Methods and compositions relating to single-stranded nucleic acid laddering by guided cleavage

Abstract

The present disclosure relates to methods, compositions, and kits for preparing cleaved single-stranded nucleic acids, which can be used to prepare ladder compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 406,513, filed September 14, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to methods, compositions, and kits for preparing cleaved single-stranded nucleic acids, which can be used to prepare ladder compositions. [Background technology]

[0003] Single-stranded deoxyribonucleic acid (ssDNA) or single-stranded ribonucleic acid (ssRNA) is used in various biotechnology applications, including, but not limited to, genome editing, gene synthesis, gene therapy, and drug delivery. Detection and size analysis of single-stranded nucleic acid molecules can be achieved by various techniques, such as electrophoresis in agarose gels or polyacrylamide gels, capillary electrophoresis, microfluidic chips, etc.

[0004] Nucleic acid-based molecular weight standards (or ladders) are useful tools for estimating the quality, size, and / or quantity of nucleic acid samples. The standards are typically fractionated simultaneously (e.g., in parallel) with the sample. After detection, a comparison is made between the sample bands and the standard bands. Knowing the size (e.g., in nucleotides, i.e., nt) or other characteristics of the standards allows for the estimation of the size of unknown fragments. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,273,863 [Patent Document 2] U.S. Patent Publication No. 2019 / 0203242 [Patent Document 3] U.S. Patent Publication No. 2007 / 0178482 [Patent Document 4] International Publication No. WO2020 / 232286 [Patent Document 5] U.S. Patent No. 5,316,908 [Patent Document 6] U.S. Patent Publication No. 2019 / 0002868 [Non-patent literature]

[0006] [Non-Patent Document 1] Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY. [Non-patent document 2] Sambrook J, Fritsch EF, and Maniatis T, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989). [Non-patent document 3] Sambrook J and Russell W, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001) [Non-patent document 4] Altschul SF et al. J. Mol. Biol. 1990;215:403~10 [Non-Patent Document 5] Zhang J et al., Genome Res. 1997;7:649~56 [Non-patent document 6] Smith TFら、Adv. Appl. Math. 1981;2(4):482~9

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[0007] The present disclosure relates to methods, compositions, and kits comprising cleaved single-stranded nucleic acids. In certain embodiments, such nucleic acids may be useful as ladders containing single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), or single-stranded DNA / RNA hybrids (ssDNA / RNA).

[0008] In certain embodiments, the methods herein allow for the preparation of single-stranded nucleic acids (e.g., ssDNA, ssRNA, or ssDNA / RNA) in amounts and under conditions that result in ladder compositions. For example, typical methods for preparing double-stranded DNA (dsDNA) are not suitable for preparing ssDNA ladders. In one example, digestion of dsDNA of known length may be used to create ladder fragments, with the length of the ladder fragments determined by restriction enzyme sites. In another example, polymerase chain reaction (PCR) may be used to generate dsDNA of a predetermined length. Such processes are limited, for example, by the length of the fragments that can be generated, and long fragments (e.g., those greater than 500 bases) are difficult to generate. Furthermore, such processes may require the removal of certain components before use as a ladder. For example, if an ssDNA ladder is desired, the generated dsDNA fragments must be denatured or otherwise treated to produce the single-stranded form and / or to remove unwanted complementary sequences. In other cases, PCR requires additional reagents (eg, polymerase, salts, other excipients, etc.) and it may be preferable to remove these before use as a ladder. [Means for solving the problem]

[0009] Thus, in some non-limiting embodiments, the methods herein allow for the preparation of nucleic acids having sufficient length (e.g., greater than 500 bases in length) and / or a desired conformation (e.g., a single-stranded conformation).

[0010] The present disclosure also relates to methods for generating single-stranded nucleic acids with a predetermined or defined size (e.g., length). In some embodiments, the methods herein may employ oligonucleotide-guided cleavage of a single-stranded template with a cleavage enzyme (e.g., a restriction enzyme, a nicking enzyme, a programmable endonuclease, an Argonaute protein, a Cas enzyme, and other enzymes described herein). The guided oligonucleotide (or guide) may be ssDNA, ssRNA, or ssDNA / RNA. Using such a guide, a double-stranded structure can be formed between the guide and the template, and a cleavage enzyme can be recruited and bound to the double-stranded structure. If a cleavage site (e.g., a restriction site, a recognition sequence, a protospacer region, a protospacer adjacent motif (PAM) sequence, or a reverse complement thereof) is present in the template, the cleavage enzyme can be used to cleave the cleavage site. In this manner, the methods may result in a plurality of cleaved template molecules (e.g., a plurality of cleaved single-stranded nucleic acids).

[0011] In some embodiments, the guide oligonucleotide is sufficiently short so that separation of the guide is not necessary to use the cleaved template molecule as a ladder. Non-limiting examples of lengths of the guide oligonucleotide include less than about 40, 30, 20, or 10 nucleotides (nt), or about 10-40 nt (e.g., about 10-38 nt, 10-36 nt, 10-34 nt, 10-32 nt, 10-30 nt, 10-29 nt, 10-28 nt, 10-27 nt, 10-26 nt, 10-25 nt, 10-24 nt, 10-23 nt, 10-22 nt, 10-21 nt, 10-20 nt, 12-40 nt, 12-38 nt, 12~36nt, 12~34nt, 12~32nt, 12~30nt, 12~29nt, 12~28nt, 12~27nt, 12~26nt, 12~25nt, 12~24nt, 12~23nt, 12~22nt, 12~21nt, 12~20nt, 14~40nt, 14~38nt, 14~36nt, 14~34nt, 14~32nt, 14~30nt, 14~29nt, 14~28nt, 14~27nt, 14~26nt, 14~25nt, 14~24nt, 14~23nt, 14~22nt, 14~21nt, 14~20nt, 16~40nt, 16~38nt, 16~36nt, 16~34nt, 16~32nt, 16~30nt, 16~29nt, 16~28nt, 16~27nt, 16~26nt, 16~25nt, 16~24nt, 16~23nt, 16~22nt, 16~21nt, 16~20nt, 18~40nt, 18~38nt, 18~36nt, 18~34nt, 18~32nt, 18~30nt, Examples include 18-29 nt, 18-28 nt, 18-27 nt, 18-26 nt, 18-25 nt, 18-24 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 20-40 nt, 20-38 nt, 20-36 nt, 20-34 nt, 10-32 nt, 20-30 nt, 20-29 nt, 20-28 nt, 20-27 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, and 20-21 nt.

[0012] The present disclosure also provides nucleic acid compositions (or ladders) that can be used as standards for estimating the size (e.g., the chain length in nt of a single-stranded polynucleotide) and / or mass of nucleic acid molecules whose size and / or mass are unknown. Accordingly, the present disclosure relates to methods for producing such compositions or ladders, further details of which are provided below.

[0013] definition As used herein, the term "about" means ±10% of the stated value. As used herein, this term modifies a stated value, range of values, or one or more endpoints of a range.

[0014] As used herein, the term "cleavage" or "cleaving" refers to the breakage of the covalent backbone in a template (e.g., a target region of a template). Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-strand and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two different single-strand cleavage events. Cleavage can result in the generation of blunt ends, zigzag ends (or sticky ends), or nicked ends. Furthermore, cleavage can occur in DNA, RNA, and DNA / RNA hybrid sequences or regions. In some, but not all, embodiments, cleavage can be associated with the breakage of the covalent backbone in the guided oligonucleotide.

[0015] Terms such as "complementarity" or "complementary" refer to the ability of a nucleic acid to form hydrogen bonds, either conventionally Watson-Crick or otherwise non-conventional, with another nucleic acid sequence in a sequence-specific antiparallel manner under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength, e.g., to form Watson-Crick base pairs and / or G / U base pairs with another nucleic acid, to "anneal," or to "hybridize" (i.e., a nucleic acid can specifically bind to a complementary nucleic acid). As is known in the art, standard Watson-Crick base pairing includes adenine (A) and thymidine (T) pairing, adenine (A) and uracil (U) pairing, and guanine (G) and cytosine (C) pairing. Additionally, it is known in the art that guanine (G) base pairs with uracil (U) in hybridization between two RNA molecules (e.g., dsRNA). Complementarity refers to the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 would be 50%, 60%, 70%, 80%, 90%, and 100% complementarity). "Perfectly complementary" means that all contiguous residues in a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" or "sufficiently complementary" or "fully complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. Other features and advantages of the present disclosure will become apparent from the following detailed description, drawings, and claims.

[0016] Stringent hybridization conditions refer to conditions under which nucleic acids complementary to a target region hybridize primarily to the target region and do not substantially hybridize to non-target regions. Stringent conditions are generally sequence-dependent and vary depending on multiple factors. Generally, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to its target region. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology—Hybridization With Nucleic Acid Probes, Part 1, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assay,” Elsevier, NY.

[0017] As used herein, the term "fragment" with respect to a nucleic acid refers to a portion of a nucleic acid that is at least one nucleotide shorter than a reference sequence. This portion preferably comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule. A fragment may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 1800, or more nucleotides. In another example, any nucleic acid fragment that can include a stretch of at least about 5 (e.g., about 10, about 20, about 30, about 40, about 50, or about 100) nucleotides that is at least about 40% (e.g., about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 87%, about 98%, about 99%, or about 100%) identical to any of the sequences described herein can be utilized in accordance with the present disclosure. In yet another example, any nucleic acid fragment that can include a stretch of at least about 5 (e.g., about 7, about 8, about 10, about 12, about 14, about 18, about 20, about 24, about 28, about 30, or more) nucleotides that is at least about 40% (about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 87%, about 98%, about 99%, or about 100%) identical to any of the sequences described herein can be utilized in accordance with the present disclosure.

