents (e.g., alkylbenzene sulfonates),cationic detergents (e.g., alkylbenzene quaternary ammonium), and zwitterionic detergents.A composition or kit comprising dNTPs may include one, two, three of all four of dATP,dTTP, dGTP and dCTP. A kit comprising rNTPs may include one, two, three of all four ofrATP, rUTP, rGTP and rCTP. A kit may further comprise one or more modified nucleotides.A kit may optionally comprise one or more primers (random primers, bump primers,exonuclease-resistant primers, chemically-modified primers, custom sequence primers, orcombinations thereof).A kit may be a non-natural collection of components configured, for example, forconvenient storage, shipping, delivery, and / or use. One or more components of a kit may beincluded in one container for a single step reaction, or one or more components may becontained in one container, but separated from other components for sequential use or paralleluse. The contents of a kit may be formulated for use in a desired method or process.A kit is provided that contains: (i) a salt active nuclease variant; and (ii) a buffer. Thesalt active nuclease variant may have a lyophilized form or may be included in a buffer (e.g., astorage buffer or a reaction buffer in concentrated form). A kit may contain the salt activenuclease variant in a mastermix suitable for receiving and digesting a nucleic acid. A salt activenuclease variant may be a purified enzyme so as to contain substantially no DNA or RNA andno nucleases. The reaction buffer in (ii) and / or storage buffers containing the DNA polymerasein (i) may include non-ionic, ionic e.g. anionic or zwitterionic surfactants and crowding agents.A kit may include the salt active nuclease variant and the reaction buffer in a single tube or indifferent tubes.A subject kit may further include instructions for using the components of the kit topractice a desired method. The instructions may be recorded on a suitable recording medium.For example, instructions may be printed on a substrate, such as paper or plastic, etc. Assuch, the instructions may be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with the packaging orsubpackaging) etc. Instructions may be present as an electronic storage data file residing on asuitable computer readable storage medium (e.g. a CD-ROM, a flash drive). Instructions maybe provided remotely using, for example, cloud or internet resources with a link or otheraccess instructions provided in or with a kit.MethodsSalt active nuclease variants disclosed herein may be useful in many molecular,cellular, and therapeutic applications, processes, methods, and / or workflows. For example,salt active nuclease variants may be use in methods and / or workflows that include, forexample, strand displacement, nick translation, in vitro transcription, in vitro proteinexpression (e.g., cell free protein expression, PURExpress®) DNA fragmentation,footprinting, PCR (e.g., RT-PCR), RNA sequencing, and RNA purification. Salt activenuclease variants may be used, in some embodiments, to digest DNA (e.g., dsDNA and / orssDNA) and / or RNA (e.g., dsRNA and / or ssRNA) where its presence may impair orcomplicate analysis (e.g., of other components of a sample). The present disclosure provides,in some embodiments, methods comprising contacting a salt active nuclease variant with amolecule comprising a polydeoxyribonucleotide. For example, a method may comprisecontacting a salt active nuclease variant with a composition comprising DNA and at least onenon-DNA species to digest the DNA present. Such action on the DNA present may leave thenon-DNA species unmodified or substantially unmodified (e.g., as to structure, composition,and / or concentration).The present disclosure relates, in some embodiments, to methods comprisingcontacting a salt active nuclease variant with a composition comprising a polynucleotide andat least one material of interest to form polynucleotide cleavage products without modifyingthe material of interest. For example, a method may comprise contacting a salt activenuclease variant with a composition comprising DNA and / or RNA molecules and a protein(or