MRNA analysis using restriction enzymes

Abstract

The present disclosure describes methods, kits, and systems for digesting polyribonucleotides. The methods involve selectively forming an oligonucleotide (e.g., DNA: RNA or RNA: RNA) duplex with a single-stranded target RNA, and then selectively cleaving the target RNA into smaller fragments using a sequence-specific nuclease that acts only on the RNA within the duplex. Additional sequence-specific ribonucleases can be used to provide additional cleavage of the target RNA at a predetermined site. By forming double strands to increase the availability of nuclease enzymes that can be used to cleave the single-stranded target RNA and to selectively control the location at which the target RNA is cleaved, the target RNA can be digested into fragments within a controllable size range that are optimal for polynucleotide analysis, such as by liquid chromatography and mass spectrometry.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 395,978, filed on August 8, 2022, entitled “mRNA ANALYSIS USING RESTRICTIONENZYMES,” the entire contents of which are hereby incorporated by reference herein in their entirety. Technical Field

[0003] The present disclosure relates to the use of enzymes that cleave intraduplex RNA to selectively cleave large RNA molecules into fragments of predetermined sizes for use in polynucleotide analysis. Background Art

[0004] mRNA is being used as a new therapeutic approach, including use in vaccines and protein replacement therapies. During the enzymatic manufacturing process of mRNA therapeutics, incomplete mRNA products are generated along with other potential impurities such as double-stranded RNA (dsRNA). In addition, during manufacturing and storage, RNA can degrade due to exposure to heat, hydrolysis, oxidation, light, and ribonucleases. Batch manufacturing can also introduce variability to treatment. Therefore, in order to ensure quality, it is necessary to analyze the manufactured mRNA.

[0005] Typical mRNA length is about 2,000-5,000 nucleotides (0.6MDa-1.5MDa). Such macromolecules are difficult to characterize by traditional polynucleotide analysis methods (comprising oligonucleotide separation and mass spectrometry). Such characterization and quantitatively may be necessary for evaluating the purity of synthesis and determining the pharmacokinetic and pharmacodynamic parameters of therapeutic polynucleotides. In order to overcome the characterization limitation of larger polynucleotides, usually first they are digested into smaller (shorter) fragments for analysis. However, very small fragments may be difficult to accurately map, and therefore may provide less information, particularly for primary sequence. Therefore, there may be an optimal range of fragment sizes that can promote polynucleotide analysis. However, there are currently not enough methods that can be used for larger polyribonucleotides (for example, mRNA) to be segmented into suitable or optimal size fragments, for polynucleotide analysis.

[0006] Therefore, need to improve the method for characterizing large polyribonucleotide.More specifically, need to effectively can polyribonucleotide be digested into the polyribonucleotide digestion method with the fragment of more controlled length distribution, such as being more suitable for polynucleotide analysis (comprising for example by liquid chromatography and / or mass spectrometry) and being enough to be applicable to those methods of polyribonucleotide (for example, mRNA) with variable sequence identity. Summary of the invention

[0007] The disclosure herein generally relates to improved methods, kits and systems for digesting and characterizing large RNA molecules. Large RNA molecules are difficult to characterize by conventional methods (including liquid chromatography and mass spectrometry). Digestion of large RNA molecules into smaller fragments, particularly with sequence-specific nuclease digestion, can allow easier characterization and mapping of RNA fragments, but unlimited cutting of RNA, particularly with less selective ribonucleases (recognition of short motifs) for cutting may result in fragments that are too small to effectively map and / or multiple isobaric fragments that are difficult to distinguish. The technology described herein relates to selectively forming oligonucleotide duplexes with single-stranded RNA, so that nucleases that are not conventionally used to cut single-stranded RNA (including traditionally known nucleases that only cut double-stranded DNA) can be reused to selectively cut large RNA molecules into fragments of controllable / predetermined size in a site-specific manner. Therefore, the precise size range of the RNA fragments from digestion can be customized for polynucleotide analysis, including by liquid chromatography and / or mass spectrometry.

[0008] According to one aspect of the present disclosure, a method is provided herein for digesting an RNA molecule with a known reference sequence into smaller RNA fragments. The method requires forming one or more oligonucleotide duplexes with the RNA molecule along a specific portion of the reference sequence. The RNA molecule is then digested into fragments with one or more sequence-specific nucleases, which cut the RNA molecule at a plurality of predetermined sequence-specific sites. One or more sequence-specific nucleases are duplex-dependent nucleases that act only on RNA within the duplex. Each of the one or more duplexes formed with the RNA molecule has a motif recognized by a duplex-dependent nuclease in one or more duplex-dependent nucleases. An embodiment of the method may include one or more of the following features.

[0009] The method can use multiple sequence-specific nucleases to digest RNA molecules. The multiple nucleases can be multiple duplex-dependent nucleases. The sequence-specific duplex-dependent nuclease can be a restriction endonuclease, a CAS protein, an artificial site-specific RNA endonuclease (ARSE), an enzyme or a deoxyribozyme including an RNase III domain. The duplex-dependent nuclease as a restriction endonuclease can be AvaII, AvrII, BanI, TaqI, HinfI or HAEIII. Other sequence-specific nucleases used can be RNase T1, RNase A, colicin E5 or MazF.

[0010] The RNA molecule may have a length greater than about 1,000mer. In some embodiments, the RNA fragment may be about 6mer to 1,000mer in length, more specifically about 6mer to 500mer in length, even more specifically about 6mer to 50mer in length, or further specifically about 6mer to 20mer in length. In some embodiments, the RNA fragment may be about 10mer to 1,000mer in length, more specifically about 10mer to 500mer in length, even more specifically about 10mer to 50mer in length, or further specifically about 10mer to 20mer in length. In some cases, the RNA fragment may be about 20mer in length.

[0011] One or more duplexes can be multiple duplexes. Each duplex in one or more duplexes can be formed by another oligonucleotide of about 10mer to 50mer in length. Each duplex in one or more duplexes can be formed by hybridizing an exogenous oligonucleotide with an RNA molecule. One or more duplexes can be formed with DNA oligonucleotides.

[0012] At least one of the sequence-specific nucleases is immobilized on a solid support. The immobilized nuclease can be provided in the form of an immobilized enzyme reactor (IMER) that allows RNA molecules to flow through and digest. The nuclease immobilized in the IMER may not be a duplex-dependent nuclease and can be used to further digest selected fractions of RNA fragments that have been digested with a duplex-dependent nuclease.

[0013] The RNA molecule may be an mRNA molecule.The plurality of predetermined sequence-specific sites may include a site within about 100 nucleotides of the proximal end of the 3' poly(A) tail and / or a site within about 100 nucleotides of the 5' cap.

[0014] The method may also require the use of liquid chromatography to separate one or more RNA fragments in the RNA fragments based on length. The method may also require the use of mass spectrometry to measure the mass of one or more RNA fragments in the RNA fragments. The method may also require mapping the RNA fragments to a reference sequence.

[0015] According to another aspect of the present disclosure, a test kit for digesting an RNA molecule with a reference sequence into smaller RNA fragments is provided herein. The test kit includes a variety of oligonucleotides. Each oligonucleotide is configured to hybridize with a single unique portion of an RNA molecule and has a motif recognized by a sequence-specific duplex-dependent nuclease that acts only on RNA within the duplex. The embodiment of the test kit may include one or more of the following features.

[0016] The length of each oligonucleotide in the oligonucleotide can be about 10mer to 50mer. In some embodiments, the length of each oligonucleotide in the oligonucleotide is about 15mer to 25mer. The multiple oligonucleotides can include at least two motifs recognized by different sequence-specific duplex-dependent nucleases. The kit can also include one or more sequence-specific duplex-dependent nucleases.

[0017] According to another aspect of the present disclosure, another kit for digesting RNA molecules into smaller RNA fragments is provided herein. The kit includes multiple sequence-specific nucleases, at least one of which is a duplex-dependent nuclease that acts only on RNA within a duplex, and at least one of which is a ribonuclease that acts on single-stranded RNA. At least one duplex-dependent nuclease may be or may include a restriction endonuclease.

[0018] According to another aspect of the present disclosure, a system for mapping RNA fragments to a reference sequence is provided herein. The system has: a detector configured to quantify the amount of RNA oligonucleotides having a length of about 20mer to 1,000mer; and a processor operably connected to the detector. The processor is programmed to map the detected RNA oligonucleotide to a reference sequence of the RNA molecule based at least in part on the length or mass of the detected RNA oligonucleotide. Mapping the detected RNA oligonucleotide to the reference sequence requires the processor to determine the length of the fragment that should be produced by digesting the RNA molecule into smaller fragments according to any embodiment of the aforementioned method. Embodiments of the system may include one or more of the following features.