[0018] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. The hydrogen bonds may occur by Watson-Crick base pairing, by Hoogsteen binding, or in some other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a more extensive process, such as denaturation at high temperatures (e.g., above 90°C) and / or annealing at a specific cooling rate (e.g., ramping rate). A sequence capable of hybridizing to a given sequence is said to be the "complement" of the given sequence. Hybridization and washing conditions are well known and are exemplified in Sambrook J, Fritsch EF, and Maniatis T, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), especially Chapter 11 and Table 11.1 thereof, and Sambrook J and Russell W, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0019] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions suitable for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, variables well known in the art. The higher the degree of complementarity between two nucleotide sequences, the higher the melting temperature (T m) becomes larger. For hybridization between nucleic acids having short stretches of complementarity (e.g., complementarity over 35 or fewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides), the location of mismatches becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of a hybridizable nucleic acid is at least about 10 nucleotides. Exemplary minimum lengths for a hybridizable nucleic acid are at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides, and at least about 30 nucleotides. Furthermore, as will be appreciated by those of skill in the art, the temperature and salt concentration of the hybridization solution can be adjusted as needed depending on factors such as the length of the complementary region and the degree of complementarity.

[0020] It is understood in the art that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. Furthermore, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop or hairpin structure). A polynucleotide may contain at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity with a target region within its target nucleic acid sequence. For example, an antisense nucleic acid in which 18 out of 20 nucleotides of an antisense compound are complementary to a target region and therefore specifically hybridizes corresponds to 90 percent complementarity. In this example, the remaining non-complementary nucleotides may be clustered or may be interposed by complementary nucleotides, and the remaining non-complementary nucleotides need not be contiguous with each other or with complementary nucleotides. The percent complementarity between specific regions of a nucleic acid sequence in a nucleic acid can be routinely determined using BLAST (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul SF et al., J. Mol. Biol. 1990;215:403-10; Zhang J et al., Genome Res. 1997;7:649-56), or by using the Gap program (Wisconsin Sequence Analysis Package, Unix version 8, Genetics Computer Group, University Research Park, Madison Wis.) with default settings using the algorithm of Smith TF et al., Adv. Appl. Math. 1981;2(4):482-9.

[0021] As used herein, "ladder" or "size standard" may be used interchangeably and generally refer to a set of standards used to identify the approximate size and / or mass of a molecule. In certain embodiments, the size identification may involve electrophoresis using the principle that molecular weight is inversely proportional to the rate of migration through a gel matrix. In some embodiments, a ladder, when used in electrophoresis, may be a linear or logarithmic scale for estimating the size or mass of unknown fragments (provided the fragment sizes of the markers are known).

[0022] "Linker" refers to any useful multivalent (e.g., bivalent) moiety that is useful for attaching various moieties or segments. Exemplary linkers include nucleic acid sequences, chemical linkers, and the like. In one example, a linker can have a length of about 3 nucleotides (nt) to 100 nt. For example, a linker can have a length of about 3 nt to 90 nt, 3 nt to 80 nt, 3 nt to 70 nt, 3 nt to 60 nt, 3 nt to 50 nt, 3 nt to 40 nt, 3 nt to 30 nt, 3 nt to 20 nt, or 3 nt to 10 nt. In another example, the linker may have a length of 3 nt to 5 nt, 5 nt to 10 nt, 10 nt to 15 nt, 15 nt to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 35 nt, 35 nt to 40 nt, 40 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, or 90 nt to 100 nt.

[0023] The terms "nicking enzyme" or "nicking endonuclease" or "nickase" are sometimes used interchangeably and generally refer to an enzyme that cleaves one strand of double-stranded DNA at a specific recognition nucleotide sequence known as a restriction site. Such enzymes hydrolyze (cut) only one strand of the DNA duplex, thereby generating a DNA molecule that is "nicked" or cut in only one strand (e.g., only the top strand or only the bottom strand).

[0024] The terms "nuclease" and "endonuclease" are used interchangeably herein to refer to an enzyme with catalytic activity for DNA and / or RNA cleavage. Examples of nucleases include restriction enzymes, nicking enzymes, programmable endonucleases, Argonaute proteins, Cas enzymes, or variants thereof. Variants can include mutants of wild-type nucleases (e.g., substitutions in one or more amino acids, such as conservative amino acid substitutions) that have increased, decreased, or retain, but do not eliminate, catalytic activity for DNA and / or RNA cleavage. Generally, various sites and motifs (e.g., double-stranded structures) useful for recruiting nucleases to a complex containing a template-bound (or hybridized) guiding oligonucleotide are described herein. Such sites and motifs can include one or more cleavage sites resulting from nuclease cleavage and / or can include one or more sites for binding to a nuclease. Non-limiting examples of such recruitment sites or motifs include restriction sites (e.g., for binding and / or cleavage by a restriction enzyme or a nicking enzyme), recognition sites (e.g., for binding and / or cleavage by a nuclease such as a programmable endonuclease, including an Argonaute protein), protospacer regions (e.g., for cleavage by a nuclease such as a programmable endonuclease, including a Cas enzyme), and / or protospacer adjacent motif (PAM) sequences (e.g., for binding by a nuclease such as a programmable endonuclease, including a Cas enzyme).

[0025] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded (e.g., sense or antisense), double-stranded, or multi-stranded ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof, genomic DNA, cDNA, or DNA / RNA hybrids, as well as polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Polynucleotides can have any useful two- or three-dimensional structure or motif, such as regions containing one or more duplex, triplex, quadruplex, hairpin, and / or pseudoknot structures or motifs. When N is used in a nucleic acid sequence, it can be represented by any nucleic acid (eg, G, C, A, T, or U, and modified forms thereof).

[0026] The term "modified" as used with respect to nucleic acids means a nucleic acid sequence that includes one or more modifications to the nucleobase, nucleoside, nucleotide, phosphate group, sugar group, and / or internucleoside linkage (e.g., phosphodiester backbone, linked phosphate, or phosphodiester linkage).

[0027] Nucleoside modifications include pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl -4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine cytidine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, ze Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-Diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wyobutosine, 7-deaza-guanosine, 7- These may include, but are not limited to, deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

[0028] The sugar modification is Locked Nucleic Acid (LNA: 2'-hydroxyl is C 1~6 Alkylene bridge or C 1~6connected to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge), replacement of oxygen in ribose (e.g., with S, Se, or an alkylene such as methylene or ethylene), addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., to form a cyclobutane or oxetane four-membered ring), ring expansion of ribose (e.g., anhydrohexitols, altritols, mannitols, cyclohexanyls, cyclohex ... Nucleic acids may include, but are not limited to, nucleotides containing 6- or 7-membered rings, such as hexenyl and morpholino, polycyclic forms (e.g., tricyclic), and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, in which the ribose is replaced by a glycol unit linked to a phosphodiester bond), threose nucleic acids (TNAs, in which the ribose is replaced by aL-threofuranosyl-(3'→2')), and peptide nucleic acids (PNAs, in which a 2-amino-ethyl-glycine bond replaces the ribose and phosphodiester backbone). The sugar group may also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, polynucleotide molecules may include nucleotides containing, for example, arabinose as the sugar.

[0029] The backbone modification may include, but is not limited to, a 2'-deoxy modification or a 2'-O-methyl modification. The phosphate group modification may include, but is not limited to, a phosphorothioate, a phosphoroselenate, a boranophosphate, a boranophosphate ester, a hydrogen phosphonate, a phosphoramidate, a phosphorodiamidate, an alkyl or aryl phosphonate, a phosphotriester, a phosphorodithioate, a bridged phosphoramidate, a bridged phosphorothioate, or a bridged methylene phosphonate.

[0030] The terms "protein," "peptide," or "polypeptide," used interchangeably, refer to a chain of more than two amino acids that constitutes all or part of a naturally occurring polypeptide or peptide, regardless of post-translational modification (e.g., glycosylation or phosphorylation), or that constitutes a non-naturally occurring polypeptide or peptide, which may contain coded amino acids, non-coded amino acids, modified amino acids (e.g., chemically and / or biologically modified amino acids), and / or modified backbones.

[0031] The terms "restriction enzyme" or "restriction endonuclease" or "restrictase" are sometimes used interchangeably and generally refer to enzymes that cut double-stranded DNA into fragments at or near specific recognition sites in the molecule known as restriction sites. Restriction enzymes are a type of enzyme within the broader family of endonucleases. To cut DNA, all restriction enzymes make two cuts, once in each of the sugar-phosphate backbones (i.e., each strand) of the DNA double helix.

[0032] The following drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way. Like reference numerals in the drawings indicate like elements. [Brief explanation of the drawings]

[0033] [Figure 1]