vitamins or saccharides or polysaccharides or lipids or cellular metabolites or any othermolecule of interest other than polynucleotides) to digest the DNA and / or RNA moleculespresent without modifying the protein (or another non-polynucleotide molecule of interest).Such contacting may be included in methods for cleaning up protein after isolation from acell or tissue, after in vitro transcription, prior to elution from a solid support, prior to aminoacid sequencing, for preparing protein samples for separation on 2-D gels, and / or foridentifying protein binding sequences on DNA (salt active nuclease footprinting). In someembodiments, the salt active nuclease variant may be included in any desirable degree ofpurity (e.g., as cell paste, crude extract, partially purified, and / or purified preparations). Theprotein of interest may remain unaltered or substantially unaltered. For example, contacting asalt active nuclease variant with a composition comprising a protein (or another non-polynucleotide molecule of interest) and DNA and / or RNA may result in a productcomposition comprising over 50%, over 80%, over 85%, over 90%, over 95%, or over 99%of the starting, intact protein (or another non-polynucleotide molecule of interest) and / or lessthan 50%, less than 25%, less than 10%, less than 5%, less than 1%, less than 0.5%, or lessthan 0.1% of the starting, intact DNA and less than 50%, less than 25%, less than 10%, lessthan 5%, less than 1%, less than 0.5%, or less than 0.1% of the starting, intact RNA. Amethod may include fracturing one or more cells to form the composition comprising theprotein (or another non-polynucleotide molecule of interest) and DNA and / or RNA, whereinat least a portion of the protein (or another non-DNA molecule of interest) comprises cellularprotein (or another cellular, non-DNA molecule of interest), at least a portion of the DNAcomprises cellular DNA, and at least a portion of the RNA comprises cellular RNA. The sizeof digestion products and / or degree of digestion may be managed, for example, by selectingthe salt active nuclease variant with a desired activity, increasing or decreasing theconcentration of the selected salt active nuclease variant, increasing or decreasing theincubation time or temperature, increasing or decreasing magnesium concentration,increasing or decreasing salt concentration, and / or increasing or decreasing the pH. In someembodiments, methods may be adapted to digest DNA as fully as practicable or digest DNA(non-specifically) into fragments within a selected range of sizes.A salt active nuclease variant may be useful in digesting and / or removing unwantedpolynucleotides from a composition comprising liposomes, micelles, phage particles, and / orviral particles. For example, a desirable polynucleotide (e.g., a therapeutic RNA or DNA)may be packaged in a micelle, liposome, phage particle, or viral particle using a protocolwhich does or may result in a packaged composition comprising some packagedpolynucleotide and some residual unpackaged polynucleotide (or fragments thereof).According to some embodiments, a method may comprise contacting a salt active nucleasevariant with such packaged composition (e.g., comprising unpackaged polynucleotides) toproduce a product composition, wherein the product composition comprises less unpackagedpolynucleotide than the packaged composition.In some embodiments, a salt active nuclease variant may be contacted with acomposition comprising DNA (e.g., genomic fragments or other long (≥10kb) DNAfragments) to digest such DNA. Such contacting may be included in methods for creating afragmented DNA library and methods of cell culture preparation (e.g., tissue disaggregation),cultivation, manipulation, and storage to reduce or prevent cell clumping.A salt active nuclease variant may be used in connection with in vitro transcription(IVT), according to some embodiments. IVT methods may include contacting a DNAtemplate (e.g., a double stranded DNA comprising a coding sequence and an expressioncontrol sequence operably linked to the coding sequence) with an RNA polymerase (e.g., T7RNA