[0019] The processor may also be configured to automatically identify a motif within a reference sequence for which a cleavage with a sequence-specific nuclease will produce a fragment of about 20mer to 1,000mer in length. The sequence-specific cleavage may be or may include one or more selective cleavages with one or more duplex-dependent nucleases of the aforementioned method. The processor may be operably connected to one or more databases having a plurality of sequence-specific nucleases and motifs corresponding to each of the sequence-specific nucleases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present technology will be more fully understood through the following detailed description made in conjunction with the accompanying drawings, in which:

[0021] Figure 1 is an HPLC chromatogram of a ladder of oligodeoxythymidine from 15mer to 100mer;

[0022] Figure 2A is a simulated HPLC chromatogram of RNA fragments generated by digestion of a COVID-19 mRNA vaccine with a TaqI restriction site; and

[0023] Figure 2B is a simulated HPLC chromatogram of RNA fragments generated by digestion of the COVID-19 mRNA vaccine with selected TaqI, AvaII, and BanI restriction sites. DETAILED DESCRIPTION

[0024] Polynucleotide analysis and fragment size

[0025] Disclosed herein is a method for digesting RNA (polyribonucleotides), which is easier to produce RNA fragments with more controllable size ranges than standard RNA digestion methods. It should be understood that in the methods, kits, and systems described herein, other types of oligonucleotides (e.g., DNA) can easily replace the RNA to be digested, and the selection of the enzyme for digestion depends on the specific oligonucleotide composition. In a specific implementation, the size range is optimal for polynucleotide analysis. Polynucleotide analysis can be performed to determine or confirm the length, molecular weight, purity, capping state, and / or primary sequence of a polynucleotide sample. When the sample is heterogeneous, the analysis can be used to characterize the distribution of any one or more variables. Competitive factors about polynucleotide size can complicate polynucleotide analysis, including polynucleotide mapping such as by liquid chromatography and mass spectrometry (including tandem mass spectrometry). Generally, larger (longer) polynucleotides are more difficult to characterize by separation methods (such as liquid chromatography). Larger polynucleotides are also generally more difficult to characterize by mass spectrometry, while smaller (shorter) oligonucleotides are easier to analyze. Without being limited by theory, smaller oligonucleotides are more likely to produce complete mass measurements. Larger oligonucleotides are also more prone to salt addition, multiple charge states, and ionization and segmentation efficiency reduction, thereby complicating mass spectrometry. However, the characterization of shorter oligonucleotides (e.g., 2mer, 3mer, 4mer, 5mer and 6mer oligonucleotides) may have less information, because shorter sequences are unlikely to occur uniquely in large oligonucleotide sequences (e.g., especially greater than 1,000mer, 1,500mer, 2,000mer, etc. oligonucleotide sequences), so they can be mapped to multiple positions in the target oligonucleotide. Moreover, compressing all primary sequence information into very short oligonucleotide fragments of a small range increases the possibility of producing isobaric fragments, particularly considering that the selection of available nucleotides is limited, they can not be distinguished via mass spectrometry or can only be distinguished via the complex and difficult analysis using tandem mass spectrometry (MS / MS).

[0026] In some embodiments, digestion methods as described herein are used to generate one or more fragments from larger oligonucleotides, and these fragments can be uniquely mapped to the larger oligonucleotides. In some cases, the length of the fragment is at least about 10mer, 15mer, 20mer, 25mer, 30mer, 35mer, 40mer, 45mer or 50mer. In some embodiments, digestion methods as described herein are used to generate one or more fragments (for example, by liquid chromatography) that are easy to separate from larger oligonucleotides. In some embodiments, digestion methods as described herein are used to generate one or more fragments from larger oligonucleotides, and the size of these fragments is best for accurately determining the quality by mass spectrometry and / or tandem mass spectrometry. In some cases, the length of the fragment is not more than about 2,000mer, 1,500mer, 1,000mer, 500mer, 200mer or 100mer. In some cases, the length of one or more fragments may be no greater than about 90mer, 80mer, 70mer, 60mer, 50mer, 45mer, 40mer, 35mer, 30mer, 25mer, 20mer, 15mer or 10mer. In certain specific embodiments, the length of one or more fragments may be about 10mer to 100mer, 10mer to 50mer, 10mer to 25mer, 15mer to 100mer, 15mer to 50mer, 15mer to 25mer, 20mer to 100mer, 20mer to 50mer, 25mer to 100mer, 25mer to 50mer, 30mer to 100mer, 30mer to 50mer, 35mer to 100mer, 30mer to 50mer, 40mer to 100mer or 40mer to 50mer. In various specific implementations, the fragments characterized only by liquid chromatography may be longer than the fragments characterized by mass spectrometry. In various embodiments, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the generated fragments (by number or mass percentage) or all of the generated fragments fall within one or more preselected size ranges, including any one or more of the ranges described herein.

[0027] In various embodiments, the digestion methods described herein are performed on a target oligonucleotide (target RNA) comprising ribonucleotides, or on an analyte sample (which may be referred to as an "RNA sample") comprising a target RNA (e.g., with potential impurities). In some cases, the RNA sample is a synthetically manufactured RNA (e.g., mRNA), such as for therapeutic purposes. The target RNA can be a large RNA molecule with a reference RNA sequence, and for polynucleotide analysis, it is useful to divide the target RNA molecule into smaller fragments for analysis. The target RNA molecule can be at least about 500met, 600met, 700met, 800met, 900met, 1,000met, 1,100met, 1,200met, 1,300met, 1,400met, 1,500met, 1,600met, 1,700met, 1,800met, 1,900met, 2,000met, 2,100met, 2,200met, 2,300met, 2,400met, 2,500met, 3,000met, 3,500met, 4,000met, 4,500met or 5,000met. In certain embodiments, the target RNA molecule is at least about 1,000met. In certain other embodiments, the target RNA molecule is at least about 2,000met. Also, in certain other embodiments, the target RNA molecule is at least about 5,000met. In some embodiments, the use of the digestion methods described herein on the target RNA molecule can result in multiple cuts within the target RNA molecule (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cuts per target molecule). In some embodiments, cutting can result in the distribution of fragments with unique lengths, which may be advantageous for polynucleotide analysis. In certain embodiments, each fragment analyzed or mapped may have a length that is at least about 2met, 3met, 4met, 5met, 6met, 7met, 8met, 9met, 10met, 15met, 20met, 25met, 30met, 35mer, 40mer, 45mer, 50mer, 60mer, 70mer, 80mer, 90mer, or 100mer different from any other fragment. In various embodiments, the methods described herein will allow mapping of at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (percent sequence coverage) of a target RNA molecule.

[0028] Nucleases for digestion of RNA

[0029] Digestion methods as described herein can be used to digest large target RNA molecules into fragments within the optimal size range for polynucleotide analysis, which is described elsewhere herein. The digestion of RNA can be performed with one or more enzymes (nucleases) (e.g., 1, 2, 3, 4, 5 or more nucleases) that cut the phosphodiester bonds between ribonucleotides. As will be appreciated by those of ordinary skill in the art, some nucleases may only cut single-stranded nucleic acids, and may or may not have specificity for DNA or RNA. Some nucleases may only cut double-stranded nucleic acids (duplexes), and may or may not have specificity for DNA / DNA duplexes (i.e., DNA duplexes), RNA / RNA duplexes (i.e., RNA duplexes), or DNA / RNA duplexes (i.e., heteroduplexes). As used herein, "duplexes" may refer to a region of a nucleic acid molecule in which two oligonucleotide chains are reversibly bound to each other by Watson-Crick base pairing (hydrogen bonds between complementary nucleotides within the duplex). The duplex may be formed by complete complementarity of base pairs along the length of the double-stranded region, or by partial (e.g., substantially) complementarity (e.g., allowing one or more mismatches, not excluding hybridization of two oligonucleotide chains under relevant hybridization conditions). In some embodiments, the duplex acted upon by the digestion method described herein may be an RNA duplex, wherein both chains are RNA. In some embodiments, the duplex may be a heteroduplex, wherein an RNA chain (such as an RNA chain of a target RNA molecule) is bound to a DNA chain. In some embodiments, the RNA molecule and / or other oligonucleotides within the duplex may include modified nucleotides, including, for example, oligonucleotides including both deoxyribonucleotides and ribonucleotides. The duplex may not extend over the entire length of the molecule. The portion of the RNA molecule of the duplex may be relatively small (e.g., not exceeding about 30%, 20%, 10%, 5% of the length of the molecule). For example, as described in detail herein, a single large RNA molecule may simultaneously form a variety of duplexes with a plurality of smaller oligonucleotides at various positions along the length of the RNA molecule. In some embodiments, the portion of the RNA molecule that will be protected from cleavage by a ribonuclease (e.g., RNase T1) acting on single-stranded RNA is a duplex. In such embodiments, the digestion methods described herein can be modified to abandon the use of duplex-dependent nucleases so that the duplexes allow for (negative) selection of portions of the RNA molecule that are sensitive to cleavage by sequence-specific ribonucleases used for single-stranded RNA.

[0030] Nucleases described herein can cut nucleic acids at sequence-specific sites. As used herein, "sequence specificity" indicates that for a given nuclease and a given target sequence, the precise cleavage site (if any) will be determined under appropriate reaction conditions (and assuming, for example, that there is no secondary structure preventing cleavage). Such sequence-specific nucleases can recognize sequence "motifs", which accurately determine where the target oligonucleotide will be bound and cut by the nuclease. As used herein, "DNA motifs" can refer to motifs recognized in DNA chains, and "RNA motifs" can refer to motifs recognized in RNA chains. Depending on a specific nuclease (wherein the nuclease has catalytic activity on each chain of a duplex), a motif in one chain of a duplex can be understood to have a complementary motif in the other chain of the duplex. It should be understood that when the nuclease is active on both DNA and RNA, an equivalent RNA motif can be easily determined from a DNA motif (i.e., when present in the motif, uracil (U) can easily replace thymine (T)). Therefore, when referring to such nucleases, reference to the nuclease motif may be understood to refer to the motif of either chain, depending on the context. Similarly, it should be understood that, unless otherwise specified, a modified oligonucleotide may replace the corresponding unmodified nucleotide within the motif (as recognized by the natural enzyme), or more generally, replace any place within the oligonucleotide acted by the nuclease without substantially hindering enzymatic activity. For example, the modified N1-methyl-pseudouridine (Ψ) found in various mRNA vaccines can generally replace uridine without loss of activity.