[0023] Figure 1 is a schematic diagram of a non-limiting method for preparing a ladder composition. As shown, non-limiting preparation of a single-stranded ladder by guided cleavage of a nucleic acid (e.g., ssDNA) template is presented. The oval represents a single-stranded template, which may be either circular or linear. The guiding oligonucleotide (dashed line) may be (A) pre-annealed with the template, (B) pre-assembled with the cleavage enzyme (gray triangle), or (C) directly mixed with the cleavage enzyme and template. In all cases, the guiding oligonucleotide and cleavage enzyme can bind to a target region of the template and also cleave the template to generate single-stranded nucleic acid fragments of different sizes. Such fragments can then be separated and detected by electrophoresis (e.g., displayed as a gel image or electropherogram), as appropriate. The guiding oligonucleotides may be of the same or different sequences. [Figure 2] Figure 1 shows a ssDNA ladder prepared by oligonucleotide-guided restriction enzyme digestion. (A) A map of the restriction enzyme positions in the M13mp18 ssDNA template and (B) an electropherogram analysis of ssDNA size by different combinations of restriction enzyme digestion are shown. The x-axis indicates fragment migration time, and the y-axis is fluorescence. (C) A simulated gel image of the results generated by LabChip® Reviewer is also shown. The size of the intact circular ssDNA is 7246 nt. Enzyme (-) was used as a negative control. A lower marker was used for sample alignment. [Figure 3]Schematic diagrams of non-limiting guided oligonucleotides for use in restriction enzyme-based template (e.g., ssDNA) cleavage. (A) A guided oligonucleotide that results in a blunt end is shown. The guided oligonucleotide may contain a target binding region (N in italics) that is sufficiently complementary to the target region (N) and a restriction sequence (YYYY) that is sufficiently complementary to a restriction sequence (XXXX) within the target region. (B) A guided oligonucleotide that results in a sticky or zigzag end is also shown. The guided oligonucleotide may contain a target binding region (N in italics) that is sufficiently complementary to the target region (N) and a restriction sequence (YYYYYY) that is sufficiently complementary to a restriction sequence (XXXXXX) within the target region. Potential cleavage locations (dashed triangles) are shown as non-limiting examples only. [Figure 4] Schematic diagrams of non-limiting guided oligonucleotides for use in CRISPR-based template (e.g., ssDNA template) cleavage. The target region can be downstream (e.g., approximately 20 nt downstream) of the reverse complement of the protospacer adjacent motif (PAM) sequence within the template. Here, this reverse complement has a sequence of CCN (5' to 3'), such that the PAM is NGG (5' to 3'). (A) A single-guide RNA is shown, including a target binding region (N in italics) that is sufficiently complementary to the target region (N), a CRISPR ribonucleic acid (crRNA) region, a linker, and a trans-acting CRISPR RNA (tracrRNA) region. (B) Also shown is a bipartite guided RNA, including a guided RNA with a first portion (a target binding region that is sufficiently complementary to the target sequence region, where the target binding region is linked to the crRNA region) and a second portion (the tracrRNA region). The first and second portions may form a double-stranded structure by hybridization between portions of the crRNA and tracrRNA regions (dashed lines). Potential cleavage sites (dashed triangles) are shown as non-limiting examples only. [Figure 5]1 is a map of M13mp18 with non-limiting restriction sites. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure encompasses methods for producing size or mass standard ladders and compositions comprising such ladders. In some non-limiting embodiments, the compositions can include single-stranded nucleic acid size standards. As described herein, ladders can be prepared by using guided cleavage of a single-stranded template. Any useful template can be used; for example, the single-stranded template can be derived from natural sources, or can be a synthetic construct derived from natural sources or an engineered construct (e.g., engineered M13 DNA containing one or more inserts, such as an insert having 1000 nucleotides). Furthermore, the template can be a linear or circular construct. In some embodiments, the template (e.g., an ssDNA template) can be either linear or circular, and there is no size limit.

[0035] 1 presents a non-limiting method for preparing a ladder composition, which can include incubating a template (oval) in the presence of a guiding oligonucleotide (dashed line) and a cleavage enzyme (triangle), thereby generating a ladder composition comprising a plurality of cleaved single-stranded nucleic acids.

[0036] The template can be a single-stranded nucleic acid (e.g., ssDNA, ssRNA, or ssDNA / RNA). Consequently, the ladder composition can include multiple cleaved templates (e.g., cleaved ssDNA, ssRNA, or ssDNA / RNA). Furthermore, the target region within the template can include a restriction sequence, a recognition sequence, a protospacer region, a protospacer adjacent motif (PAM) sequence, or a reverse complement thereof. Such sites can be locations within the target region that are cleaved by a cleavage enzyme upon binding to a guide oligonucleotide (or guide).

[0037] The guide oligonucleotide can be configured to bind to a target region of the template. In one example, the guide oligonucleotide (or guide) can include a target binding region configured to bind (or hybridize) to a target region within the template. In some embodiments, the guide includes a restriction sequence, a recognition sequence, a protospacer region, a PAM sequence, or a reverse complement thereof. The template and guide can be designed to bind (or hybridize) to each other. For example, if the template includes a restriction sequence, the guide can include a reverse complement of the restriction sequence to ensure hybridization between the template and guide, thereby forming a hybridized double-stranded structure. Such a double-stranded structure can then recruit a cleavage enzyme and facilitate cleavage at a location within the target region of the template. Non-limiting examples of such sites are described herein.

[0038] In certain embodiments, a template can include multiple target regions, and a guide can be designed to bind (or hybridize) to each target region in the template. Thus, the method can include the use of multiple guides, such that a first guide is configured to bind to a first target region in the template and a second guide is configured to bind to a second target region in the template. In the presence of a cleaving enzyme, a single template can be cleaved n times, where n is the number of times determined by the n guides that can bind to the template (e.g., n can be 1, 2, 3, 4, 5, or more).

[0039] The cleavage enzyme can be configured to bind to the guide and cleave at locations within the target region, thereby generating a ladder composition comprising a plurality of cleaved single-stranded nucleic acids, non-limiting examples of which are described herein.

[0040] The template, guide, and cleavage enzyme can be provided in any useful manner. For example, these components can be provided sequentially or simultaneously, or introduced to provide a complex. As can be seen in Figure 1A, a guide can be first provided to the template to provide a complex containing the bound template. One or more cleavage enzymes can then be provided to the bound template to generate cleaved nucleic acids. Such cleavage products can be separated and / or detected as appropriate.

[0041] Other complexes can also be formed. As can be seen in Figure 1B, a pre-assembled complex containing a guide bound to the enzyme can be prepared by first providing the guide to the cleavage enzyme. A template can then be provided to generate a cleaved nucleic acid derived from the template. In yet other embodiments, the components are provided separately. As can be seen in Figure 1C, a cleaved nucleic acid can be generated by adding the guide, cleavage enzyme, and template separately to a reaction mixture.

[0042] The guide and cleavage enzyme can be any combination that results in guided cleavage of the template. In one example, the guide can include ssDNA, and the cleavage enzyme can include a restriction enzyme, a nicking enzyme, or an Argonaute protein. In yet another example, the guide can include ssRNA, and the cleavage enzyme can include a Cas enzyme. In yet other embodiments, an initial enzyme that preferentially cleaves ssDNA can be modified so that it also cleaves ssRNA, or vice versa. Additionally, cleavage fragments obtained from one or more methods (e.g., any of those described herein) can be mixed together to form the final ladder preparation.

[0043] In some embodiments, ssDNA size standards are generated by guided cleavage of an ssDNA template (see, e.g., FIG. 1). For example, the guide may be pre-annealed with the target ssDNA template (see, e.g., FIG. 1A), pre-assembled with the cleavage enzyme before providing the target ssDNA template (see, e.g., FIG. 1B), or directly mixed with the cleavage enzyme and ssDNA template (see, e.g., FIG. 1C). Here, the guide and cleavage enzyme can be configured to bind to a target region of the ssDNA template, thereby causing the enzyme to cleave the ssDNA template to generate ssDNA fragments of different sizes. The sizes are determined by where the guide binds or hybridizes to the template, thereby providing a cleavage site for the cleavage enzyme. Fragments can be optionally separated and / or detected by electrophoresis (e.g., as displayed in FIG. 1 as a gel image or electropherogram). Non-limiting characterization of library preparations is presented in FIG. 2 and Example 1 herein.

[0044] Methods involving specific cleavage enzymes are also described. In some embodiments, a template (e.g., an ssDNA template) can be cleaved with a restriction enzyme or nicking enzyme in conjunction with a short, single-stranded guide oligonucleotide. Restriction enzymes recognize short DNA sequences (or restriction sites) and cleave double-stranded DNA at specific sites within or adjacent to these sequences. Approximately 3,000 restriction enzymes have been discovered that recognize over 230 different DNA sequences. Restriction enzymes are present in bacteria, viruses, archaea, and eukaryotes. Nicking enzymes cleave only one strand of double-stranded DNA, leaving the other strand intact. Over 200 nicking enzymes have been studied, some of which are commercially available and routinely used in research and commercial products. Any useful restriction enzyme and nicking enzyme can be used in the methods herein, and non-limiting examples of such enzymes are described herein.

[0045] In some embodiments, a template (e.g., an ssDNA template) can be cleaved by a restriction enzyme or nicking enzyme with the ssDNA as a guide. A non-limiting example is provided in Example 1. In this method, the guide can have the minimum size (or length) required for the restriction enzyme / nicking enzyme to bind and cleave. A non-limiting length of a guide for use with a restriction enzyme or nicking enzyme is about 10 nt to 30 nt. The guide can be designed as the reverse complement of the target region in the template. The guide can then be annealed with the template (e.g., by a denaturing and annealing process) to form a short double-stranded region that can be recognized and cleaved by the restriction enzyme / nicking enzyme. Non-limiting denaturation and annealing conditions can include any of those described herein, for example, denaturation at high temperature (e.g., denaturation at about 85°C or higher for any useful time, such as about 2 minutes or more, including heating to 70°C for 2 minutes followed immediately by cooling on ice for about 5 minutes before analysis), and / or annealing at high temperature and cooling to a temperature below 37°C (e.g., about 90°C for 4 minutes, then annealing at 70°C for 10 minutes, and finally ramping down to 37°C at 0.1°C / sec over 20 minutes).

[0046] One or more guides (e.g., short ssDNA oligonucleotides) can be annealed to the template simultaneously or in a sequential manner. One or more corresponding restriction enzymes / nicking enzymes can be applied for cleavage simultaneously or in a sequential manner. Any commercially available or previously published restriction enzymes or nicking enzymes (natural sources or engineered) can be used for ladder preparation. After cleavage, the restriction enzymes / nicking enzymes can be inactivated by the use of heat or chemical chelation (e.g., by adding ethylenediaminetetraacetic acid (EDTA)).

[0047] Figure 3 provides a schematic diagram of non-limiting guides and templates for use with restriction enzymes. As can be seen in Figure 3A, the guide can include a target binding region (italicized), which further includes a restriction sequence (YYYY, corresponding to one strand of a double-stranded restriction site). Furthermore, the target binding region is configured to bind to (or hybridize with) a target region of the template. The cleavage site can be located within the restriction site such that cleavage between the two strands can result in blunt ends. As can be seen in Figure 3B, cleavage can also result in sticky or zigzag ends.