polymerase) optionally in the presence of NTPs, salt, and / or a reaction buffer to form atranscription product composition comprising a transcription product (e.g., RNA) and theDNA template. The resulting RNA may be translated into protein by any available method,for example, PURExpress® (New England Biolabs, Inc.). IVT methods, in someembodiments, may comprise contacting a salt active nuclease variant with the DNA templateto digest the DNA template and form a digested composition comprising DNA templatedigestion products and / or RNA digestion products. IVT methods may comprise separatingthe protein product from one or more of the other components of the transcription / translationproduct composition or the digested composition, for example, the DNA template, DNAtemplate digestion products, the RNA polymerase, NTPs, salt, and reaction buffer.Techniques for such separation include column purification, phase separation (e.g., phenol-chloroform), and fractionation (e.g., size, charge, hydrophobicity, polarity, and ratios thereof)among others.According to some embodiments, contacting a DNA and / or RNA with a salt activenuclease variant may (further) comprise contacting the polydeoxyribonucleic acid with thesalt active nuclease variant in the presence of total salt ≥50 mM, ≥100 mM, ≥150 mM, ≥200mM, ≥250 mM, ≥300 mM, ≥350 mM, ≥400 mM, ≥450 mM, ≥500 mM, ≥600 mM, ≥700mM, ≥800 mM, ≥900 mM, or ≥ 1 M salt. In each case, salt present may be a single saltspecies or a mixture of salts. In each case, salt present may comprise monovalent and / ordivalent salts.The present disclosure further relates to methods of making a salt active nucleasevariant. For example, a salt active nuclease variant may be produced by in vitro transcriptionand / or in vitro translation (e.g., PURExpress®, New England Biolabs, Inc.). In someembodiments, a salt active nuclease variant may be produced in vivo. For example, a methodmay include (a) culturing a host cell comprising an expression vector or expression cassettecomprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least93%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence (e.g., acodon optimized sequence) encoding the amino acid sequence of SEQ ID NO: 1, 2, 3, and / or4 operably linked to an expression control sequence to produce a cultured host cellcomposition comprising the salt active nuclease variant, and (optionally) isolating the saltactive nuclease variant from the cultured host cell composition (e.g., culture supernatant,culture lysate, culture cell paste).EXAMPLESSome specific example embodiments may be illustrated by one or more of the examplesprovided herein.EXAMPLE 1: Nuclease Activity on dsDNA, ssDNA, and RNA Substrates in NaClExample endonucleases (e.g., vsEndA and vsEndA-Q52S) were tested with substratesand under conditions disclosed in this and subsequent examples and the resultingperformance compared. vsEndA and vsEndA-Q52S were expressed in E. coli. For this, the 2codon optimized genes vsendA and vsEndA-Q52S were cloned in a high copy plasmid undercontrol of an inducible promoter.For testing vsEndA and vsEndA-Q52S activity on dsDNA, ssDNA and RNAfluorescent substrates, both enzymes were diluted to a working concentration of 0.1 µg / mL in25 mM Tris-HCl pH 8.5, 5 mM MgCl2 supplemented with NaCl at 0, 100, 250, 500, 750 and1000 mM NaCl and mixed with 5 µM of a dsDNA substate (a 35mer FAM-BHQ1 labeledhairpin oligonucleotide (SEQ ID NO:7)) or 5 µM of a ssDNA substrate (a linear 15mer oligosubstrate (SEQ ID NO:8)) or 0.2 µM of RNA fluorescent substrate, and with1 mg / mL of calf-thymus DNA. Each 50 µL reaction was loaded into a well of a 96 well plate half area blackbottom and incubated at 25°C on a SpectraMax (Molecular Devices) to follow the kinetic atEx 485 nm - Em 525 nm cutoff 515 nm every 30 sec for 30 min. As measurements wereperformed 2 times, the released fluorescence values chosen at a specific endpoint in the linearrange were averaged and converted to percentages of activity using the highest value as100%. Results are shown in FIGURE 1.On DNA substrates (dsDNA and ssDNA), the activities of both enzymes peak at 500mM NaCl under