[0031] The method for digesting RNA disclosed herein can be performed with sequence-specific nucleases only, so that the sequence and length of the fragments produced by one or more nucleases digesting RNA molecules with known sequences can be predicted with certainty (assuming that each targeted cleavage site is actually cut). Some sequence-specific nucleases can cut oligonucleotides at fixed positions within the recognition motif. Some nucleases can cut oligonucleotides at fixed positions (e.g., defined by many nucleotides) outside the recognition motif, so that for the purposes of this disclosure, the nuclease can be considered to be sequence-specific because the exact cleavage site can be predicted from a known target sequence with a recognition motif. In some embodiments, RNA is digested using a variety of sequence-specific nucleases (e.g., 2, 3, 4, 5 or more sequence-specific nucleases).

[0032] Various sequence-specific nucleases may have different degrees of selectivity relative to potential target oligonucleotides, wherein the nucleases with lower selectivity are more inclined to produce more cutting in the target oligonucleotide than the nucleases with higher selectivity. This is especially true for large target oligonucleotides, which are generally more likely to show higher sequence diversity (at least on a smaller scale) relative to smaller oligonucleotide targets. Motif length (i.e., the number of nucleotides in the motif) may be related to nuclease selectivity, wherein nucleases that recognize longer motifs are generally more selective than nucleases that recognize shorter motifs. In some embodiments, more selective nucleases are preferred so as to produce less targeted cutting in the target RNA molecule, and thus produce longer fragments on average for at least one nuclease used in digestion. In some embodiments, for a variety of nucleases used in digestion (e.g., each nuclease), more selective nucleases are preferred. In some embodiments, nucleases may recognize motifs of 3, 4, 5, 6, 7 or more nucleotides in length. Such nucleases may be used in combination with nucleases that recognize shorter motifs (e.g., 1 and / or 2 nucleotides in length). Combinations of nucleases with varying degrees of selectivity may be used, including in a targeted manner, as described elsewhere herein.

[0033] According to the methods disclosed herein, at least one nuclease used in digestion is a sequence-specific "duplex-dependent nuclease of RNA" (i.e., a nuclease that cuts RNA molecules only when bound to a duplex formed within an RNA molecule). In some embodiments, the duplex must be a heteroduplex. In some embodiments, the duplex must be an RNA duplex. In some embodiments, the duplex can be a heteroduplex or an RNA duplex. In some embodiments, the duplex-dependent nuclease of RNA will produce two blunt ends. In some embodiments, the duplex-dependent nuclease of RNA will produce two overhangs or "sticky ends". In some embodiments, multiple sequence-specific duplex-dependent nucleases (e.g., 2, 3, 4, 5 or more) of RNA are used in digestion. In some embodiments, one or more sequence-specific duplex-dependent nucleases of RNA can be used in combination with one or more sequence-specific nucleases (e.g., standard ribonucleases (RNases)) that are not duplex-dependent nucleases of RNA to digest RNA.

[0034] Considering that naturally occurring RNAs generally exist in single-stranded form (although most can self-hybridize in secondary structures such as loops and stems via Watson-Crick base pairing), many such sequence-specific duplex-dependent nucleases for RNA are deoxyribonucleases (DNases), which are found in nature to cleave DNA duplexes, but have been found to exhibit sufficient catalytic activity for other types of duplexes (e.g., heteroduplexes) including RNA. Because sequence-specific forms of deoxyribonucleases (including some nucleases for DNA duplexes) are generally known to exhibit higher sequence selectivity than the most selective sequence-specific forms of ribonucleases, the use of compatible sequence-specific deoxyribonucleases on target RNAs (particularly double-stranded RNAs) can achieve higher selectivity in RNA digestion than the use of typical ribonucleases, thereby allowing for more targeted cleavage, which can more easily produce RNA fragments within the desired size range. However, as described elsewhere herein, the selective formation of duplexes can advantageously increase the selectivity of sequence-specific duplex-dependent nucleases for RNA, even if the selectivity of the nuclease is generally lower (allowing for more targeted cleavage). The formation of duplexes within the target RNA prior to cleavage can further facilitate digestion and analysis of the target RNA, as the duplexes can prevent / destroy the formation of secondary structures in the sample RNA, which otherwise may result in missed cleavage using standard ribonucleases on single-stranded target RNAs. Although partial digestion with ribonucleases used for cleavage of single-stranded RNA and therefore prone to missed motifs within secondary structures can advantageously produce longer fragments than would be expected from complete digestion with ribonucleases, as described, for example, in Vanhinsbergh et al., Anal Chem. 2022 May 24; 94(20): 7339-7349 (doi: 10.1021 / acs.analchem.2c00765), RNA mapping can become very complicated given the large number of putative fragments that may be formed by partial digestion, and the reproducibility of mRNA mapping experiments may be hampered. Thus, use of RNA duplex-dependent nucleases may offer advantages in predictability of cleavage compared to use of sequence-specific ribonucleases that act on single-stranded RNA under conditions that promote missed cleavage to induce larger fragment sizes.

[0035] Representative sequence-specific duplex-dependent nucleases of RNA

[0036] Various nucleases, including natural enzymes and engineered enzymes, are known in the art and can be used as sequence-specific duplex-dependent nucleases of RNA according to the methods described herein. In some embodiments, the sequence-specific duplex-dependent nuclease of RNA can be a restriction endonuclease (restriction enzyme). Restriction endonucleases are nucleases that cut DNA duplexes into fragments at or near specific recognition sites within the molecule called restriction sites. All restriction endonucleases cut the sugar-phosphate backbone of both strands of the DNA double helix. Various examples of restriction endonucleases (including their respective motifs) are described in REBASE TM Database (available at re3data.org / repository / r3d100012171), which is a public database. Restriction endonucleases are generally divided into five types, which differ in structure, whether they cut their DNA substrate at their recognition site, or whether the recognition site and the cleavage site are separate from each other. In some embodiments, the restriction endonuclease is a Type II restriction endonuclease. Type II restriction endonucleases generally cut each strand of the duplex at a specific site within the recognition motif itself. Their activity does not use ATP or AdoMet. They generally only require Mg. 2+ Acts as a cofactor. Some Type II restriction endonucleases cleave the duplex to form two blunt ends, while others form two overhangs or "sticky ends".

[0037] In some embodiments, the sequence-specific duplex-dependent nuclease of RNA can be an IIP type restriction endonuclease. In some embodiments, the duplex-dependent nuclease of RNA can be a member of a structural class that uses a classical PD-(E / D)XK catalytic motif to affect cutting (e.g., AvaII, AvrII, BanI, TaqI, or HinfI). In some embodiments, the duplex-dependent nuclease of RNA can be MvaI or BcnI. IIP type restriction endonucleases form homodimers and recognize palindromic motifs of 4-8 nucleotides in length. They are usually cut within the recognition motif. In some embodiments, the duplex-dependent nuclease of RNA recognizes palindromic motifs. In some embodiments, the duplex-dependent nuclease of RNA recognizes motifs of 4-8 nucleotides in length. The IIP type enzyme with specificity for 6bp-8bp sequence mainly acts as a homodimer, which is composed of two identical protein chains that are bound to each other in opposite orientations (for example, EcoRI, HindIII, BamHI, NotI, PacI). Each protein subunit binds roughly half of the recognition sequence and cuts a DNA chain. Because the two subunits are identical, the enzyme is symmetrical, so the entire recognition sequence and the cutting position are also symmetrical. Usually, these enzymes cut two DNA chains simultaneously, and each catalytic site works independently of each other. The IIP type enzyme that recognizes a shorter 4-bp sequence usually acts as a monomer (for example, MspI, HinP1I, BstNI, NciI) composed of a single protein chain. They only have a catalytic site, and only cut a chain after binding. However, because they recognize palindromic sequences, they can bind in any orientation, and finally cut two chains, first cutting a chain and then cutting another chain. The conversion of the enzyme orientation that occurs is usually very fast, and there is almost no accumulation of "nicked" intermediate molecules, which are only cut in the first chain. Other types of IIP enzymes (eg, SfiI, NgoMIV) act as complex homotetramers, homodimers, dimers, or higher order oligomers that simultaneously bind and cleave two or more recognition sequences.

[0038] Depending on the proximity of the subunits of the IIP-type homodimer to each other, the recognized sequence can be continuous (e.g., EcoRI: GAATTC), or discontinuous, with one unspecified bp (e.g., HinfI: GANTC), two unspecified bp (e.g., Cac8I: GCNNGC), three unspecified bp (e.g., AlwNI: CAGNNNCTG), four unspecified bp (e.g., PshAI: GACNNNNGTC), five unspecified bp (e.g., BglI: GCCNNNNNGGC), or more unspecified bp (N), up to nine unspecified bp (e.g., XcmI: CCANNNNNNNNNTGG). IIP-type enzymes cut their recognition sequences at various positions, which depend on the position of the catalytic site relative to the sequence recognition residues in the protein. Some generate 5′-overhangs (“staggered ends”) of four bases (e.g., HindIII: A / AGCTT) or two bases (e.g., NdeI: CA / TATG). Others generate 3′-overhangs of four bases (e.g., SacI: GAGCT / C) or two bases (e.g., PvuI: CGAT / CG). Still others generate blunt ends (e.g., EcoRV: GAT / ATC). Enzymes with ambiguous base pairs in their recognition sequences can generate ends with an odd number of bases, including one base (e.g., NciI: CC / SGG), three bases (e.g., TseI: G / CWGC), five bases (e.g., PspGI: / CCNGG), or more.