[0048] In other embodiments, a template (e.g., an ssDNA template) can be cleaved by a DNA-guided programmable endonuclease with ssDNA as a guide. For example, the prokaryotic Argonaute (pAgo) protein is a programmable endonuclease involved in cellular defense against foreign genetic elements. In this method, Argonaute proteins from natural sources or engineered proteins can be used for ladder preparation. Thermus thermophilus pAgo (TtAgo) can use short single-stranded DNA (ssDNA) guides to bind to target DNA. When used with an Argonaute protein, the guide can include a target binding region and a recognition sequence. Non-limiting recognition sequences for use with Argonaute proteins are described herein. One or more guides can be used simultaneously or sequentially with the programmable endonuclease protein, buffer, and other components for guided cleavage. Cleavage can be terminated by heat or chemical inactivation (e.g., by adding EDTA).

[0049] In yet another embodiment, a template (e.g., an ssDNA template) can be cleaved by a clustered regularly interspaced short palindromic repeats-associated (CRISPR-associated) Cas (Cas) enzyme with a guide ssRNA. The CRISPR-Cas system has been shown to confer adaptive immunity against mobile genetic elements. Double-stranded DNA cleavage by the Cas9 enzyme is a hallmark of the type II CRISPR-Cas immune system. The Cas9-guide RNA complex recognizes a 20-bp sequence in DNA and generates a site-specific double-strand break. Furthermore, ssDNA cleavage is an intrinsic function of the Cas9 enzyme family, which requires unique substrate binding and catalytic domain organization. DNA-cleaving Cas enzymes (e.g., Cas9, Cas12a, or others described herein) are of great interest for genome editing. The specificity of these DNA nucleases is determined by the guide, allowing for flexible targeting. In addition to this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics that can be exploited for a variety of applications.

[0050] In some embodiments, a template (e.g., an ssDNA template) can be cleaved by a CRISPR-Cas enzyme with ssRNA as a guide. In this method, DNA-cleaving Cas enzymes, such as, but not limited to, Cas9 and Cas12a proteins derived from natural sources or engineered proteins, can be used for ladder preparation. The design of the RNA guide is based on the specific sequence of the target region (within the template) and the corresponding requirements from the Cas enzyme. One or more ssRNA guides can be used simultaneously or sequentially in conjunction with the CRISPR-Cas enzyme, buffer, etc. for guided cleavage. CRISPR-Cas enzyme cleavage can be terminated by heat inactivation or chemical inactivation (e.g., by adding EDTA).

[0051] Figure 4 provides a schematic of non-limiting guides and templates for use with Cas enzymes. As can be seen in Figure 4A, a guide can include a target binding region (italicized), a CRISPR ribonucleic acid (crRNA) region, an optional linker, and a trans-acting CRISPR RNA (tracrRNA) region. The sequence within the PAM motif is associated with the corresponding Cas enzyme. Furthermore, the target binding region is configured to bind (or hybridize) to a target region of the template. Cleavage sites can be located within the target region and the target binding region. In some embodiments, the Cas enzyme can be modified to confer nickase activity.

[0052] In certain embodiments, ladders can be prepared from one or more methods described herein. Using such methods, separate populations of cleaved nucleic acids can be prepared, which can then be mixed together as a final product. In one non-limiting embodiment, the ssDNA template is M13mp18 (a single-stranded circular DNA having approximately 7.2 kilobases (kb); see, e.g., Figure 5). Such templates can be used to generate the following linear size standards: a population comprising at least one cleaved nucleic acid having 7.2 kb (for example, by linearizing the circular initial template by cleavage with a restriction enzyme (for example, HindIII)), a population comprising a first cleaved nucleic acid having a length of 2.1 kb and a second cleaved nucleic acid having a length of 5.1 kb (e.g., by cleavage with two restriction enzymes (e.g., HindIII and PacI)), a population comprising a first cleaved nucleic acid having 3.2 kb and a second cleaved nucleic acid having 4 kb (e.g., by cleavage with two restriction enzymes (e.g., HindIII and AfeI)), and / or A population comprising a first cleaved nucleic acid having 1.0 kb and a first cleaved nucleic acid having 6.2 kb (e.g., by cleavage with a CRISPR-Cas enzyme). In some embodiments, ladder compositions can be prepared by combining the above fragments to generate size standards of approximately 1.0 kb, 2.1 kb, 3.2 kb, 4 kb, 5.1 kb, 6.2 kb, and 7.2 kb. In other embodiments, ladder compositions can be prepared by combining the above fragments to generate size standards of approximately 1.1 kb, 2.1 kb, 3.2 kb, 4 kb, 5.1 kb, 6.2 kb, and 7.2 kb.

[0053] In some embodiments, size standards (e.g., ssDNA size standards) prepared by the methods herein can be separated and detected in electrophoresis platforms, such as, but not limited to, agarose gels or polyacrylamide gels, capillary electrophoresis, microfluidic chips, high performance liquid chromatography, mass spectrometry, and the like.

[0054] In some embodiments, the methods herein enable the preparation of ladder compositions with high efficiency. While not wishing to be limited by mechanism, the methods utilize a reduced number of reagents (e.g., short guiding oligonucleotides and cleavage enzymes) and a reduced number of reaction events (e.g., binding and cleavage events). By selecting process conditions that facilitate efficient binding and cleavage, ladders can be produced in a cost- or process-efficient manner. Furthermore, as described herein, the ladder preparations produced do not require purification and / or dilution prior to use, potentially eliminating downstream processing steps.

[0055] In some embodiments, the methods herein allow for the preparation of ladder compositions having nucleic acids of increased size. For example, PCR can be used to prepare nucleic acids for use as ladders, but relatively long nucleic acids are difficult to prepare using PCR. Using the methods herein, nucleic acids having lengths of 500 bases or more can be prepared. Further non-limiting lengths can include greater than about 500 bases (0.5 kilobases or kb), greater than 1000 bases (1 kb), greater than 2000 bases (2 kb), greater than 3000 bases (3 kb), greater than 4000 bases (4 kb), greater than 5000 bases (5 kb), greater than 6000 bases (6 kb), greater than 7000 bases (7 kb), or greater than 8000 bases (8 kb). In other embodiments, the length is about 0.5 to 3 kb, 0.5 to 5 kb, 0.5 to 10 kb, 0.5 to 20 kb, 0.5 to 50 kb, 0.5 to 100 kb, 0.5 to 200 kb, 0.5 to 500 kb, 0.5 to 750 kb, 1 to 3 kb, 1 to 5 kb, 1 to 10 kb, 1 to 20 kb, 1 to 50 kb, 1 to 100 kb, 1 to 200 kb, 1 to 500 kb, 1 to 750 kb, 2 to 3 kb, 2 to 5 kb, 2 to 10 kb, 2 to 20 kb, 2 to 50 kb, 2 to 1 The length may be 00 kb, 2 to 200 kb, 2 to 500 kb, 2 to 750 kb, 3 to 5 kb, 3 to 10 kb, 3 to 20 kb, 3 to 50 kb, 3 to 100 kb, 3 to 200 kb, 3 to 500 kb, 3 to 750 kb, 5 to 10 kb, 5 to 20 kb, 5 to 50 kb, 5 to 100 kb, 5 to 200 kb, 5 to 500 kb, 5 to 750 kb, 10 to 20 kb, 10 to 50 kb, 10 to 100 kb, 10 to 200 kb, 10 to 500 kb, or 10 to 750 kb.

[0056] In other embodiments, the methods herein enable the preparation of ladder compositions with high purity. For example, and not by way of limitation, the methods herein may use a single-stranded template to generate cleaved single-stranded nucleic acids. Starting with a highly pure template allows for the production of cleaved nucleic acids with high purity that would otherwise require additional purification steps. The methods herein may also be adapted to avoid components that reduce the purity of the resulting ladder composition. For example, PCR or other amplification reactions may be avoided, and reagents to facilitate amplification (e.g., polymerase, additional salts, additional buffers, buffer components, etc.) may be avoided. In another example, template cleavage may be adapted to avoid excess or additional components (e.g., bovine serum albumin (BSA), denaturants, etc.).

[0057] Purity can be determined in any useful manner. In one non-limiting example, purity can be based on the homogeneity of single-stranded fragments (e.g., homogeneity assessed by fragment size or fragment sequence). In certain embodiments, homogeneity can be determined by the efficiency of an enzymatic cleavage reaction. In another example, the purity of a buffer in a ladder composition can be assessed. For example, a buffer in the final ladder composition can be identified as being useful for long-term storage (e.g., one month, three months, six months, one year, or longer storage that does not damage single-stranded nucleic acids or affect other properties of the nucleic acids, such as the presence of undesired double-stranded structures or undesired fragment sizes). However, in some cases, double-stranded structures may form in the buffer, and the ladder composition may be denatured before use. By way of example and not limitation, the ladder and test sample can be exposed to a denaturing process (e.g., any described herein) prior to analysis, such as before electrophoresis. In another example, buffers in the final ladder composition can be specified as not interfering with one or more downstream processes (eg, assays, purification processes, separation processes, etc., such as electrophoresis).

[0058] Methods for determining purity can include any useful methodology, including, but not limited to, the use of a spectrophotometer, an electrophoresis analyzer, an ssDNA assay, etc. Non-limiting examples include analysis of various absorbance measurements (e.g., absorbance at 260 nm (A260), absorbance at 280 nm (A280), or the ratio of A260 and A280, such as with a NanoDrop™ spectrophotometer from Thermo Fisher Scientific, Waltham, MA), the amount of ssDNA (e.g., with a Qubit™ ssDNA Assay Kit from Thermo Fisher Scientific), the amount of dsDNA (e.g., with a Qubit™ dsDNA HS Assay Kit), and fragment analysis using electrophoresis (e.g., with a LabChip® from PerkinElmer, Waltham, MA).

[0059] In some embodiments, conditions during cleavage can be optimized to be compatible with downstream electrophoresis methods without the need for post-cleavage purification. One such condition can include the presence of a blocking agent, such as bovine serum albumin (BSA) or recombinant albumin (rAlbumin). Typically, BSA or rAlbumin is a standard component in buffers used during cleavage with restriction or nicking enzymes. In use, albumin-containing components can be used to stabilize proteins in the reaction mixture and / or to prevent adhesion of reaction products or reagents (e.g., primers, proteins, etc.) to the surfaces of reaction tubes or pipettes. When such buffers are used, cleaved products (e.g., as ssDNA ladders) can be used directly for agarose gel or PAGE gel analysis. However, such products may need to be diluted or purified before performing electrophoresis analysis (e.g., in the LabChip® platform, which limits BSA concentrations to 0.05 mg / mL). Using the methods herein, such blocking agents can be avoided during cleavage.