the conditions tested. When tested on dsDNA (FIGURE 1A), vEndA-Q52Sexhibits 4x increased activity at 100 mM NaCl, 2x at 500 mM and 1.6x 750 mM NaClcompared to vsEndA. On a ssDNA substrate (FIGURE 1B), vEndA-Q52S shows 2ximprovement compared to vsEndA at 100 mM, 4x at 500 mM and 750 mM NaCl. Finally, onthe RNA substrate (FIGURE 1C), vEndA-Q52S is ~2x more performant than vsEndA inNaCl <350 mM. Overall, these data show that, when tested on fluorescent substrates(dsDNA, ssDNA and RNA) in presence of calf-thymus DNA, vsEndA-Q52S outperformsvsEndA wild-type by 2-4x between 250 and 750 mM NaCl in a Tris buffer pH 8.5.EXAMPLE 2: Nuclease Activity of a SAN Variant and 2 Other Endonucleases in NaClActivities of an example salt active nuclease variant and 2 commercially availableendonucleases were compared in the presence of NaCl. vsEndA-Q52S, an endonucleasefrom Supplier A and an endonuclease from Supplier B were each diluted to a workingconcentration of 0.1 µg / mL in their respective buffer (vsEndA-Q52S and Supplier A: 25 mMTris-HCl pH 8.5, 5 mM MgCl2, Supplier B: 50 mM Tris-HCl pH 8, 1 mM MgCl2)supplemented with 100, 250, 500, 750 and 1000 mM NaCl, and mixed with 5 µM of thedsDNA substrate of Example 1 and 0.5 mg / mL of calf-thymus DNA, in 50 µL final. Eachreaction was loaded on a 96 well plate half area black bottom and incubated at 25°C in aSpectraMax (Molecular Devices) to follow the kinetics as described in Example 1.The endonuclease from Supplier B shows decreased activity as the concentration ofNaCl is increased. On the other hand, both vsEndA-Q52S and Supplier A activity increasesas the NaCl concentration increases, to a peak at 500 mM NaCl followed by a slight decrease(not less than 50%) between 500 mM and 1000 mM NaCl. vsEndA-Q52S is consistently 2xmore active than the Supplier A endonuclease (FIGURE 2). Overall, vsEndA-Q52Soutperforms Supplier A and Supplier B endonucleases by 2x to 4x between 300 mM and1000 mM NaCl.EXAMPLE 3: pH Spectrum of Nuclease Activity on dsDNA, ssDNA, and RNA SubstratesNuclease activity was assessed in 25 mM Tris-HCl, 500 mM NaCl, 5 mM MgCl2buffer across a pH range of 6.5 to 9.5. Enzymes vsEndA and vsEndA-Q52S were diluted to aworking concentration of 0.1 µg / mL in each buffer and mixed with 5 µM of the dsDNAsubstrate of Example 1 or 5 µM of the linear ssDNA substrate of Example 1 or 0.2 µM ofRNA fluorescent substrate, the RNA oligonucleotide substrate v2 from the Kit RNaseAlertMQC System v2 (Thermofisher), along with 1 mg / mL of calf-thymus DNA. The 50 µLreactions were loaded on a 96 well plate half area black bottom and incubated at 25°C in aSpectraMax (Molecular Devices) to follow the kinetics as described in Example 1. Resultsare shown in FIGURE 3 and TABLE 1.When tested in Tris buffer pH 6.5 to 9.5 with 500 mM NaCl and 5 mM MgCl2 on adsDNA substrate and calf-thymus DNA, vsEndA-Q52S showed similar activity to vsEndA,except at pH 8.5, where vsEndA-Q52S is 40% more active than vsEndA (FIGURE 3).TABLE 1: Evaluation of vsEndA and vsEndA-Q52S activity at diverse pHs on varioussubstrates. The respective activity of each enzyme was evaluated independently as follows+++>80% activity, ++>50% activity, +>10% activity, - <10% activity measured.pHdsDNASSDNARNA6.57++7.5++++8+++++++8.5+++++++++9++++++9.5++++vsEndA and vsEndA-Q52S activities are both optimal at pH 8.5 on all 3 substrates.On dsDNA and ssDNA, the activity is over 5% between pH 7 and 9.5, while on RNA, thepeak is narrower between 8 and 9 in the conditions tested (TABLE 1).EXAMPLE 4: Thermal Spectrum of Nuclease Activity on dsDNA, ssDNA, and RNAA temperature screen was carried out by mixing vsEndA or vsEndA-Q52S with DNAsubstrates in 25 mM Tris-HCl pH 8.5, 500 mM NaCl, 5 mM MgCl2 followed by anincubation at 4°C, 20°C, 40°C, 50°C or 60°C for 10 min. Both enzymes were diluted at 0.1µg / mL in 25 mM Tris-HCl pH 8.5, 500 mM NaCl, 5 mM MgCl2 and mixed with 5 µM of thedsDNA substrate of Example 1 and 1 mg / mL of calf-thymus DNA. The 50 µL reactions wereset up in PCR strip tubes and incubated at 4°C, 20°C, 40°C, 50°C and 60°C in a PCRinstrument (Bio-Rad) for 10 min. The samples were immediately