[0039] Most IIP-type enzymes recognize unique sequences in which only one specific base pair can be present at each position (e.g., BglII: AGATCT). However, some IIP-type enzymes recognize "degenerate" or "ambiguous" sequences in which alternative bases may be present. The most common degenerate nucleotides are Y (pyrimidine, C or T) and R (purine, A or G) (e.g., ApoI: RAATTY). Others are M (modifiable bases, A or C) and K (non-modifiable bases, G or T) (e.g., AccI: GTMKAC); W (weak hydrogen bond, A or T) (e.g., BstNI: CCWGG); and S (strong hydrogen bond, C or G) (e.g., NciI: CCSGG). The atomic structure of the enzyme's binding site determines which base pairs can be recognized at each position.

[0040] Murray et al., Nucleic Acids Res. 2010 Dec; 38(22): 8257-68 (doi: 10.1093 / nar / r / gkq702) provides several examples of DNA endonucleases that have been found to exhibit activity against RNA in heteroduplexes, which is incorporated herein by reference in its entirety. See also Kisiala et al., Nucleic Acids Res. 2020 Jul 9; 48(12): 6954-6969 (doi: 10.1093 / nar / gkaa403), which is incorporated herein by reference in its entirety. Table 1 below depicts the motifs and functionality of several DNA endonucleases that exhibit such activity. In some embodiments, one or more nucleases (e.g., 1, 2, 3, 4, 5, or 6) from the nucleases of Table 1 are used as sequence-specific duplex-dependent nucleases of RNA in the digestion methods described herein. In some specific implementations, one or more sequence-specific duplex-dependent nucleases of RNA can be selected as needed to obtain fragments within the desired size range in such duplexes in the order of relative activity to RNA, wherein the nuclease with higher activity is selected first, and the nuclease with lower activity is selected later. For example, in some cases, some such nucleases can be selected in the relative order of TaqI, AvaII, AvrII, BanI, Hinf1. In some cases, some such nucleases can be selected in the relative order of AvaII, MvaI, BcnI. Other DNA endonucleases that can be used as sequence-specific duplex-dependent nucleases of RNA include, for example, MvaI (motif: CC / WGG) and BcnI (motif: CC / SGG), which are all IIP-type endonucleases.

[0041] Table 1: Exemplary sequence-specific DNA endonucleases that exhibit duplex-dependent cleavage of RNA

[0042]

[0043]

[0044] In some embodiments, the sequence-specific duplex-dependent nuclease of RNA can be a CRISPR-associated system (Cas) protein. CRISPR (clustered regularly spaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotes (such as bacteria and archaea), which provides immunity to plasmids and phages by using exogenous DNA stored as CRISPR spacer sequences together with Cas nucleases to prevent infection. Recently, the CRISPR-CAS system has been effectively reused for gene editing. Like restriction endonucleases, most CRISPR-CAS systems catalyze the cutting of double-stranded DNA or occasional single-stranded DNA. CRISPR technology is well known in the art. In short, CAS proteins, such as CAS9 proteins that are more commonly used for gene editing, generally require CRISPR RNA (crRNA) that recognizes the target sequence via Watson-Crick binding and a trans-activating RNA (tracrRNA) that allows the formation of a duplex region with a portion of crRNA that allows the Cas protein to be compounded. CrRNA and tracrRNA can be connected to a single guide RNA (sgRNA), typically with a hairpin loop. Thus, crRNA / sgRNA provides sequence specificity for Cas nucleases. Many Cas proteins also require recognition of a protospacer adjacent motif (PAM) sequence adjacent to the target sequence. However, this is ultimately not too restrictive, as it is typically a very short, non-specific sequence that occurs frequently in many places throughout the genome (e.g., the SpCas9 PAM sequence is 5'-NGG-3' and occurs approximately every 8 to 12 base pairs in the human genome).

[0045] Recently, it has been discovered that the CAS13 protein targets single-stranded RNA rather than DNA for cleavage and can be programmed to be sequence-specific. CAS13 is described in more detail in Wessels et al., Nat Biotechnol. 2020 Jun;38(6):722-727 (doi:10.1038 / s41587-020-0456-9); Abudayyeh et al., Science. 2016 Aug 5;353(6299):aaf5573 (doi:10.1126 / science.aaf5573); East-Seletsky et al., Nature. 2016 Oct 13;538(7624):270-273 (doi:10.1038 / nature19802); Mol. Cell. 2015 Nov 5; 60(3): 385-97 (doi: 10.1016 / j.molcel.2015.10.008), each of which is incorporated herein by reference in its entirety. It has also been shown that short DNA oligomers (PAMmers) containing PAM sequences can be provided to Streptococcus pyogenes (SpyCas9) to induce single-stranded RNA (ssRNA) binding and cleavage. In addition, it has recently been shown that Cas9 enzymes from two subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) through an RNA-guided mechanism that is independent of the PAM sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific. Cas9 cleavage of RNA is described in more detail in Strutt et al., Elife. 2018 Jan 5; 7: e32724 (doi: 10.7554 / eLife.32724), which is incorporated herein by reference in its entirety. In some embodiments, the sequence-specific duplex-dependent nuclease of RNA can be a Cas protein (e.g., Cas13 or Cas9 protein). In some embodiments, the nuclease is a subtype II-A or II-C Cas9 protein. In some embodiments, the nuclease is a Staphylococcus aureus (S. aureus) Cas9 (SauCas9) or Campylobacter jejuni (C..jejuni) Cas9 (CjeCas9) protein. In various embodiments using CAS proteins, crRNA or sgRNA can be effectively used as exogenous nucleotides, as described elsewhere herein, which form duplexes for promoting sequence-specific duplex-dependent cleavage of RNA.

[0046] In some embodiments, the sequence-specific duplex-dependent nuclease of RNA can be an artificial nuclease. For example, in some embodiments, one or more sequence-specific duplex-dependent nucleases of RNA can be an artificial site-specific RNA endonuclease (ASRE), as described in Choudhury et al., Nat Commun. 2012; 3: 1147 (doi: 10.1038 / ncomms2154), which is incorporated herein by reference in its entirety. In short, the ASRE can include an RNA-binding PUF domain that can be engineered to specifically bind to any 8-nucleotide RNA motif, connected to a PIN domain for cutting RNA. Other artificial nucleases, including nucleases that use a PUF domain to recognize a specific RNA motif, can be used in the methods described herein.

[0047] In some embodiments, the sequence-specific duplex-dependent nuclease of RNA can be an enzyme in the RNase III family, characterized by an RNase III catalytic domain. These enzymes recognize and cut double-stranded RNA (dsRNA) at specific sites. They are ubiquitous enzymes in cells, playing a major role in pathways such as RNA precursor synthesis, RNA silencing, and pnp self-regulatory mechanisms. The enzyme can be a class 1, class 2, class 3, or class 4 RNase III. In some embodiments, the RNase III enzyme can be Dicer. Dicer cuts dsRNA and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments in vivo, referred to as small interfering RNA and microRNA, respectively. These fragments are about 20-25 base pairs long and have an overhang of two bases at the 3′ end. Dicer promotes the activation of RNA-induced silencing complexes (RISC), which is essential for RNA interference. Human dicer includes two RNase III domains, two double-stranded RNA binding domains (DUF283 and dsRBD), a helicase domain, and PAZ (Piwi / Argonaute / Zwille). Current studies have shown that the PAZ domain is able to bind to the 3′ overhang of 2 nucleotides of dsRNA, while the RNase III catalytic domain forms a pseudodimer around the dsRNA to initiate chain cleavage. Dicer can work in conjunction with other regulatory proteins to effectively position the RNase III domain, thereby controlling the specificity of the sRNA product. In some embodiments, additional regulatory proteins are used in combination with Dicer. Dicer is described in more detail in Paturi et al., Front Mol Biosci. May 7, 2021; 8: 643657 (doi: 10.3389 / fmolb.2021.643657), which is incorporated herein by reference in its entirety. In some embodiments, the RNase III enzyme can be Drosha. Drosha is the main nuclease that performs the initial steps of miRNA processing in the nucleus. It works closely with DGCR8 in vivo and is associated with Dicer. In some embodiments, additional regulatory proteins are used in combination with Drosha. Drosha and Dicer are described in more detail in Leitao et al., Noncoding RNA. 2022 Jan 18;8(1).10 (doi: 10.3390 / ncrna8010010), which is incorporated herein by reference in its entirety.

[0048] Other sequence-specific nucleases for dsRNA may be able to be engineered from RNase III-type ribonucleotides, such as Dicer and Drosha. et al., Nucleic Acids Res. 2015 Mar 11; 43(5): 2864-73 (doi: 10.1093 / nar / gkv009) (the document is incorporated herein by reference in its entirety) describes how BsMiniIII can cut long dsRNA in a sequence-specific manner with different preferences for specific motifs (including AC / UC, preferentially AC / CU or AG / GU) in a short time frame, as well as non-specific cutting that occurs in a longer time frame, proposing that the enzyme is a prototype for engineered sequence-specific ribonucleases. In some implementations, if the nuclease only performs a sequence-specific reaction within the time frame of the digestion reaction, the nuclease can be considered sequence-specific.

[0049] In some embodiments, one of the one or more sequence-specific nucleases used to digest RNA can be a deoxyribozyme (also known as a DNA enzyme, DNAzyme, or catalytic DNA). A deoxyribozyme is a DNA oligonucleotide that is capable of performing a specific, usually catalytic, chemical reaction. The most abundant class of deoxyribozymes is ribonucleases, which catalyze the cleavage of ribonucleotide phosphodiester bonds by transesterification to form a 2'3'-cyclic phosphate end and a 5'-hydroxyl end. Most, but not all, of these deoxyribozymes require a divalent metal ion cofactor such as Mg. 2+ Although the first discovered DNAzymes generally recognized R / Y and A / G motifs, where R represents a purine (A or G) and Y represents a pyrimidine (U or C), a series of variants have been discovered that allow cleavage of most dinucleotide sequences N / N at reasonable rates in vitro.