[0060] In the cleavage reaction for ladder production, the template (e.g., ssDNA) and enzyme are typically provided in sufficient amounts to minimize the risk of lost performance due to surface binding. Thus, in some embodiments, the cleavage buffer may be modified to remove or minimize the amount of BSA or rAlbumin. As a result, the fragments obtained from enzymatic cleavage (e.g., cleaved ssDNA fragments) can be subjected to electrophoresis without the need for purification and / or dilution.

[0061] In another example, the presence of double-stranded (ds) nucleic acids may be undesirable, and the present method eliminates the presence of excess ds nucleic acids. If a double-stranded structure exists when the guide oligonucleotide binds to the template, such a structure can be designed to have a minimal effect on the ladder that is generated. For example, a shortened guide oligonucleotide (or guide) can be used, such that the length of the guide can be 10, 20, 30, 40 nt, or even shorter than the truncated nucleic acid provided in the ladder composition.

[0062] In yet another example, the methods herein enable the preparation of ladder compositions having primarily single-stranded and cleaved nucleic acids. As described herein, the methods herein can avoid the continuous formation of double-stranded structures (e.g., as in PCR) to yield cleaved single-stranded nucleic acids. Thus, the resulting cleaved nucleic acids can be considered single-stranded. The degree of single-strandedness can be determined in any useful manner, such as by measuring absorbance at 260 nm (basochromicity results in an increase in absorbance upon denaturation of double-stranded structures), using an intercalating dye capable of binding to double-stranded structures, etc. The degree of single-strandedness can be determined for a population of cleaved single-stranded nucleic acids, and can be at least 80%, 90%, or more of the population is single-stranded.

[0063] In yet other embodiments, the present disclosure can be used to overcome current limitations for preparing ssDNA size standards, particularly for large-sized ladders (e.g., 0.5 kb, 1 kb, 2 kb, or larger). Such large-sized ladders can be useful in applications that analyze relatively long nucleic acid fragments. Applications can include, for example, gene therapy, mRNA-based vaccines, or other therapeutic approaches. In certain embodiments, the ladder composition can include one or more of the following features: The starting template can be a natural source of single-stranded nucleic acid (e.g., ssDNA). In a non-limiting embodiment, the template can be commercially available in large quantities at low cost. Desired size truncated nucleic acids can be generated by sequence-specific cleavage. In theory, there is no limit to the required standard size. The cleaved nucleic acid can be used directly, for example, without the need for further purification to remove one strand from the double-stranded template or the double-stranded cleavage product. Such an approach can simplify the production process and reduce production costs. The cleavage method herein can be scaled up to large vessels because the cleavage temperature can be performed at a single temperature, compared to PCR, which may require thermocycling. The avoidance of thermal ramping and PCR reagents simplifies the production process.

[0064] For any of the methods herein, advantages can include control of the size of the cleaved ladder by the user or intended use, not limited by the availability of commercially available cleavage enzymes.

[0065] In some embodiments, the present disclosure encompasses methods for single-stranded nucleic acid (e.g., ssDNA) ladder compositions that are independent of PCR, isothermal amplification that can generate double-stranded products, or DNAzyme cleavage. In certain embodiments, such methods may enable large-scale production.

[0066] In other embodiments, the ladder composition can include a first population of first cleaved single-stranded nucleic acids. This population can have any characteristics. In one example, the population can include nucleic acids having a particular length. In some embodiments, at least 50%, 60%, 70%, 80%, or more of the population are 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more kb in length, or between about 0.5 and 750 kb (e.g., 0.5-3 kb, 0.5-5 kb, 0.5-10 kb, 0.5-20 kb, 0.5-50 kb, 0.5-100 kb, 0.5-200 kb, 0.5-500 kb, 1-3 kb, 1-5 kb, 1-10 kb, 1-20 kb, 1-50 kb, 1-100 kb, 1-200 kb, 1-500 kb, 1-750 kb, or more). kb, 2 to 3 kb, 2 to 5 kb, 2 to 10 kb, 2 to 20 kb, 2 to 50 kb, 2 to 100 kb, 2 to 200 kb, 2 to 500 kb, 2 to 750 kb, 3 to 5 kb, 3 to 10 kb, 3 to 20 kb, 3 to 50 kb, 3 to 100 kb, 3 to 200 kb, 3 to 500 kb, 3 to 750 kb, 5 to 10 kb, 5 to 20 kb, 5 to 50 kb, 5 to 100 kb, 5 to 200 kb, 5 to 500 kb, 5 to 750 kb, 10 to 20 kb, 10 to 50 kb, 10 to 100 kb, 10 to 200 kb, 10 to 500 kb, or 10 to 750 kb). In other embodiments, a single population may have a plurality of first cleaved single-stranded nucleic acids and a plurality of second cleaved single-stranded nucleic acids, where the first cleaved single-stranded nucleic acids have a first length and the second cleaved single-stranded nucleic acids have a second length, and the first length and the second length may be different.

[0067] In some embodiments, the ladder composition can include a first population and a second population, each population comprising a plurality of cleaved single-stranded nucleic acids, hi other embodiments, the ladder composition can include multiple populations, each population comprising a respective plurality of cleaved single-stranded nucleic acids.

[0068] Furthermore, in some embodiments, each population can differ from another population. Differences between populations can include, for example, differences in size, mass, and / or sequence. Such differences between populations can be characterized by a certain percentage of each population having a particular characteristic. For example, and not by way of limitation, at least 90% of a first population can have a length greater than at least 90% of a second population.

[0069] In other embodiments, each population may include a common feature. For example and not limitation, at least 80%, 90%, or more of a population (e.g., or each population in a plurality of populations) may be single-stranded. In another example, at least 50%, 60%, 70%, 80%, 90%, or more of a population (e.g., or each population in a plurality of populations) have a length of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more kb, or a length of about 0.5 to 750 kb (e.g., any range described herein).

[0070] When multiple populations are present, each population may have a common characteristic (e.g., a length of about 0.5 to 750 kb). However, each population may still have different characteristics (e.g., a first population may have a length of about 0.5 kb, a second population may have a length of about 1 kb, a third population may have a length of about 2 kb, etc.). Those skilled in the art will understand how desirable characteristics of such ladder compositions and populations within the compositions can be determined, and how the methods herein can be designed to prepare such compositions.

[0071] The ladder composition may include other additional features, such as those described in U.S. Pat. No. 8,273,863, U.S. Patent Publication No. 2019 / 0203242 or U.S. Patent Publication No. 2007 / 0178482, International Publication No. WO2020 / 232286, Bush et al., Molecules 2020 Jul 26;25(15):3386, and / or Gu et al., Biotechniques 2013 Jun;54(6):337-43, each of which is incorporated herein by reference in its entirety.

[0072] template Any useful template can be used. In some embodiments, the template can be obtained from a natural source (e.g., a bacteriophage, viral nucleic acid, adeno-associated viral nucleic acid, etc.). Non-limiting examples of templates (e.g., ssDNA templates) include M13 bacteriophage (single-stranded circular DNA having approximately 7.2 kb), single-stranded viral DNA isolated from ΦX174 bacteriophage (e.g., ΦX174 virion DNA) (single-stranded circular DNA having approximately 5.4 kb), or single-stranded linear AAV DNA (single-stranded linear DNA having approximately 4.7-4.8 kb), such as adeno-associated virus (AAV) serotypes 1, 2, 4, or 7-11. Such templates can be obtained from any useful bacteriophage or virus, and such templates can comprise DNA and / or RNA.

[0073] In some embodiments, the template (e.g., ssDNA template) can be a genetically engineered form of a nucleic acid sequence obtained from a natural source. In other embodiments, the template (e.g., ssDNA template) comprises a synthetic construct.

[0074] In some embodiments, the template (e.g., ssDNA template) can be prepared from an in vitro process, including, but not limited to, the production of ssDNA from double-stranded plasmids in vitro. Non-limiting examples of in vitro processes are provided, for example, in Thermo Fisher Scientific, "Production of Single-Stranded Circular DNA Molecules from Supercoiled Double-Stranded Plasmids in Vitro," 2012, p. 1 (accessible at tools.thermofisher.com / content / sfs / brochures / 76-circular-ssdna-from-supercoiled-double-stranded-plasmids-in-vitro.pdf), which is incorporated herein by reference in its entirety. In other embodiments, the template (e.g., ssDNA template) can be either a natural source or a synthetic construct, or the product of an in vitro amplification from an in vitro process. The amplification method can be, but is not limited to, rolling circle amplification (RCA).

[0075] Guided Oligonucleotides The guided oligonucleotide (or guide) can be a single-stranded structure formed from DNA, RNA, or both. In certain embodiments, the guide is a synthetic construct. Based on the desired target region and known target region in the template, the guide can be designed to include a target binding region configured to bind (or hybridize) to the target region in the template. Furthermore, the guide can include a region that binds (or hybridizes) to a portion of the cleavage enzyme. Upon binding to the template bound to the guide, the cleavage enzyme can cleave the template (e.g., within the target region of the template). In this way, cleavage of the template is guided by the guide.

[0076] The target binding region of a guide can include one or more restriction sequences, recognition sequences, protospacer regions, PAM sequences, or their reverse complements. Each of these restriction sequences, recognition sequences, protospacer regions, PAM sequences, or their reverse complements can be single-stranded. When such a sequence binds to a template, a double-stranded structure is formed, and this double-stranded structure forms a site or motif for the cleavable enzyme to bind. Thus, a guide can include a strand of any of the restriction motifs described herein, any of the recognition motifs described herein, or any of the reverse complements thereof.