transferred on a 96 wellplate half area black bottom to measure the fluorescence released on a SpectraMax(Molecular Devices) as described in Example 1. The values of released fluorescence wereconverted to percentages of activity using the highest value as 100% (FIGURE 4).The activity of both enzymes vsEndA and vsEndA-Q52S peaks at 50°C when testedon DNA in a Tris buffer pH 8.5 with 500 mM NaCl. vsEndA-Q52S systematicallyoutperforms vsEndA by 1.2 to 2x at all temperature tested (FIGURE 4).EXAMPLE 5: Nuclease Activity on ADNASerial dilutions of vsEndA and vsEndA-Q52S from 10 ng / mL to 3.125 pg / mL wereprepared in 25 mM Tris-HCl pH 8.5, 0.5 M NaCl, 5 mM MgCl2, 0.1 µg / mL BSA, 0.01%Triton reaction buffer containing 0.02 mg / mL of lambda DNA (New England Biolabs). The20 µL reactions were incubated at 37°C for 10 minutes and then stopped with 7 µL of loadingbuffer 6x (New England Biolabs). The final samples were loaded on a precast agarose gel1.2% (Lonza) for analysis (FIGURE 5).When tested on lambda DNA at 37°C for 10 min in a Tris buffer pH 8.5 with 500 mMNaCl, 0.05 ng of vsEndA digests 1 µg of lambda DNA (>95% digestion determined by visualobservation on the agarose gel), while only 0.01 ng of vsEndA-Q52S is necessary (FIGURE5). These data show that vsEndA-Q52S is 5 times more active than vsEndA at digestinglambda DNA in the conditions tested.EXAMPLE 6: Nuclease Activity on total RNA from HEK CellsSerial dilutions of vsEndA and vsEndA-Q52S were prepared in a 25 mM Tris-HCl pH8.5, 500 mM NaCl, 5 mM MgCl2, so that 250 ng / mL to 0.5 ng / mL of enzymes were mixedwith 50 µg / mL of total RNA extract from HEK cells. The 20 µL reactions were incubated at25°C for 10 min, then stopped with 100 mM EDTA and mixed with 2x RNA loading buffer(New England Biolabs). 10 µL of the final samples were loaded on a precast agarose gel1.2% (Lonza) for analysis (FIGURE 6).When tested on a total RNA extract from HEK cells at 25°C for 10 min in a Trisbuffer pH 8.5 with 500 mM NaCl, 3.1 ng of vsEndA digests 2.5 µg RNA, while 2 time less(1.6 ng) of vsEndA-Q52S is necessary (FIGURE 6). These data indicate that vsEndA-Q52Sis 2 times more performant than vsEndA at digesting extracted RNA when tested at 500 mMNaCl pH 8.5.EXAMPLE 7: Nuclease Activity on a Fluorescent RNA SubstrateSerial dilutions of vsEndA and vsEndA-Q52S were prepared in a 25 mM Tris-HCl pH8.5, 500 mM NaCl, 5 mM MgCl2, 0.005% Tween 20 so that 3 µg / mL to 0.05 µg / mL ofenzymes were mixed with 0.2 µM of FAM-RNA substrate (SEQ ID NO:9 with the 5' FAMlabel), 0.8 µM unlabeled oligo (SEQ ID NO:9 without the 5' FAM label) and 1 mg / mL ofcalf-thymus DNA in a total of 10 µL. The reactions were incubated at 25°C for 10 min, thenstopped with 10 µL of 2x RNA loading buffer (New England Biolabs). 12 µL of the finalsamples were loaded on a Novex™™ TBE-Urea Gel 15% (ThermoFisher). The gel wasanalyzed on Typhoon instrument (Cytiva) using Cy2 setting (FIGURE 7).FIGURE 7 shows that ~1.9 ng vsEndA-Q52S digests 0.2 µM of the fluorescent RNAsubstrate, which is 2 time less (3.8 ng) than vsEndA (FIGURE 7). This show that vsEndA-Q52S is ~2 times more performant than vsEndA at digesting this fluorescent RNA substratein the conditions tested.EXAMPLE 8: Nuclease Activity in the Presence of KCl, Mg(NO3)2, or MgCl2Nuclease activity was assessed in the presence of salts other than NaCl. Thesubstitution of NaCl with KCl or Mg(NO3)2 was tested in 25 mM Tris-HCl pH 8.5, 5 mMMgCl2 supplemented with 0 to 1000 mM KCl or 0 to 200 mM Mg(NO3)2, while thesubstitution of NaCl with MgCl2 was tested in 25 mM Tris-HCl pH 8.5 with and without 500mM NaCl supplemented with 0 to 200 mM MgCl2. 