[0050] The catalytic or random enzyme region of the deoxyribozyme oligonucleotide can be flanked by a binding arm on one or both sides, and the binding arm is targeted and combined with the RNA oligonucleotide target via Watson-Crick. Some deoxyribozymes can preferentially use several pairs of mismatched nucleotides near the cleavage site. The length of the binding arm can adjust the binding affinity to the target RNA, and the longer the binding arm, the higher the affinity. In some embodiments, long binding arms may be preferred to promote higher binding affinity and / or increased target specificity. Molar excess deoxyribozymes can be used to drive complete digestion under single conversion conditions. Although the target motif of the deoxyribozyme is usually shorter (e.g., 2bp), the use of a binding arm comprising nucleotides complementary to the target RNA sequence can effectively increase the sequence specificity of the deoxyribozyme. To some extent, specific deoxyribozymes will not catalyze available target motifs (i.e., the recognition of longer sequence motifs by deoxyribozymes) in the absence of hybridization of the binding arm, and now deoxyribozymes can be considered as sequence-specific duplex-dependent nucleases of RNA. In practice, the catalytic portion of the DNAzyme acts as a nuclease, and the binding arm acts as a duplex-forming oligonucleotide that promotes more selective sequence-specific binding of the catalytic portion of the DNAzyme. DNAzymes are described in more detail in Silverman, Nucleic Acids Res. 2005 Nov 11; 33(19): 6151-63 (doi: 10.1093 / nar / gki930), which is incorporated herein by reference in its entirety. Similar to DNAzymes, peptide nucleic acid-based nuclease systems (PNAzymes) can be used as sequence-specific, or more specifically, sequence-specific duplex-dependent nucleases for digestion of RNA. PNAzymes are described in more detail in Murtola et al., J Am Chem Soc. 2010 Jul 7;132(26):8984-90 (doi: 10.1021 / ja1008739); and Luige et al., Molecules. 2019 Feb 14;24(4):672 (doi: 10.3390 / molecules24040672). Similarly, aptamers (ribozymes fused to aptamers) as described, for example, in Peng et al., RSC Chem Biol. 2021 Jul 2; 2(5): 1370-1383 (doi: 10.1039 / d0cb00207k) (which is incorporated herein by reference in its entirety) can be considered duplex-dependent nucleases of RNA to the extent that they are engineered to cleave only available sequence-specific motifs after the aptamer recognizes a specific motif. To the extent that any of these enzymes are not duplex-dependent, they can be used as additional sequence-specific ribonucleases, as discussed below.

[0051] Representative nucleases for additional RNA digestion

[0052] In various embodiments, one or more sequence-specific nucleases of RNA that are not duplex-dependent nucleases of RNA are used in combination with one or more sequence-specific duplex-dependent nucleases of RNA to digest the target RNA. For example, 1, 2, 3, 4, 5 or more sequence-specific ribonucleases can be used to digest RNA according to the methods described herein. These sequence-specific ribonucleases can cut single-stranded RNA. A variety of sequence-specific ribonucleases are well known in the art, including but not necessarily limited to those described elsewhere herein. For example, various nucleases are described in Yang, Q Rev Biophys. February 2011; 44 (1): 1-93 (doi: 10.1017 / S0033583510000181), which is incorporated herein by reference in its entirety, including some nucleases that can be used as sequence-specific ribonucleases. In some embodiments, the sequence-specific ribonuclease can be a ribozyme, as described, for example, in Peng et al., RSC Chem Biol. 2021 Jul 2; 2(5): 1370-1383 (doi: 10.1039 / d0cb00207k), which is incorporated herein by reference in its entirety. In some embodiments, the sequence-specific ribonuclease can be a nuclease, which is described in Jiang et al., Anal Chem. 2019 Jul 2; 91(13): 8500-8506 (doi: 10.1021 / acs.analchem.9b01664), which is incorporated herein by reference, and describes digestion, characterization, and mapping of RNA with such ribonucleases.

[0053] In some embodiments, the digestion methods disclosed herein use RNase T1 as a sequence-specific ribonuclease. RNase T1 is an endoribonuclease that specifically degrades single-stranded RNA after a G residue. It cuts the phosphodiester bond between the 3'-guanylate residue and the 5'-OH residue of the adjacent nucleotide to form a corresponding intermediate 2', 3'-cyclic phosphate. The reaction products are 3'-GMP and an oligonucleotide with a terminal 3'-GMP. The activity of RNase T1 does not require metal ions.

[0054] In some embodiments, the digestion methods disclosed herein use RNase A as a sequence-specific ribonuclease. RNase A is an endoribonuclease that specifically degrades single-stranded RNA after pyrimidine residues (C or U). It effectively hydrolyzes RNA by cleaving the phosphodiester bond between the 3'-phosphate group of the pyrimidine nucleotide and the 5'-ribose of its adjacent nucleotides 1, 2, and 3. The resulting intermediate 2', 3'-cyclic phosphodiester is then further hydrolyzed to a 3'-monophosphate group.

[0055] In some embodiments, the digestion methods disclosed herein use colicin as a sequence-specific ribonuclease (e.g., colicin E5). Colicin is a type of bacteriocin produced by some Escherichia coli strains and toxic to them. Colicin is released into the environment to reduce competition from other bacterial strains and binds to outer membrane receptors, using them to transfer to the cytoplasm or cytoplasmic membrane, where they exert cytotoxic effects, some of which include RNase activity. RNase-type colicin inhibits protein synthesis in sensitive cells by cutting specific sites near the 3′ end of 16S rRNA. Colicin E5 is a known tRNase, specifically, it inhibits protein synthesis by specifically cutting tRNATyr, tRNAHis, tRNAAsn and tRNAAsp of sensitive Escherichia coli cells. Colicin E5 cleaves these tRNAs between the 34th queuosine (Q) and the 35th uridine (U), corresponding to the first and second letters of the anticodon triplet, to produce a 2', 3'-cyclic phosphate and a 5'-OH terminus. Q is a nucleoside with a unique base, queuine, which is a highly modified guanine (G) base, widely found in the above positions in the above four tRNA species of prokaryotes and eukaryotes. However, colicin E5 has been shown to exhibit RNase activity against G / U motifs as well as Q / U motifs. Colicin E5 is described in more detail in Ogawa et al., Nucleic Acids Res. 2006; 34(21): 6065-73 (doi: 10.1093 / nar / gkl629), which is incorporated herein by reference in its entirety.

[0056] In some embodiments, the digestion methods disclosed herein use MazF as a sequence-specific ribonuclease (e.g., E. coli Maz F or Mycobacterium tuberculosis MazF). MazF is a bacterial toxin that is part of the MazE-MazF toxin-antitoxin system. MazF in E. coli is an N / ACA-specific endoribonuclease that functions independently of the ribosome and RNA codon context. The 2′-OH group in the N residue of the N / ACA cleavage motif is generally required for MazF cleavage. MazF is described in more detail in Zhang et al., J Biol Chem. 2005 Feb 4; 280(5): 3143-50 (doi: 10.1074 / jbc.M411811200), which is incorporated herein by reference in its entirety. Other orthologs of MazF may recognize different 3-, 5-, or 7-residue motifs. For example, the MazF-mt6 ortholog from Mycobacterium tuberculosis recognizes and cleaves the UU / CCU motif, as described, for example, in Schifano et al., Proc Natl Acad Sci US A. 2013 May 21; 110(21):8501-6 (doi: 10.1073 / pnas.1222031110), which is incorporated herein by reference in its entirety. MazF enzymes are generally expensive, and their recognition motifs are relatively uncommon in mRNA, such that use of MazF enzymes alone may not provide sufficient cleavage of large RNA molecules to obtain fragments within the optimal size range for polynucleotide analysis.

[0057] Exemplary sequence-specific ribonucleases and their motifs are depicted below in Table 2. In various implementations, 1, 2, 3, or 4 of the ribonucleases listed in Table 2 are combined with one or more sequence-specific duplex-dependent nucleases of RNA to digest the target RNA.

[0058] Table 2: Exemplary sequence-specific ribonucleases

[0059] RNase Motif (5′-3′) RNase T1 G / Colicin E5 G / U MazF (E. coli) N / ACA MazF-mt6 UU / CCU

[0060] In some embodiments, non-specific 3 'and / or 5 'exonucleases can be used in combination with sequence-specific duplex-dependent nucleases of RNA, and optionally in combination with sequence-specific ribonucleases that are not duplex-dependent, as described elsewhere herein. Exonucleases are enzymes that work by hydrolyzing the phosphodiester bond at the 3 'end or 5 'end from the end of the polynucleotide chain, cutting one nucleotide at a time. By using non-specific exonucleases, fragments from previous digestion can be further digested to generate a ladder of partially digested fragments. For example, the separated fragments from the initial digestion can be subjected to varying degrees of degradation (e.g., by longer reaction times with exonucleases) of one or more exonucleases. Differentially degraded fragments can be characterized, for example, by mass spectrometry, as described elsewhere herein. The molecular weight of the differentially degraded fragments that make up the ladder can be used to elucidate the sequence of the original fragment.