[0077] Thus, a single-stranded sequence derived from any of the sites or motifs herein may be provided within the guide. For example, if the cleavage enzyme is a restriction enzyme, the guide may contain a restriction sequence derived from the corresponding restriction motif. When the restriction sequence of the guide binds to the target region of the template, a double-stranded restriction motif may be formed. Such a motif may then bind to the desired restriction enzyme.

[0078] One skilled in the art would be able to develop guides that facilitate binding to and cleavage of the template. By way of example and not limitation, the following design considerations may be taken into account:

[0079] Step 1: Identify restriction enzymes / nicking enzymes based on the sequence of the template and the target region within the template. For ssDNA templates, the locations of restriction enzymes can be identified using commercially available or free software (e.g., NEB cutter 2.0, available at nc2.neb.com / NEBcutter2 / ). A non-limiting example of a sequence map is provided in Figure 5.

[0080] Step 2: Select an appropriate enzyme or enzymes (e.g., multiple enzymes). Factors to consider when selecting a restriction enzyme / nicking enzyme (e.g., those described below or in Figure 5) include one or more of the following: The size of the cleavage products meets requirements (e.g., size requirements). Cost of the enzyme. For example and not limitation, if one were to choose only one enzyme for a single cut of M13mp18, one could choose HindIII as it is significantly cheaper than other enzymes; and / or If multiple enzymes are used for simultaneous cleavage, the cleavage buffer should be compatible with the different enzymes. Non-limiting examples of software for determining buffer compatibility include, for example, nebcloner.neb.com / #! / redigest.

[0081] Step 3: Design guides based on restriction / nicking enzyme selection. Factors to consider when designing guides include one or more of the following: · Include a sequence (for the target binding region) that can bind (complement or hybridize) to the restriction / nicking site of the target. inclusion of additional sequences, e.g., 6 nt added to either side of the restriction / nicking site (see, e.g., www.neb.com / tools-and-resources / usage-guidelines / cleavage-close-to-the-end-of-dna-fragments), which may be configured to bind to (complement or hybridize with) the target region of the template; and / or Consider using standard desalted oligonucleotides, which may reduce manufacturing costs.

[0082] Non-limiting examples of guides are provided in Table 1 of Example 1 (restriction sequences are highlighted in bold).

[0083] Guides for other cleavage enzymes can be designed to include target binding regions and other restriction sequences (e.g., for use with nicking enzymes), recognition sequences (e.g., for use with Argonaute proteins), protospacer regions (e.g., for use with Cas enzymes), PAM sequences (e.g., for use with Cas enzymes), or reverse complements thereof.

[0084] In particular, guides for use with Cas enzymes can include single-stranded guides or double-stranded guides (bipartite guides). Typically, Cas enzymes are recruited to targets by two RNA regions: a tracrRNA region and a crRNA region. Using synthetic guides, these two regions can be provided in a single, contiguous nucleic acid. Thus, in some embodiments, a single-stranded guide includes a target binding region, a crRNA region, and a tracrRNA region. Optionally, a linker may be present between the crRNA region and the tracrRNA region. In other embodiments, the guide is a double-stranded guide, including a first strand having a target binding region and a cRNA region and a second strand having a tracrRNA region. The first and second strands can combine to form a double-stranded structure.

[0085] Those skilled in the art will be able to develop guides that facilitate template binding and template cleavage using Cas enzymes. By way of example and not limitation, the following design considerations may be made (which may be performed manually or using software, including free or commercially available solutions):

[0086] Step 1: Identify candidate reverse complement PAM sequences in the ssDNA template. The PAM sequence is typically "NGG," although other PAM sequences are described herein. It may be in other formats depending on the CRISPR enzyme. The template (e.g., ssDNA template) may contain the reverse complement "CCN" (5' to 3') of the PAM sequence "NGG."

[0087] Step 2: Select candidate PAM positions within the template based on the desired cleavage product size.

[0088] Step 3: Design a guide based on the selected PAM sequence. In some embodiments, the target region is approximately 20 nt downstream of the 5'-CCN sequence in the template. Non-limiting examples of guide design are provided, for example, at takarabio.com / learning-centers / gene-function / gene-editing / gene-editing-tools-and-information / how-to-design-sgrna-sequences, snapgene.com / guides / design-grna-for-crispr, or idtdna.com / pages / community / blog / post / guide-rna-design-be-on-target!, the disclosures of each of which are incorporated herein by reference in their entirety.

[0089] Non-limiting examples of guide designs are shown in Figure 4. As can be seen in Figure 4A, a guide can comprise a single guide RNA, which can comprise (from the 5' end to the 3' end): a target binding region comprising a complementary sequence to a target region within the ssDNA template (e.g., a target region that can conveniently be about 20 nt); crRNA sequence, a linker sequence (e.g., any linker described herein) connecting the crRNA and tracrRNA, and · tracrRNA sequence.

[0090] 4B shows a bipartite guide comprising a first portion and a second portion. The first portion may comprise a nucleic acid (e.g., RNA) having: a target binding region comprising a complementary sequence to a target region within the ssDNA template (e.g., a target region that can conveniently be about 20 nt); and crRNA sequence. The second portion can include a nucleic acid (e.g., RNA) having a tracrRNA sequence. A portion of the tracrRNA sequence can be complementary to the crRNA sequence, as indicated by the vertical dashed line. The guide (e.g., at the 5' and / or 3' end of the RNA) can include one or more nucleotide modifications, such as, but not limited to, 2'OMe bases, phosphonothioate, or phosphorothioate linkage modifications. Additional modified nucleotides are described herein.

[0091] cleavage enzyme Any useful cleavage enzyme can be used, which may include one or more restriction enzymes, nicking enzymes, programmable endonucleases, Argonaute proteins, Cas enzymes, any useful nuclease (e.g., endonuclease), or variants thereof.

[0092] In one embodiment, the cleavage enzyme is a restriction enzyme, which may include a blunt-end restriction endonuclease and / or a sticky-end restriction endonuclease. Such enzymes may be used in conjunction with a guide oligonucleotide having a restriction sequence. In some embodiments, the restriction sequence is approximately 4 nt to 10 nt in length. Non-limiting examples of restriction sequences include 5'-AAGCTT-3' (e.g., for HindIII), 5'-TTAATTAA-3' (e.g., for PacI), 5'-AGCGCT-3' (e.g., for AfeI), or their reverse complements. Those skilled in the art will be able to determine the restriction site and corresponding restriction sequence of a particular restriction enzyme, as well as develop a guide oligonucleotide that facilitates template binding and template cleavage.

[0093] Non-limiting examples of restriction enzymes may include one or more of Acc65I, AccI, AfeI, AleI, AlwNI, AvaII, BaeGI, BamHI, BglI, BglII, BmrI, BsaHI, BseRI, BsmBI, BsmI, BspHI, BsrFI, BsrGI, Bsu36I, BtsI, CspCI, DraIII, DrdI, Eco53KI, EcoRI, Esp3I, FspI, HincII, HindIII, KasI, KpnI, MscI, NarI, NgoMIV, NaeI, PacI, PstI, PvuI, SacI, SalI, SbfI, SfoI, SmaI, SnaBI, SphI, SwaI, TspMI, PluTI, XbaI, XmaI, and the like.

[0094] Still other enzymes are AluI, AvaI, BamHI, BanII, BglII, ClaI, DraI, Eco47 III, EcoRI, EcoRV, FspI, HindIII, HpaI, HpaII, KpnI, MscI, MseI, NcoI, NdeI, NotI, NruI, PstI, PvuI, PvuII, RsaI, ScaI, SmaI, SspI, SstI, StuI, ThaI, XbaI, or XhoI. Still other enzymes are AatII, AbsI(x), AcuI, AflII, AgeI, AhdI, AjuI(x), ApaI, ApaLI, AscI, AsiSI, AvrII, BbsI, BcgI, BciVI, BclI, BlpI, BmgBI, BmtI, BplI(x), Bsa I, BsgI, BsiWI, BspEI, BspQI, BssHII, BssSI, BstAPI, BstBI, BstEII, BstXI, BstZ17I, EagI, EcoNI, EcoO109I, EcoRV, FseI, FspAI(x), HpaI, KflI(x), Ma Restriction sites for such enzymes include uBI(x), MfeI, MluI, MreI(x), MteI(x), NcoI, NheI, NmeAIII, NotI, NruI, NsiI, PaeR7I, PaqCI, PasI(x), PflFI, PflMI, PfoI(x), PmeI, PmlI, PpuMI, PshAI, PspOMI, PspXI, PsrI(x), RsrII, SacII, SanDI, SapI, ScaI, SexAI, SfiI, SgrAI, SgrDI(x), SpeI, SrfI(x), StuI, StyI, Tth111I, XcmI, XhoI, ZraI, etc. One of skill in the art would know how to identify restriction sites and corresponding restriction sequences for such enzymes and how to use such sites to locate cleavage in the target region of a template and / or in a guiding oligonucleotide.

[0095] The restriction enzymes described herein, and others that can similarly be used in the present disclosure, are commercially available, for example, from Thermo Fisher Scientific (Waltham, MA) or New England Biolabs (Ipswich, MA). For example, although particular restriction endonucleases are recited herein, it will be recognized that isorestriction enzymes, i.e., enzymes that have the same recognition site but cut differently, may be substituted and the same result will be achieved. For other examples of restriction enzymes and their cleavage sites, see also Roberts, Nucl. Acids Res. 1989;17(Suppl):r347-r387, which is incorporated herein by reference in its entirety.

[0096] The cleavage enzyme can be a nicking enzyme (e.g., a nicking endonuclease). Such a nicking enzyme can be used to cleave only one strand of a double-stranded structure. For example, a guided oligonucleotide can be configured to coordinate guided cleavage by binding to a target region of the template. However, cleavage by the nicking enzyme can occur only in the template strand. Such enzymes can be used in conjunction with a guided oligonucleotide having a restriction sequence. In some embodiments, the restriction sequence has a length of about 4 nt to 12 nt. Non-limiting examples of restriction sequences include 5'-NNNNNGACTC-3' (e.g., for Nt.BstNBI) or 5'-GCAGTGNN-3' (e.g., for Nb.BtsI), where N can be any nucleic acid (e.g., G, C, A, T, or U, or modified forms thereof). One skilled in the art would be able to determine the restriction site and corresponding restriction sequence for a particular nicking enzyme (e.g., a top-strand-specific or bottom-strand-specific nicking enzyme), as well as develop guiding oligonucleotides that facilitate template binding and template cleavage.