0.1 µg / mL vsEndA-Q52S was mixedwith 5 µM of the dsDNA substrate of Example 1 and 1 mg / mL of calf-thymus DNA in eachbuffer. Kinetic reactions were followed on a SpectraMax (Molecular Devices) at Ex 485 nmEm 525 nm cutoff 515 nm every 30 sec for 10 min at 25°C using 96 well plate half area blackbottom. Values of released fluorescence in the linear range were chosen to compare eachcondition tested. The percentages of activity were calculated based on the highest value set at100% (FIGURE 8).FIGURE 8A shows that KCl can substitute NaCl at an equivalent concentrationwithout impacting the enzyme activity. MgCl2 can also replace NaCl when used between 25to 150 mM, while in presence of 500 mM NaCl, MgCl2 is required between 5 and 50 mM tomaintain an activity >50% (FIGURE 8B). In FIGURE 8C, Mg(NO3)2 used between 75 mMto 250 mM can substitute 500 mM NaCl and provide >40% enzyme activity.EXAMPLE 9: Nuclease Activity at High Substrate:Enzyme Ratios20 ng of vsEndA-Q52S, the endonuclease of Supplier A or the endonuclease ofSupplier B was mixed with 150 µg of calf thymus DNA in their respective buffer (25 mMTris-HCl pH 8.5, 5 mM MgCl2 for vsEndA-Q52S and Supplier A, 50 mM Tris-HCl pH 8, 1mM MgCl2 for Supplier B) supplemented with 500 mM NaCl. The 150 µL reaction wasincubated either at 25°C or 4°C. 20 µL of the samples at 25°C were harvested at 0, 5, 10, 15,30, and 60 min, and every hour for the samples at 4°C, and mixed with 4 µL EDTA 500 mM(100 mM final) to stop the reaction. All samples were then mixed with loading buffer 6x(New England Biolabs) and loaded on a precast agarose gel 1.2% (Lonza) for analysis(FIGURE 9).FIGURE 9 shows that 20 ng of vsEndA can digest 150 µg of calf thymus DNA in 30min at 25°C in a Tris buffer pH 8.5 with 500 mM NaCl. In comparison, the endonucleasessupplier A and B needs twice (60 min) and more than 60 min, respectively. At 4°C, 20 ng ofvsEndA can digest 150 µg of calf thymus DNA in about 5h, while the endonucleases ofsupplier A and B both need >5h.EXAMPLE 10: Nuclease Activity at Various Substrate:Enzyme RatiosvsEndA and vsEndA-Q52S activity was tested on dsDNA and ssDNA fluorescentsubstrates at concentrations from 0.005 µg / mL to 0.2 µg / mL in 25 mM Tris-HCl pH 8.5, 500mM NaCl, 5 mM MgCl2 buffer in presence of 5 µM of the Example 1 dsDNA substrate or 5µM of the Example 1 linear ssDNA substrate, and with 0.5 mg / mL of calf-thymus DNA.Each 50 µL reaction was loaded on a 96 well plate half area black bottom and incubated at25°C on a SpectraMax (Molecular Devices) to follow the kinetic at Ex 485 nm Em 525 nmcutoff 515 nm every 10 sec for 5 min. The fluorescence released (in RFU) in the linear rangefor all conditions were plotted versus enzyme concentration. Linear trendline and R-squaredvalues are displayed on both graphs (FIGURE 10).FIGURE 10 shows the linear response for each enzyme on the fluorescence releasedfrom ssDNA and dsDNA substrates as they are degraded versus their concentration. vsEndA-Q52S is about 2x more active on dsDNA and 4x on ssDNA compared to vsEndA.EXAMPLE 11: Nuclease activity on mononucleosomesNuclease activity of vsEndA-Q52S and (for comparison) the Supplier B enzymes weretested on mononucleosomes. Enzymes were diluted at 1 µg / mL in their respective buffer(vsEndA-Q52S: 25 mM Tris-HCl pH 8.5, 5 mM MgCl2, Supplier B: 25 mM Tris-HCl pH 8, 1mM MgCl2) supplemented with 100, 250, 500 mM NaCl. Two microliters (2 ng) of eachenzyme were mixed with 2 µg of HeLa purified mononucleosomes (EpiCypher), in a 30 µLreaction using each respective buffer. After an incubation at 20°C for 5 min, all reactions werestopped with 6 µL of EDTA 500 mM. All samples were then mixed with loading buffer 6x(New England Biolabs) and loaded on a precast 20% TBE gel (Invitrogen) for analysis(FIGURE 11).FIGURE 11 shows that the mononucleosomal DNA, resolving at 150 pb (marked), isprogressively degraded with vsEndA-Q52S as the concentration of NaCl increases, to reachalmost full digestion at 500 mM NaCl. On the other hand, the mononucleosomal DNA is moreresistant to degradation with the endonuclease of Supplier B at the 3 NaCl concentrationstested. Nucleosomal DNA may dissociate from nucleosomal proteins (e.g., histones) withincreasing salt concentrations. Without limiting any embodiment to any particular mechanismof action, at lower salt concentrations the association of nucleosomal DNA and histones mayinfluence or limit enzyme-DNA interaction (visible DNA smears at 100 and 250 mM for bothenzymes) whereas the DNA may be more accessible at high salt (500 mM) but only vsEndA-Q52S retained sufficient activity to digest DNA under these conditions.

