Compositions, methods, kits, and instruments for analyzing RNA structure

Abstract

The present disclosure relates, according to some embodiments, to compositions and methods for assessing polynucleotide (e.g., RNA) structure (e.g.. secondary and tertiary structure).

Description

[0001]PATENT APPLICATION NEB-481-PCT COMPOSITIONS, METHODS, KITS, AND INSTRUMENTS FOR ANALYZING RNA STRUCTURE CROSS-REFERENCE TO RELATED APPLICATIONS This application also claims priority to U.S. Provisional Application No. 63 / 612,776 filed December 20, 2023, the entire contents of which are hereby incorporated in their entirety by reference. SEQUENCE LISTING STATEMENT This disclosure includes a Sequence Listing submitted electronically in .xml format under the file name “NEB-481.xml” created on December 19, 2024, and having a size of 29,790 bytes. This Sequence Listing is incorporated herein in its entirety by this reference. INTRODUCTION RNA is a dynamic, multifunctional molecule capable of a variety of biological roles ranging from protein translation to gene regulation. An RNA molecule may be described or defined by its primary structure (its linear sequence of ribonucleotides), secondary structure (its base-paired stems and single-stranded loops), and / or tertiary structure (higher order intramolecular interactions which shape the overall RNA fold). An RNA molecule’s structure may have an important impact on the behavior and function of RNA molecules in vitro and in vivo. SUMMARY Accordingly, needs have arisen for improved tools for analyzing RNA structure. The present disclosure relates to systems, apparatus, compositions, and / or methods for analyzing RNA structure using, according to some embodiments, deaminases including adenosine deaminases and cytosine deaminases. In some embodiments, adenosine deaminases convert adenosine in RNA to inosine, which may be detected, for example, by contacting the RNA with a reverse transcriptase (e.g., a reverse transcriptase that predictably incorporates a cytosine across from the inosine(s) in the RNA) to produce a cDNA copy of the RNA and sequencing the cDNA. In some embodiments, cytosine deaminases may convert cytidine in RNA to uridine, which may be detected, for example, by contacting the RNA with a reverse transcriptase (e.g., a reverse transcriptase that incorporates an adenosine across from each uridine in the RNA) to produce a cDNA copy of the RNA and sequencing the cDNA. In some PATENT APPLICATION NEB-481-PCT embodiments the converted base may be detected by direct RNA sequencing (e.g., Nanopore direct RNA sequencing). A method for deaminating a nucleic acid, in some embodiments, may comprise (a) contacting (i) a deaminase (e.g., an adenosine deaminase or a cytosine deaminase); and (ii) a nucleic acid (A) comprising one or more nitrogenous bases selected from adenine and cytosine, and (B) having a nucleotide sequence to produce a deamination product comprising one or more deaminated adenines and / or one or more deaminated cytosines; (optionally) (b) determining the position in the nucleotide sequence of (i) the one or more deaminated adenosines and / or (ii) the one or more deaminated cytosines; and (optionally) (c) determining the secondary structure context of one or more of the nitrogenous bases corresponding to the one or more deaminated adenines and / or the one or more deaminated cytosines. A nucleic acid to be analyzed using the disclosed methods may comprise or consist of, according to some embodiments, DNA or RNA or a both DNA and RNA. One or more nucleotides in a nucleic acid may be modified nucleotides. In some embodiments, a deaminase may be an AMP deaminase (e.g., a Helix pomatia AMP deaminase. A deaminase may have an amino acid sequence at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any of SEQ ID NOS:1-3. A deaminase may be an adenosine deaminase that acts on RNA. In some embodiments, deaminated adenosines are inosines. A deaminase, in some embodiments, may be an APOBEC cytosine deaminase. Deaminated cytosines are uracils, according to some embodiments. Contacting may occur at any desired temperature, for example, at a temperature of 20°C-50°C. According to some embodiments, a method may include ligating an adapter to the 5’ end of the deamination product, ligating an adapter to the 3’ end of the deamination product, or ligating an adapter to the 5’ end of the deamination product and ligating an adapter to the 3’ end of the deamination product, in each case to form an adapter ligation product and, optionally, ligating an adapter to the 5’ end of the deamination product and ligating an adapter to the 3’ end of the deamination product and wherein the adapter ligated to the 5’ end is different from the adapter ligated to the 3’ end of the deamination product. Determining the position of a deaminase adenosine and / or a deaminated cytosine may include sequencing the adapter ligation product. A method may comprise, according to some embodiments, contacting the deamination product with a reverse transcriptase to copy the deamination product into a cDNA, wherein the cDNA comprises cytosines at positions corresponding to the deaminated adenosines of the PATENT APPLICATION NEB-481-PCT deamination product. Examples of revere transcriptases include Gka reverse transcriptase, Tbr reverse transcriptase, M-MuLV reverse transcriptase (including, for example, a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability (ProtoScript® reverse transcriptase; New England Biolabs, Ipswich, MA)), a group II intron- encoded reverse transcriptase with high processivity, increased thermostability, and increased tolerance of inhibitors in the synthesis of cDNA (e.g., Induro® reverse transcriptase (New England