[0097] Non-limiting examples of nicking enzymes include Nb.BbvCI, Nb.BpulIOI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, Nb.Mva1269I, Nt.AlwI, Nt.BbvCI, Nt.BpulIOI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, etc. In other embodiments, the nicking enzyme can be a restriction enzyme (e.g., any described herein) that has been modified to nick the bottom strand or the top strand.

[0098] The cleavage enzyme can be a programmable endonuclease. In one embodiment, a programmable endonuclease is an enzyme that recognizes a user-defined nucleic acid sequence (e.g., a nucleic acid sequence in a template or guided oligonucleotide), and its recognition site can be significantly larger than the average restriction site of a restriction enzyme (e.g., a programmable endonuclease recognition site can comprise about 15 base pairs (bp) to 25 bp). In some embodiments, the recognition sequence is about 15 nt to 25 nt in length. Programmable endonucleases, such as RNA-guided CRISPR-based systems and DNA-guided prokaryotic Argonautes, are found throughout the prokaryotic kingdom and can vary significantly in activity and specificity.

[0099] In some embodiments, the cleavage enzyme can be an Argonaute protein. In certain embodiments, Argonaute proteins can be used for DNA- or RNA-guided programmable template cleavage. Such enzymes can be used with a guide oligonucleotide (e.g., a guided DNA) having a recognition sequence or its reverse complement. Non-limiting examples of recognition sequences include 5'-TTACCGCTAATGGTGTG-3' (SEQ ID NO: 1) or 5'-TNNNNNNNNNNXNNNNN-3', where X is G, C, T, or U, and N is any nucleic acid (e.g., G, C, A, T, or U, or modified forms thereof), optionally with a GC content of about 10% to 70% or 20% to 60%. The guide oligonucleotide can include a recognition sequence, and the template can be configured to bind (or hybridize) to the recognition sequence. In some embodiments, the cleavage site in the template can be located within a target region corresponding to, for example, bases 10 and 11 of the guide oligonucleotide. Those skilled in the art will be able to determine the restriction sites and corresponding restriction sequences for specific Argonaute proteins, as well as develop guided oligonucleotides that facilitate template binding and template cleavage. Methods and compositions for RNA-guided programmable template cleavage are described in Doxzen KW and Doudna JA, "DNA recognition by an RNA-guided bacterial Argonaute," PLoS ONE 12(5):e0177097 (page 14), which is incorporated herein by reference in its entirety.

[0100] Non-limiting examples of Argonaute proteins include Argonaute RISC catalytic component 2 (Ago2), prokaryotic Argonaute (pAgo), Thermus thermophilus Argonaute (TtAgo), Methanocaldococcus jannaschii Argonaute (MjAgo), or Pyrococcus furiosus Argonaute (PfAgo).

[0101] In other embodiments, the cleavage enzyme can be a Cas enzyme. In certain embodiments, Cas enzymes can be used for RNA-guided programmable template cleavage. Such enzymes can be used with guided oligonucleotides (e.g., guided RNAs) having a protospacer adjacent motif (PAM) sequence or its reverse complement. Non-limiting examples of PAM sequences include 5'-NGG and its reverse complement, 5'-CCN. Further examples of PAM sequences include 5'-NGCG, 5'-NGAG, 5'-NGNG, 5'-NAGN, 5'-NG, 5'-GAA, 5'-GAT, 5'-NNGRRT, 5'-NNGRR(N), 5'-TTTV, 5'-TYCV, 5'-TATV, 5'-NNNNRYAC, 5'-NNNNGATT, 5'-NNAGAAW, 5'-NAAAAC, and the reverse complement of any of these. For any of these sequences, each N can independently be G, C, A, T, or U, and modified forms thereof.

[0102] In the guided oligonucleotide, the PAM sequence can be located approximately 2 to 6 nt downstream of the cleavage site. The reverse complement of the PAM sequence can be located approximately 2 to 6 nt upstream of the target region in the template. One skilled in the art would be able to determine the PAM sequences and the location of these sequences for a particular Cas enzyme, as well as develop guided oligonucleotides that facilitate template binding and template cleavage.

[0103] Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14), Cas12g, Cas12h, Cas12i, Cas12j (CasΦ), Cas12k (C2c5), Cas13a (C2c6), Cas13b (C2c7), Cas13c (C2c8), Cas13d (CasY), Cas13e (CasX), Cas13f (Cas14), Cas13g, Cas13h, Cas13i, Cas13j (CasΦ), Cas13k (C2c9), Cas13a (C2c10), Cas13b (C2c11), Cas13c (C2c12), Cas13d (CasY), Cas13e (CasX), Cas13f (Cas14), Cas13g, Cas13h, Cas13i, Cas13j (CasΦ), Cas13k (C2c13), Cas13c (C2c14), Cas13d (CasY), Cas13e (CasX), Cas13f (Cas14), Cas13g, Cas13h, Cas13i, Cas13j (CasΦ), Cas13k (C2c15), Cas13k (C2c16), Cas13k (C2c17), Cas13k (C2c18), Cas13k (C2c19 ... c2), Cas13b, Cas13c, Cas13d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified Cas enzyme, like Cas9, has DNA cleavage activity.In some embodiments, the Cas enzyme is Cas9 and is selected from the group consisting of Streptococcus pyogenes (e.g., UniProtKB accession number Q99ZW2), Streptococcus thermophilus (e.g., UniProtKB accession number G3ECR1), Streptococcus pneumoniae (e.g., UniProtKB accession number A0A111NJ61), Staphylococcus aureus (e.g., UniProtKB accession number J7RUA5), Neisseria meningitidis (e.g., UniProtKB accession number A1IQ68), Campylobacter jejuni (e.g., UniProtKB accession number B1IQ68), and Campylobacter jejuni (e.g., UniProtKB accession number C1IQ68). The Cas9 may be derived from Bacillus jejuni (e.g., UniProtKB accession number Q0P897), Rhodopseudomonas palustris (e.g., UniProtKB accession number Q13CC2), Rhodospirillum rubrum (e.g., UniProtKB accession number Q2RX87), Actinomyces naeslundii (e.g., UniProtKB accession number J3F2B0), Francisella tularensis subsp. novicida (e.g., UniProtKB accession number A0Q5Y3), or Corynebacterium diphtheriae (e.g., UniProtKB accession number Q6NKI3). In some embodiments, the Cas enzyme induces cleavage of one or both strands at the location of the target region, such as within the target region and / or within the complementary sequence of the target region.In some embodiments, the Cas enzyme induces cleavage of one or both strands at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target region.

[0104] The nuclease can be a homolog or ortholog of Cas9. In some embodiments, the nuclease induces one or two strand cleavage at the location of the target region. In some embodiments, the nuclease lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter.

[0105] Any useful Cas enzyme or complex can be used. Exemplary Cas enzymes or complexes include those involved in Class 1 or Class 2 CRISPR / Cas systems, and Type I, II, III, IV, V, or VI CRISPR / Cas systems, including those requiring a single Cas protein during crRNA interference (e.g., those in Class 1 systems), Cas9 (formerly Csn1 or Csx12, e.g., those in Type II systems, including Type II-A and II-C systems), Cas12 (e.g., those in Type V systems), Cas13 (e.g., those in Type VI systems), CRISPR-associated complexes for antiviral defense (including Cascade, RAMP proteins), and those requiring multiple Cas proteins during crRNA interference. Examples of Cas enzymes or complexes include, but are not limited to, those that require an As protein (e.g., those of Class 2 systems), Cas3, Cas7, Cas6, Cas5, Cas11, and / or Cas8 (e.g., those of Type I systems such as Type IE systems), Csm (e.g., those of Type III-A systems), Cmr (e.g., those of Type III-B systems), Cas7, Cas5, Cas11, Cas10, and / or Cas6 (e.g., those of Type III systems), Cas7, Cas5, Cas11, Cas8, DinG (or CysH), and / or Cas6 (e.g., those of Type IV systems), and subassemblies or subcomponents thereof and assemblies comprising such Cas enzymes or complexes. Additional Cas enzymes and complexes are described in Makarova KS et al., "Evolution and classification of the CRISPR-Cas systems," Nat. Rev. Microbiol. 2011; 9:467-77, and Wang JY et al., "Structural biology of CRISPR-Cas immunity and genome editing enzymes," Nat. Rev. Microbiol. 2022; doi.org / 10.1038 / s41579-022-00739-4 (page 16), each of which is incorporated herein by reference in its entirety.

[0106] In some embodiments, the Cas enzyme is a variant that lacks the ability to cleave both strands of a double-stranded structure compared to the corresponding wild-type enzyme. For example, an aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase that cleaves a single strand. Other examples of mutations that render Cas9 a nickase include, but are not limited to, H840A, N854A, and / or N863A.