Claims

What is claimed is:

1. A salt active nuclease variant having an amino acid sequence that is at least 90%identical to one or more of SEQ ID NOS:1-4, having at its position corresponding to position52 of SEQ ID NO:1 an amino acid other than Q, and having catalytic activity as anendonuclease and / or an endoribonuclease at a total salt concentration of 250 mM to 1 M.

2. A salt active nuclease variant according to Claim 1, wherein the amino acidsequence is identical to SEQ ID NO:1 at its position corresponding to S52 of SEQ ID NO:1.

3. A salt active nuclease variant according to Claim 1 or Claim 2, wherein the aminoacid sequence that is at least 95% identical to one or more of SEQ ID NOS:1-4.

4. A salt active nuclease variant according to any preceding claim, wherein the aminoacid sequence is identical to SEQ ID NO:2.

5. A salt active nuclease variant according to any preceding claim, wherein the aminoacid sequence is identical to SEQ ID NO:3, wherein (a) X1 is a signal peptide, a purificationtag, or a linker and X215 is any amino acid or absent, or (b) X1 is any amino acid or absentand X215 is a purification tag or a linker or X215.

6. A salt active nuclease variant according to any preceding claim, wherein allsubstitutions are conservative substitutions.

7. A salt active nuclease variant according to any of Claims 1-5, wherein allsubstitutions are conservative substitutions except one, which is a non-conservativesubstitution.

8. A salt active nuclease variant according to any of Claims 1-5, wherein allsubstitutions are conservative substitutions except two, which are independent non-conservative substitutions.

10. A salt active nuclease variant according to any preceding claim, wherein theamino acid sequence is at least 98% identical to any of SEQ ID NOS:1-4.

11. A salt active nuclease variant according to Claim 10, wherein the amino acidsequence is at least 98% identical to SEQ ID NO:2 and wherein X47-X51 and X53-X56 areeach, independently, any amino acid.