[0061] Methods for digesting RNA

[0062] The method of digesting single-stranded target RNA using sequence-specific duplex-dependent nuclease of RNA includes forming one or more duplexes with target RNA and one or more other oligonucleotides. In some specific implementations, for each sequence-specific duplex-dependent nuclease in one or more sequence-specific duplex-dependent nucleases of RNA, one or more candidate RNA motifs are identified in the reference sequence of target RNA. Candidate RNA motifs can be selected based on expected fragment size to induce cutting, thereby producing fragments within the expected size range or size distribution. Therefore, the selective formation of the duplex with the target RNA provides a mechanism for selectively avoiding the cutting of certain available cutting sites in the target RNA (otherwise these cutting sites will be cut by the sequence-specific duplex-dependent nuclease of RNA), thereby allowing further precise control of the length of the digested fragment. In some embodiments, only a candidate RNA motif of a specific sequence-specific duplex-dependent nuclease of RNA can be selected for inducing cutting. In some embodiments, multiple candidate RNA motifs of a specific sequence-specific duplex-dependent nuclease of RNA can be selected for inducing cleavage (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more candidate RNA motifs). In some embodiments, each available candidate RNA motif of a specific sequence-specific duplex-dependent nuclease of RNA can be selected for inducing cleavage.

[0063] For each selected RNA motif of the sequence-specific duplex-dependent nuclease of RNA, a duplex can be formed with a target RNA including the selected RNA motif. The duplex may include an exogenous oligonucleotide that is capable of Watson-Crick binding to the target RNA to form a sufficiently stable duplex that is capable of binding to the specific sequence-specific duplex-dependent nuclease of RNA and promoting the cutting of the selected site. The exogenous oligonucleotide may be a DNA molecule. The exogenous oligonucleotide may be an RNA molecule. The exogenous oligonucleotide may include deoxyribonucleotides, ribonucleotides and / or modified nucleotides. As will be understood by those of ordinary skill in the art, the exogenous oligonucleotide may have a length sufficient to form a sufficiently stable duplex to allow digestion under specific digestion conditions. As will be understood by those of ordinary skill in the art, the exogenous oligonucleotide may have a length sufficient to allow the specific sequence-specific duplex-dependent nuclease of RNA to bind to the duplex in a sufficiently stable manner to promote cutting. The exogenous oligonucleotide may have a length sufficient to provide sufficient sequence selectivity so as to hybridize with the selected RNA motif without hybridizing with any non-selected RNA motif. Therefore, the exogenous oligonucleotide for forming a duplex with a given selected RNA motif can have a sequence length fully complementary to the unique region of the RNA target sequence comprising the selected RNA motif, or a sequence length at least not fully complementary to the region of the RNA target sequence comprising a non-selected RNA motif. In some embodiments, the length of every kind of exogenous oligonucleotide is at least about 10,15,20,25 or 30 nucleotides. The exogenous oligonucleotide can include nucleotides complementary to each nucleotide in the selected RNA motif. The whole sequence of the exogenous oligonucleotide can be completely complementary to the target RNA. In some embodiments, a separate duplex is formed for each selected RNA motif (that is, the region of the expected single-stranded target RNA can split the duplex formed for each selected RNA motif).

[0064] In some embodiments, a single duplex includes two or more adjacent selected RNA motifs, regardless of whether the adjacent selected RNA motifs are targeted by duplex-dependent nucleases of the same or different RNAs (i.e., a single exogenous oligonucleotide forms a duplex with an RNA target sequence region including two or more adjacent RNA motifs). In some embodiments, the length of the one or more oligonucleotides used to form one or more duplexes with the target RNA is short enough so that after digestion, the exogenous oligonucleotide will not interfere with the polynucleotide analysis of the digested RNA. Therefore, in some embodiments, the length of the one or more oligonucleotides is selected so that after digestion, the oligonucleotide will be shorter than the shortest length of any target RNA fragment to be analyzed or mapped. In some embodiments, the length of the oligonucleotide fragment may be less than or no more than about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides. In some embodiments, the exogenous nucleotides are about 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-30, 30-50, 30-45, 30-40, or 30-35 nucleotides in length.

[0065] In some embodiments, the digestion method includes using multiple sequence-specific duplex-dependent nucleases of RNA to digest the target RNA (e.g., at least 2, 3, 4, or 5 sequence-specific duplex-dependent nucleases). In some embodiments, the digestion method includes using a combination of one or more nucleases that are not duplex-dependent nucleases of RNA and one or more duplex-dependent nucleases of RNA. For example, the digestion method may include additional cutting using a sequence-specific ribonuclease. In some embodiments, a sequence-specific nuclease that is not a duplex-dependent nuclease of RNA is used for selected fragments of the target RNA (e.g., for separation of collected fractions).

[0066] Methods for in vitro enzymatic digestion of oligonucleotides, including methods in sample preparation for polynucleotide analysis (such as by liquid chromatography and / or mass spectrometry), are well known in the art. Briefly, for any given digestion reaction, a sample, a nuclease, and any suitable reaction buffer for driving the enzymatic reaction (e.g., including a metal ion for catalysis, such as Mg) can be prepared. 2+) of the reaction mixture. The digestion reaction may be carried out for a predetermined amount of time before quenching the reaction, starting the sequential reaction, or starting the polynucleotide analysis procedure (e.g., separation via liquid chromatography). The digestion reaction may be temperature controlled. A predetermined high temperature may be used to promote the digestion reaction and may be applied during the predetermined reaction time. As required, regulation of the reaction time, reaction temperature, reaction pH, or enzyme amount (e.g., concentration) may be used to control the nuclease reaction kinetics and drive complete or partial digestion. In some specific implementations, the reaction time may be controlled by quenching the enzymatic reaction by any suitable means known in the art (e.g., temperature or pH changes).

[0067] In the digestion reaction involving the sequence-specific duplex-dependent nuclease of RNA, an exogenous oligonucleotide can be introduced into the reaction mixture to form one or more duplex substrates, which will be acted upon by one or more duplex-dependent nucleases of RNA. The exogenous oligonucleotide can be introduced independently, introduced together with the duplex-dependent nuclease of RNA, introduced together with the RNA sample, introduced together with other reaction buffer components, or introduced as a combination thereof. The amount of the exogenous oligonucleotide can be used to control the reaction kinetics of the sequence-specific duplex-dependent cleavage reaction. For example, a molar excess of the exogenous oligonucleotide can be used, combined with a suitable reaction time, reaction temperature and nuclease amount, to ensure complete digestion of the selected RNA motif. Submolar amounts of exogenous oligonucleotides can be used to drive partial digestion of the selected RNA motif, as desired and known in the art. In some embodiments, equimolar amounts of target RNA and oligonucleotides are used.

[0068] The reaction mixture can be incubated or mixed (e.g., via flow) during the digestion reaction. Suitable conditions for oligonucleotide annealing are known in the art. In some implementations, the exogenous oligonucleotide is annealed to the sample oligonucleotide (e.g., target RNA), which is achieved by generating a reaction mixture with both, then heating the reaction mixture to a high temperature (e.g., 95° C.), and then allowing the reaction mixture to cool. The annealing process may destroy the secondary structure, as described elsewhere herein. In some cases, a nuclease may be added to the reaction mixture after the exogenous oligonucleotide is annealed to avoid nuclease denaturation.

[0069] In some embodiments, exogenous oligonucleotides are added to the sample RNA by in vitro reverse transcription. Methods for reverse transcription are well known in the art. Single-stranded cDNA can be directly synthesized onto the sample RNA via a single round of reverse transcription to produce one or more heteroduplexes for the cutting of the duplex-dependent nuclease of the instructive RNA, as described elsewhere herein. Therefore, the reaction mixture may include sample RNA, primers (3' primers), dNTPs and reverse transcriptase. In some embodiments, primers may be selected to ensure the reverse transcription of all selected RNA cleavage sites (e.g., 3' primers partially complementary to the target RNA sequence at the 3' end of the desired duplex). Nucleases may be added to the reaction mixture after reverse transcription is complete.

[0070] In various embodiments, one or more nucleases for digestion described herein are fixed to a solid insoluble support for enzymatic reaction. Various suitable supports are known in the art, and include, for example, beads (which may optionally be filled into a column or which may be separated from a reaction solution by processes such as centrifugation), other particles and membranes. In some cases, nucleases may be fixed on magnetic beads or particles, which may be magnetically separated from a reaction solution. Nucleases may be fixed on a solid matrix to promote the control of enzymatic reaction by removing nucleases from a reaction mixture including a nucleotide substrate. Fixation may also prevent nucleases from accumulating in a subsequent analytical device (e.g., a liquid chromatography column) from a treated reaction mixture, which may ultimately result in undesirable sample digestion during analysis. In some embodiments, one or more nucleases are used in an immobilized enzyme reactor (IMER). IMER is a flow-through device containing enzymes, which are physically restricted or positioned and retain their catalytic activity. IMER can be used repeatedly and continuously, and has been applied to (bio) polymer degradation, proteomics, biomarker discovery, inhibitor screening and detection. The online integration of IMER with analytical instruments (such as high performance liquid chromatography (HPLC) systems) reduces the time required for multi-step workflows, reduces the need for sample handling, and achieves automation. When using multiple nucleases, one or more nucleases can be fixed. In some cases, two or more nucleases are fixed on the same support. In some cases, two or more nucleases are fixed on two or more different supports. In some cases, some nucleases are fixed, while others are not fixed (used in solution).