[0107] Further cleavage enzymes and cleavage conditions are described in U.S. Pat. No. 5,316,908 or U.S. Pat. No. 8,273,863, U.S. Patent Publication No. 2019 / 0203242, U.S. Patent Publication No. 2007 / 0178482, or U.S. Patent Publication No. 2019 / 0002868, International Publication No. WO2020 / 232286, Bush et al., Molecules 2020 Jul 26;25(15):3386, Gu et al., Biotechniques 2013 Jun;54(6):337-43, Cooney, Mol. Biotechnol. 1994;2(2):119-127, Parker et al., Proc. Nat'l Acad. Sci. USA 1977;74(3):851-855, Polyarush et al., Chem. Natural Compounds 2003;39(6):592-594, Lan et al., J. Nucleic Acids 2012; 2012:254630, Wang et al., J. Nucleic Acids 2010; 2010:421803 (page 3), Amills et al., Genet Anal. 1996;13:147-9, Abdel-Fattah et al., Biotechnology. 2006;5:166-172, Chang et al., J. Biochem. Biophys. Methods 2008;70:1199-202, Wang et al., J. Nucleic Acids 2010; 2010:421803, Wu et al., Mol. Biol. Rep. 2011;38:2729-31, Swartjes et al., Biochem. Soc. Trans. 2020 Feb 28;48(1):207-219; Ma et al., Mol Cell. 2015 Nov 5;60(3):398-407; Hunt et al., Front. Mol. Biosci. 2021 Apr 29 (accessible at doi.org / 10.3389 / fmolb.2021.670940); Makarova et al., Nat. Rev. Microbiol. 2011;9:467-77; Wang et al., Nat. Rev. Microbiol. 2022;doi.org / 10.1038 / s41579-022-00739-4 (page 16), and / or Thermo Fisher Scientific, "Production of Single-Stranded Circular DNA Molecules from Supercoiled Double-Stranded Plasmids in Vitro," 2012, page 1 (accessible at tools.thermofisher.com / content / sfs / brochures / 76-circular-ssdna-from-supercoiled-double-stranded-plasmids-in-vitro.pdf), each of which is incorporated herein by reference in its entirety.

[0108] kit The present disclosure also provides single-stranded nucleic acid ladder (e.g., ssDNA ladder) kits. Such kits can include single-stranded nucleic acid (e.g., ssDNA) size standards, including one or more standards having a particular size.

[0109] The kit may include instructions for practicing any of the methods described herein. The instructions provided in the kits of the present disclosure are typically instructions on a label or package insert. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, containers, bottles, vials, and flexible packaging. The kit may include additional components such as instructional information. [Example]

[0110] Example 1 Preparation of ssDNA ladders based on restriction enzyme cleavage guided by DNA oligonucleotides A LabChip® GX Touch™ instrument (model number CLS137031, PerkinElmer, Waltham, MA) microfluidic chip system was used in this example. The oligonucleotides used as guided oligonucleotides (guides) here were synthesized by Integrated DNA Technologies (Coralville, IA). The sequences of the guides are listed in Table 1, where restriction sequences are highlighted in bold. M13mp18 single-stranded DNA, restriction enzymes, and buffers were purchased from New England Biolabs (Ipswich, MA).

[0111] [Table 1]

[0112] 1 μg of M13mp18 DNA and 1 μM of a short guide oligonucleotide in 1x NEB rCutSmart™ buffer were first annealed at 90°C for 4 minutes, then at 70°C for 10 minutes, and finally ramped down to 37°C at 0.1°C / s over 20 minutes in a 50 μL volume. Next, 20 units (20 U) of HindIII and 12.5 U of PacI or AfeI were added to these annealed products and incubated at 37°C for 90 minutes for cleavage under different experimental conditions according to Table 2. After cleavage, the reaction products were inactivated by incubation at 95°C for 5 minutes. The restriction enzyme digestion products were further purified using the 2x AMPure XP bead protocol (Part Number A63880, Beckman Coulter, Brea, CA). Purified samples were mixed with an equal volume of 1x sample buffer (LabChip® RNA Pico Reagent, part number CLS960012, PerkinElmer), heated to 70°C for 2 minutes, and immediately cooled on ice for a further 5 minutes before analysis using LabChip® RNA Pico Reagent on a LabChip® GX Touch™ instrument.

[0113] [Table 2]

[0114] The results are shown in Figure 2. In particular, Figure 2A shows the locations of the restriction enzyme sites: HindIII at 6281, PacI at 4135, and AfeI at 3040. The predicted cleavage patterns of different combinations of restriction enzymes are shown in Table 2.

[0115] Figure 2B shows the resulting electropherogram, and Figure 2C shows the corresponding gel image output from the LabChip® Reviewer software. The following observations are noted: Enzyme (-) condition: Without enzyme cleavage, intact circular ssDNA is barely visible around 70 seconds. This is the negative control for this experimental setup. HindIII+PacI cleavage conditions: Size 1 (2.1 kb) and Size 2 (5.1 kb) are clearly visible at around 50 seconds and 66 seconds, respectively. HindIII+AfeI cleavage conditions: Size 3 (3.2 kb) and size 4 (4.0 kb) are clearly visible at around 58 seconds and 62 seconds, respectively. HindIII+PacI+AfeI digestion condition: Size 5 (1.1 kb), size 1 (2.1 kb), and size 4 (4.0 kb) are clearly visible at 44 seconds, 50 seconds, and 62 seconds, respectively.

[0116] In summary, the electrophoresis results are consistent with the prediction of cleavage patterns based on the location of the restriction enzymes. These results demonstrate a non-limiting method for the preparation of ssDNA ladder compositions based on oligonucleotide-guided restriction enzyme cleavage.

[0117] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are included within the scope of the following claims. All references (including those mentioned above), scientific articles, patent publications, and other documents cited herein are incorporated by reference for the content of their disclosures.

Claims

1. 1. A method for preparing a ladder composition, comprising: providing a template comprising a single-stranded nucleic acid; incubating the template in the presence of a guiding oligonucleotide and a cleavage enzyme; wherein the guiding oligonucleotide is configured to bind to a target region of the template, and the cleavage enzyme is configured to bind to the guiding oligonucleotide and cleave at a location within the target region, thereby generating the ladder composition comprising a plurality of cleaved single-stranded nucleic acids.

2. 2. The method of claim 1, wherein the template comprises single-stranded deoxyribonucleic acid (ssDNA) and the ladder composition comprises a plurality of cleaved ssDNA.

3. The method of claim 1 , wherein the template comprises a natural construct, a synthetic construct, a linear construct, and / or a circular construct.

4. 10. The method of claim 1, wherein the template comprises 500, 1000, 2000, or more bases.

5. 2. The method of claim 1, wherein the target region comprises a restriction sequence, a recognition sequence, a protospacer region, or a reverse complement thereof.

6. 6. The method of claim 5, wherein the locations within the target region cleaved by the cleaving enzyme include locations within the restriction sequence, the recognition sequence, the protospacer region, or the reverse complement thereof.

7. 10. The method of claim 1, wherein the guiding oligonucleotide comprises single-stranded deoxyribonucleic acid (ssDNA), single-stranded ribonucleic acid (ssRNA), or a single-stranded DNA / RNA hybrid (ssDNA / RNA).

8. 2. The method of claim 1, wherein the guide oligonucleotide comprises a restriction sequence, a recognition sequence, a protospacer region, or a reverse complement thereof.

9. 2. The method of claim 1, wherein the template comprises a protospacer adjacent motif (PAM) sequence or its reverse complement near the target region, and / or the guide oligonucleotide comprises a PAM sequence or its reverse complement.

10. 10. The method of claim 9, wherein the guided oligonucleotide further comprises a CRISPR ribonucleic acid (crRNA) region and / or a trans-acting CRISPR RNA (tracrRNA) region.

11. 11. The method of claim 10, wherein the guided oligonucleotide further comprises a linker disposed between the crRNA region and the tracrRNA region.

12. 2. The method of claim 1, wherein the cleaving enzyme comprises a restriction enzyme, a nicking enzyme, a programmable endonuclease, an Argonaute protein, a CRISPR-associated (Cas) enzyme, a nuclease (e.g., an endonuclease), or a variant thereof.

13. The providing step, before the incubating step, providing a bound template by providing the guiding oligonucleotide to the template; further providing the cleavage enzyme to the bound template; 13. The method of any one of claims 1 to 12, comprising:

14. The providing step, before the incubating step, 13. The method of any one of Claims 1 to 12, comprising providing a preassembled complex to the template, the preassembled complex comprising the guiding oligonucleotide bound to the cleavage enzyme.

15. The providing step, before the incubating step, 13. The method of any one of claims 1 to 12, comprising providing the guiding oligonucleotide and the cleavage enzyme to the template, wherein the guiding oligonucleotide and the cleavage enzyme can be provided in any order or simultaneously.

16. 2. The method of claim 1, wherein at least 80%, 90%, or more of the population comprising the plurality of cleaved single-stranded nucleic acids in the ladder composition are single-stranded.

17. 10. The method of claim 1, wherein at least 50%, 60%, 70%, 80%, or more of the population comprising the plurality of cleaved single-stranded nucleic acids have a length of 500, 600, 700, 800, 900, 1000, 2000, or more bases.

18. 18. The method of any one of claims 1 to 17, wherein the incubating step is carried out in the absence of albumin, blocking agents, etc.

19. 19. The method of any one of claims 1 to 18, wherein the ladder composition is not further purified or diluted.

20. 1. A ladder composition comprising a population comprising a plurality of cleaved single-stranded nucleic acids, at least 80%, 90%, or more of said population are single-stranded; and / or at least 50%, 60%, 70%, 80%, 90% or more of the population have a length of 500, 600, 700, 800, 900, 1000, 2000 or more bases; Ladder composition.

21. 21. The ladder composition of claim 20, wherein the population comprises a plurality of truncated single-stranded deoxyribonucleic acid (ssDNA).

22. 22. The ladder composition of claim 20 or 21, wherein the composition is prepared by a method according to any one of claims 1 to 19.

23. a first ladder composition comprising the ladder composition of any one of claims 20 to 22; instructions for using the first ladder composition in an assay; and A kit comprising:

24. 24. The kit of claim 23, further comprising a second ladder composition comprising the ladder composition of any one of claims 20 to 22, A kit wherein the first ladder composition and the second ladder composition are different, and the instructions further comprise instructions for using the second ladder composition in the assay.

25. 25. The kit of claim 24, wherein the first ladder composition comprises a first population comprising a plurality of first cleaved single-stranded nucleic acids, and the second ladder composition comprises a second population comprising a plurality of second cleaved single-stranded nucleic acids, and at least 90% of the first population have a different length than at least 90% of the second population (e.g., at least 90% of the first population have a length that is longer or shorter than at least 90% of the second population).

26. 26. The kit of any one of claims 23 to 25, wherein the assay comprises electrophoresis.

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