12. A salt active nuclease variant according to Claim 10, wherein the amino acidsequence is at least 98% identical to any of SEQ ID NO:2 and wherein(a) X47, X49-X51, and X53-X55 are each, independently, C, D, E, H, K, N, P, Q,R, S, T, or Y, and(b) X48 and X56 are each, independently, A, L, I, M, W, F, C, G, or P.

13. A salt active nuclease variant according to any preceding claim, wherein the saltactive nuclease variant has at least 30% of its peak catalytic activity in the presence of a totalsalt concentration of 250 mM to 1 M, the salt consisting essentially of NaCl, KCl, or NaCl andKC1.

14. A salt active nuclease variant according to any preceding claim, wherein the saltactive nuclease variant has at least 50% of its peak catalytic activity in the presence of a totalsalt concentration of 250 mM to 750 mM, the salt consisting essentially of NaCl, KCl, or NaCland KCl.

15. A salt active nuclease variant according to any preceding claim, wherein the saltactive nuclease variant has at least 70% of its peak catalytic activity in the presence of a totalsalt concentration of 300 mM to 600 mM, the salt consisting essentially of NaCl, KCl, or NaCland KCl.

16. A salt active nuclease variant according to any preceding claim, wherein the saltactive nuclease variant has at least 30% of its peak catalytic activity in the presence of a totalsalt concentration of 50 mM to 150 mM, the salt consisting essentially of MgCl2, Mg(NO3)2,or MgCl2 and Mg(NO3)2.

17. A salt active nuclease variant according to any preceding claim, wherein the saltactive nuclease variant has at least 70% of its peak catalytic activity at pH > 8.

18. A salt active nuclease variant according to any preceding claim, wherein the saltactive nuclease variant has at least 70% of its peak catalytic activity at 40°C - 55°C.

19. A salt active nuclease variant according to any preceding claim, wherein the saltactive nuclease variant is an immobilized salt active nuclease variant comprising the salt activenuclease variant and a solid support.

20. A salt active nuclease variant according to Claim 19, wherein the solid support isa magnetic bead, an agarose bead, a polystyrene bead, a polyacrylamide bead or a chitin bead.

21. A composition comprising(a) a salt active nuclease variant according to any preceding claim; and(b) 250 mM to 1 M salt.

22. A composition according to Claim 21 further comprising a buffering agent.

23. A fusion protein comprising a single polypeptide chain, the single peptide chaincomprising:(a) a salt active nuclease variant according to any of Claims 1-20; and(b) a SSO7d DNA binding peptide, a transcription factor, an antibody, protein A, amaltose binding domain, a histidine tag, a chitin binding domain, an alpha matingfactor, an O6-alkylguanine-DNA alkyltransferase, and / or albumin.

24. A method for hydrolyzing DNA and RNA comprising contacting (a) acomposition comprising DNA and RNA, and (b) a salt active nuclease variant according toany of Claims 1-20 to form a reaction mixture comprising DNA hydrolysis products andRNA hydrolysis products.

25. A method according to Claim 24, wherein the composition comprising DNA andRNA and / or the reaction mixture further comprises a total salt concentration of 250 mM to 1M, the salt consisting essentially of NaCl, KCl, or NaCl and KCl.

26. A method according to Claim 24, wherein the composition comprising DNA andRNA and / or the reaction mixture further comprises a total salt concentration of 50 mM to 150mM, the salt consisting essentially of MgCl2, Mg(NO3)2, or MgCl2 and Mg(NO3)2.

27. A method according to any of Claims 24-26, wherein the reaction mixturecomprises ≤10% of the DNA that was in the composition.

28. A method according to any of Claims 24-27, wherein the reaction mixturecomprises ≤10% of the RNA that was in the composition.

29. A method according to any of Claims 24-28, wherein the composition comprisingDNA and RNA further comprises one or more DNA binding proteins.

30. A method according to any of Claims 24-29, wherein the composition comprisingDNA and RNA further comprises nucleosomes.

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