[0071] When using multiple nucleases to digest RNA samples, the digestion reaction of any two nucleases can be performed simultaneously (in parallel) or sequentially. In some embodiments, sequential digestion can be performed before and after the separation of the digested sample. For example, oligonucleotide fragments (e.g., larger fragments) can be fractions collected after separation by liquid chromatography, and additional digestion reactions are performed. In some embodiments, as described elsewhere herein, subsequent digestion may be performed with sequence-specific ribonucleases (e.g., RNase T1), due to the relatively low sequence specificity of ribonucleases and the large amount of cutting and small fragments that it may produce on larger undigested target RNAs, this situation can be preferentially avoided for the first round of pre-separation digestion. Compared with larger undigested samples, using such ribonucleases on smaller fragments will generally produce fewer small fragments. In addition, compared with the presence of smaller fragments obtained from the entire target RNA reference sequence, it is unlikely that smaller fragments are produced in the separated portion of the larger RNA reference sequence to complicate mapping. In some cases, such single-stranded ribonuclease digestion can be more easily performed online with fractionation output, because it is not necessary to introduce exogenous oligonucleotides to complete digestion. Such reactions can be injected into an IMER including an immobilized nuclease for subsequent digestion reactions.

[0072] Methods for Characterization of Digested RNA

[0073] The methods described herein may include separating the polyribonucleotide fragments digested according to length. In some embodiments, separation is performed by chromatography. Chromatographic methods for separating oligonucleotides are well known in the art. Chromatography may be liquid chromatography. Chromatography may be reverse phase chromatography. In some embodiments, chromatography is ion pairing chromatography, wherein ion pairing reagents are mixed with analytes before separation. In some specific implementations, salt gradients may be applied. In some specific implementations, anion exchange columns may be used. In some embodiments, chromatography is ultra high performance liquid chromatography (UHPLC). In various specific implementations, liquid chromatography may be 2D-LC. In some specific implementations, ultraviolet detection of fragments separated by liquid chromatography (LC-UV) may be sufficient for RNA mapping. As known in the art, other suitable detection methods for detecting fragments separated by LC may also be used. Various elution fractions (e.g., peaks) may be stored online (e.g., stored in a system loop), or stored offline for later processing. On-line processing of the sample (before and / or after the first round of liquid chromatography) can be performed on-line using one or more IMERs comprising one or more of the nucleases described herein for performing digestion.

[0074] Method described herein may include mass spectrometry analysis of the polynucleotide fragments digested. The method for analyzing polyribonucleotide fragments by mass spectrometry is well known in the art. In some embodiments, mass spectrometry analysis may include tandem mass spectrometry (MS / MS). The method for performing mass spectrometry analysis may include charge reduction and / or data deconvolution, which is well known in the art.

[0075] The methods described herein may include mapping one or more or all of the digested ribonucleotide fragments to a target RNA molecule. Methods for RNA mapping are well known in the art. See, for example, Vanhinsbergh et al., Anal Chem. 2022 May 24; 94(20): 7339-7349 (doi: 10.1021 / acs.analchem.2c00765), which is incorporated herein by reference in its entirety. Suitable methods for characterizing nucleic acids, including methods by liquid chromatography and mass spectrometry, are described in Santos et al., J Sep Sci. 2021 January; 44(1): 340-372 (doi: 10.1002 / jssc.202000833); Klont et al., Drug Discov Today Techno1. 2021 December; 40: 64-68 (doi: 10.1016 / j.ddtec.2021.10.004), each of which is incorporated herein by reference in its entirety. In some embodiments, LC-MS or LC-MS / MS can be used to determine quality information prior to RNA mapping. Tandem MS / MS analysis can be used to distinguish isobaric fragments. Liquid handling systems well known in the art (e.g., robotic automated liquid handling systems) can be used to facilitate any one or more steps involved in the digestion or characterization process.

[0076] Digestion and analysis of mRNA

[0077] As mentioned elsewhere herein, the digestion methods described herein can be used for mRNA molecules. mRNA molecules generally include a poly (A) tail at its 3' end and a 5' cap at its 5' end. The poly (A) tail and the 5' cap are generally separated from the internal coding sequence (CDS) of the mRNA molecule translated into an amino acid sequence by a 3' untranslated region (3'UTR) and a 5' untranslated region (5'UTR), respectively. The poly (A) tail is composed of multiple adenosine monophosphates, which form a sequence of variable length, which is important for the nuclear export, translation and stability of mRNA. The length of the poly (A) tail is uneven (e.g., about 60mer to 120mer), and may be difficult to control in the manufacture of mRNA. The confirmation of the length or length distribution of the poly A tail in an mRNA sample may be important, but the unevenness makes the mass spectrometry analysis of the complete mRNA difficult. The 5' cap is a specially changed nucleotide whose function is to regulate nuclear export, prevent exonuclease degradation, promote the initiation of translation, and promote the excision of 5' proximal introns. There are various 5' cap structures in nature. In eukaryotes, the 5' cap consists of a methylated guanine nucleotide at position 7 and is attached to the mRNA via an uncommon 5' to 5' triphosphate bond (i.e., a 7-methylguanylate cap). In the manufacture of mRNA, it may be important to confirm the uniformity and efficiency of 5' capping. The analysis of 5' capping is described in more detail in U.S. Publication No. 202I / 0108252 to Beverly, published on April 15, 2021.

[0078] The analysis of 5' capping, poly (A) tailing and internal mRNA sequences may complicate the analysis of other items. For example, the uneven quality / length of the 5' end or 3' end in the sample may affect the analysis of the internal fragment length. Therefore, it may be advantageous to analyze each of them separately or at least remove the analysis of one item from the analysis of other items. The digestion method described herein may include targeted cutting of the 5' end and / or 3' end from the mRNA molecule before analysis. In some embodiments, the internal mRNA sequence can be further digested and analyzed after removing the 5' end and / or 3' end. In some embodiments, the 5' end and / or 3' end can be analyzed after removing the remainder of the mRNA molecule. In various specific implementations, the remainder of the molecule will be large enough so that if it is further kept undigested before separation, it should show obvious retention behavior so that it will not interfere with the analysis of the 5' end and / or 3' end. Therefore, in some embodiments, the mRNA molecule is digested into two or three fragments before analysis. In some cases, the 5′ and / or 3′ end may be cleaved from the remainder of the mRNA molecule at a target cleavage site that is 0-10, 0-20, 0-30, 0-40, 0-50, 0-60, 0-70, 0-80, 0-90, 0-100, 0-150, 50-100, 50-150, or 100-150 nucleotides from the proximal end of the 5′ cap or poly(A) tail.

[0079] Similarly, in some implementations, polynucleotide analysis may only desire selected portions or fragments of RNA molecules. Thus, selective cleavage may be performed once or twice in the RNA molecules according to the methods described herein to isolate the fragments for analysis. Additional digestion may then be performed on the selected fragments, as described elsewhere herein. Unselected portions of the RNA molecules may be ignored to simplify analysis of the selected fragments.

[0080] For RNA digestion / point Kits and Systems for Analysis

[0081] Disclosed herein are kits and systems for carrying out methods described elsewhere herein.Kits may generally include any two or more components required for carrying out methods described herein.In some embodiments, the kit includes one or more nucleases for carrying out digestion described herein, including any nuclease described herein.In some embodiments, the kit includes multiple nucleases (e.g., 2, 3, 4, 5 or more nucleases) for carrying out digestion described herein.The kit may include at least one sequence-specific duplex-dependent nuclease of RNA.The kit may include multiple sequence-specific duplex-dependent nucleases (e.g., 2, 3, 4, 5 or more) of RNA.The kit may include at least one sequence-specific ribonuclease that is not a duplex-dependent nuclease of RNA.The kit may include multiple sequence-specific ribonucleases that are not a duplex-dependent nuclease of RNA.In some cases, the kit may include at least one sequence-specific duplex-dependent nuclease of RNA and at least one sequence-specific ribonuclease that is not duplex-dependent.

[0082] In some embodiments, the kit may include one or more solid substrates, and one or more nucleases described herein may be fixed on the one or more solid substrates. In some cases, one or more pre-loaded solid substrates (that is, one or more nucleases have been fixed thereon) may be provided. In some cases, one or more unloaded solid substrates may be provided. The solid substrate may be provided in combination with one or more nucleases for being fixed to the substrate. The kit may include one or more reagents for performing fixed chemistry. The kit may include a reagent for removing an enzyme from a solid support.

[0083] In some embodiments, the kit may include one or more reagents for performing a digestion as described herein. For example, the kit may include a suitable reaction buffer (e.g., including the necessary metal ions) for performing an enzymatic reaction and / or for quenching an enzymatic reaction (e.g., by inducing a pH change).

[0084] In some embodiments, the kit may include one or more exogenous oligonucleotides for one or more sequence-specific duplex-dependent cutting of RNA as described herein. One or more exogenous oligonucleotides may be configured to digest RNA molecules with a specific primary sequence. Oligonucleotides may be provided in combination with one or more nucleases, which recognize one or more specific motifs in one or more oligonucleotides. In some embodiments, the kit may include one or more components for reverse transcription of cDNA from RNA samples, such as primers (e.g., 3' primers), dNTPs and / or reverse transcriptase.

[0085] In some embodiments, the kit may include one or more components for performing polynucleotide analysis on RNA molecules digested according to the methods described herein. For example, the kit may include a polynucleotide ladder or standard for performing the analysis. In some embodiments, the kit may include a column suitable for separating ribonucleotides within a digestion size range by HPLC.

[0086] In some embodiments, one or more components for performing digestion as described herein (including, for example, a kit as described herein) are provided as part of a system having one or more pieces of equipment for performing polynucleotide analysis (e.g., HPLC, mass spectrometry). The system may include, for example, a detector for quantifying an analyte via HPLC or mass spectrometry. These systems or their components may include a computing component for performing the analysis, including suitable hardware and software known in the art. Various software can be used for performing polynucleotide analysis, as described, for example, in Vanhinsbergh et al., Anal Chem. 2022 May 24; 94(20): 7339-7349 (doi: 10.1021 / acs.analchem.2c00765), which is incorporated herein by reference in its entirety. The system may include a processor operably connected to a memory for performing the analysis. In various specific implementations, the system may include a processor configured to map the fragment to a reference RNA sequence based, at least in part, on the output received from one or more detectors and the target cleavage site. In some specific implementations, the system may be configured to output or provide candidate RNA motif targets for a given reference sequence based on the availability of one or more nucleases (e.g., sequence-specific duplex-dependent nucleases of RNA). The system may allow a user to select one or more candidate motifs for cutting. The system may automatically predict the sequence and size of the fragments produced by the selection of the candidate motifs. The system may provide information on whether the size distribution and / or fragment size meet any predetermined criteria, as described elsewhere herein. The system may be configured to recommend specific cutting based on the predetermined availability of nucleases. The system may provide recommended oligonucleotide sequences for sequence-specific duplex-dependent cutting as described elsewhere herein. The system may include a database of suitable nucleases (e.g., sequence-specific duplex-dependent nucleases of RNA) and corresponding motifs to automatically select cutting sites and / or nucleases for digestion.

[0087] Example

[0088] Digestion of COVID-19 mRNA vaccines

[0089] analyze Motifs of sequence-specific duplex-dependent nucleases of RNA of SARS-Cov-2 mRNA vaccines, in particular motifs of restriction endonucleases TaqI, AvaII and Ban I. Table 3 below indicates the cleavage sites of the identified candidate RNA motifs, the specific motifs available at each cleavage site, and the restriction endonucleases specific for each cleavage site.

[0090] Table 3: TaqI, AvaII, and BanI restriction in SARS-Cov-2 mRNA vaccines Location

[0091]

[0092]

[0093] Various digestion protocols were simulated by selecting certain candidate RNA motifs for cleavage and simulating the separation of the resulting digestion fragments by computer-simulated liquid chromatography. Initially, ion-pairing reversed-phase liquid chromatography (IP RP LC) was used on a BEH C18 A ladder of oligo-dT from 15mer to 100mer was chromatographed on a column (2.1 mm x 50 mm, 1.7 μm, purchased from Waters Corporation, Milford, MA) under the following conditions: column temperature 60°C; flow rate 0.4 ml / min; run time 20 min; gradient 90%-50% A / 10%-50% B (A = 25% acetonitrile, 75% 0.1 M HAA pH 8.13; B = 75% acetonitrile, 25% 0.1 M HAA pH 8.13). The results are shown in Figure 1 It can be seen that larger oligonucleotides are more difficult to selectively separate than smaller oligonucleotides. A calibration curve based on the retention time (Tr) predicted under these conditions based on the length of the oligonucleotide (L) was fitted to the experimental data as follows:

[0094] T r = b / L 2 +c / L+d (Formula I),

[0095] where b = 2,450; c = -361; and d = 17.77. Using this calibration curve, computer simulations were performed on the separation of oligonucleotide fragments from various hypothetical digests of the mRNA vaccine.

[0096] Table 4 below depicts the fragments expected to be generated by digestion of each candidate TaqI RNA motif with TaqI and the expected retention time of each fragment. A simulated chromatogram generated by TaqI digestion is shown in Figure 2A In. Figure 2AShown, 525mer / 570mer fragment and 801mer / 909mer fragment are co-eluted.It should be understood that longer columns and / or optimized separation methods should be able to improve the separation of these fragments.In some specific implementations, these peaks can be collection fractions, and additional separation is carried out, optionally with additional digestion (for example, via other restriction endonucleases and / or ribonucleases).

[0097] Table 4: Digestion of COVID-19 mRNA vaccines with TaqI restriction sites

[0098]

[0099]

[0100] Table 5 below depicts the fragments expected to be produced by alternative digestion schemes, where only selected RNA motifs are cleaved with TaqI, AvaII, and BanI, and the expected retention time for each fragment. The simulated chromatogram produced by the digestion is shown in Figure 2B In. Figure 2B As shown, the peaks corresponding to most fragments are distinguishable from each other.

[0101] Table 5: Digestion of COVID-19 mRNA vaccine with selected TaqI, AvaII and BanI restriction sites

[0102]

Claims

1. A method for digesting an RNA molecule having a known reference sequence into smaller RNA fragments, the method comprising: forming one or more oligonucleotide duplexes with the RNA molecule along a specific portion of the reference sequence; The RNA molecule is digested into the fragments using one or more sequence-specific nucleases that cleave the RNA molecule at a plurality of predetermined sequence-specific sites, wherein the one or more sequence-specific nucleases include one or more duplex-dependent nucleases that act only on RNA within a duplex, and wherein each of the one or more duplexes formed with the RNA molecule includes a motif recognized by one of the one or more duplex-dependent nucleases.

2. The method of claim 1, wherein the one or more sequence-specific nucleases comprises a plurality of nucleases.

3. The method of claim 2, wherein the plurality of nucleases comprises a plurality of duplex-dependent nucleases.

4. The method of any one of the preceding claims, wherein the one or more duplex-dependent nucleases comprise one or more of a restriction endonuclease, a Cas protein, an artificial site-specific RNA endonuclease (ARSE), an enzyme comprising an RNase III domain, or a deoxyribozyme.

5. The method of claim 4, wherein the one or more duplex-dependent nucleases comprise one or more restriction endonucleases selected from the group consisting of AvaII, AvrII, BanI, TaqI, HinfI and HAEIII.

6. The method of any one of the preceding claims, wherein the one or more sequence-specific nucleases comprise one or more of RNase T1, RNase A, colicin E5, and MazF.

7. The method of any of the preceding claims, wherein the RNA molecule has a length greater than about 1,000mer.

8. The method of any one of the preceding claims, wherein the RNA fragment is about 10mer to 1,000mer in length.

9. The method of claim 8, wherein the RNA fragment has a length of about 10mer to 500mer.

10. The method of claim 9, wherein the RNA fragment has a length of about 10mer to 100mer. The method according to claim 10 , wherein the RNA fragment has a length of about 10mer to 50mer.

12. The method according to any one of the preceding claims, wherein the RNA fragment is about 20mer in length.

13. The method of any one of the preceding claims, wherein the one or more duplexes comprises a plurality of duplexes.

14. The method according to any one of the preceding claims, wherein each of the one or more duplexes is formed by the RNA molecule and another oligonucleotide having a length of about 10mer to 50mer.

15. The method according to any one of the preceding claims, wherein each of the one or more duplexes is formed by hybridization of an exogenous oligonucleotide to the RNA molecule.

16. The method according to any one of the preceding claims, wherein the one or more duplexes are formed using DNA oligonucleotides.

17. The method according to any one of the preceding claims, wherein at least one of the sequence-specific nucleases is immobilized on a solid support.

18. The method of claim 17, wherein the at least one immobilized nuclease is provided in the form of an immobilized enzyme reactor (IMER) that allows flow-through digestion of the RNA molecules.

19. The method of claim 18, wherein the nuclease immobilized within the IMER is not a duplex-dependent nuclease and is used to further digest the selected fraction of RNA fragments after digestion with a duplex-dependent nuclease.

20. The method of any one of the preceding claims, wherein the RNA molecule is an mRNA molecule and the plurality of predetermined sequence-specific sites include sites within about 100 nucleotides of the proximal end of the 3' poly(A) tail and / or sites within about 100 nucleotides of the 5' cap.

21. The method of any one of the preceding claims, further comprising separating one or more of the RNA fragments based on length using liquid chromatography.

22. The method of any one of the preceding claims, further comprising measuring the mass of one or more of the RNA fragments using mass spectrometry.

23. The method of any one of the preceding claims, further comprising mapping the RNA fragments to the reference sequence.

24. A kit for digesting an RNA molecule having a reference sequence into smaller RNA fragments, the kit comprising a plurality of oligonucleotides, wherein each oligonucleotide is configured to hybridize to a single unique portion of the RNA molecule and comprises a motif recognized by a sequence-specific duplex-dependent nuclease that acts only on RNA within the duplex.

25. The kit of claim 24, wherein each of the oligonucleotides is about 10mer to 50mer in length.

26. The kit of claim 24 or 25, wherein the plurality of oligonucleotides comprises at least two motifs recognized by different sequence-specific duplex-dependent nucleases.

27. The kit according to any one of claims 24 to 26, further comprising one or more of said sequence-specific duplex-dependent nucleases.

28. A kit for digesting RNA molecules into smaller RNA fragments, the kit comprising a plurality of sequence-specific nucleases, the plurality of sequence-specific nucleases comprising one or more duplex-dependent nucleases that act only on RNA within a duplex and one or more ribonucleases that act on single-stranded RNA.

29. The kit of claim 28, wherein the one or more duplex-dependent nucleases comprises a restriction endonuclease.

30. A system for mapping RNA fragments to reference sequences, the system comprising: a detector configured to quantify the amount of RNA oligonucleotides having a length of about 20mer to 1,000mer; and A processor operably connected to the detector, wherein the processor is programmed to map the detected RNA oligonucleotide to a reference sequence of the RNA molecule based at least in part on the length or mass of the RNA oligonucleotide, wherein mapping the detected RNA oligonucleotide to the reference sequence comprises determining the length of the fragments expected to be produced by digesting the RNA molecule into smaller fragments according to any one of claims 1 to 23.

31. A system according to claim 30, wherein the processor is further configured to automatically identify a motif within the reference sequence, for which cleavage with the sequence-specific nuclease will produce a fragment having a length of about 20mer to 1,000mer, wherein the sequence-specific cleavage includes one or more selective cleavages performed with the one or more duplex-dependent nucleases.

32. The system of claim 30 or 31, wherein the processor is operably connected to one or more databases comprising a plurality of sequence-specific nucleases and motifs corresponding to each of the sequence-specific nucleases.

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