Biolabs, Ipswich, MA)), ProtoScript® reverse transcriptase (New England Biolabs, Ipswich, MA), an RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits RTx activity below 40°C (e.g., WarmStart® RTx reverse transcriptase (New England Biolabs, Ipswich, MA)), AMV reverse transcriptase, or combinations thereof. A method may comprise, in some embodiments, amplifying the cDNA to produce an amplified cDNA and, optionally, sequencing the cDNA or the amplified cDNA to produce one or more sequence reads. A method, in some embodiments, may comprise comparing a sequence read to a reference sequence (e.g., the nucleotide sequence of the ribonucleic acid or another comparator sequence) (x) to identify guanosines in the sequence read that correspond to adenosines in the reference sequence, and / or (y) to identify thymidines in the sequence read that correspond to cytosines in the reference sequence. For example, a method may comprise comparing a sequence read to a reference sequence (e.g., the nucleotide sequence of the ribonucleic acid or another comparator sequence) to identify adenosines and deaminated adenosines in the sequence read that correspond to adenosines in the reference sequence. For example, a method may comprise comparing a sequence read to a reference sequence (e.g., the nucleotide sequence of the ribonucleic acid or another comparator sequence) to identify cytosines and deaminated cytosines in the sequence read that correspond to cytosines in the reference sequence. A method optionally may include assigning a reactivity metric to each determined position of (i) the one or more deaminated adenosines and / or (ii) the one or more deaminated cytosines. An assigned reactivity metric may be combined with a predicted structure of the ribonucleic acid to produce a modified predicted structure of the ribonucleic acid, in some embodiments. Examples of a predicted structure may include a covariation analysis structure arising from a multiple sequence alignment of RNA homologs or related molecules, a computational structure arising from thermodynamic models, computational structures arising from machine learning and hybrid techniques, computational structures arising from deep learning methods, an RNA structure arising from RNA other biochemical structure probing techniques (e.g., dimethyl PATENT APPLICATION NEB-481-PCT suflate (DMS), selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagents, diethyl pyrocarbonate (DEPC), RNAse. Optionally, an assigned reactivity metric may be combined with a structure arising from biophysical experimental data including, for example, an NMR structure, a crystallographic structure, and / or a cryo-electron microscopy structure. The present disclosure also relates to systems and methods of predicting a structure (e.g., secondary structure) of an RNA molecule comprising at least one adenosine and / or at least one cytosine. In some embodiments, a method may comprise (a) determining at least one thermodynamic free energy of the RNA molecule based on the primary structure (nucleotide sequence) of the RNA molecule, (b) generating an initial set of potential secondary structures for the RNA molecule, wherein the initial set of potential secondary structures for the RNA molecule comprises a plurality of predicted candidate secondary structures the RNA molecule based on the primary structure and the determined thermodynamic free energy; (c) determining a deamination reactivity of at least one of the adenosines and / or at least one of the cytosines (e.g., by (I) contacting (x) a deaminase (e.g., an adenosine deaminase or a cytosine deaminase); and (y) the RNA molecule, wherein the RNA molecule comprises adenosine and cytosine, to produce a deamination product comprising one or more deaminated adenosines and / or one or more deaminated cytosines; and (II) determining the position in the nucleotide sequence of (x) the one or more deaminated adenosines and / or (y) the one or more deaminated cytosines); (d) correlating the deaminase reactivity of the RNA molecule with one or more of the candidate secondary structures from the initial set; and (e) ranking at least two (and optionally up to all) of the candidate structures on the basis of consistency with the determined deaminase reactivity. The present disclosure further relates to systems and methods of improving the output of RNA structure (e.g., secondary structure) prediction systems. For example, a method may comprise (a) identifying a set of RNA molecules, the set comprising a plurality of individual RNA molecules, each with different primary structures, and each comprising at least one adenosine and / or at least one cytosine; (b) generating a set of candidate secondary structures (optionally, tertiary structures in addition to or instead of secondary structures) for each RNA molecule of the set with a secondary structure prediction system (optionally, a tertiary structure prediction system in addition to or instead of a secondary structure prediction system) and inputs to the secondary structure prediction system; (c) determining for each RNA molecule within the set a deamination reactivity of at least one (and optionally up to all) of the adenosines PATENT APPLICATION NEB-481-PCT in the RNA molecule and / or at least one (and optionally up to all) of the cytosines in the RNA molecule (e.g., by (I) contacting (x) a deaminase (e.g., an adenosine deaminase or a cytosine deaminase); and (y) the RNA molecule to produce a deamination product comprising one or more deaminated adenosines and / or one or more deaminated cytosines; and (II) determining the position in the nucleotide sequence of (x) the one or more deaminated adenosines and / or (y) the one or more deaminated cytosines); (d) for each RNA molecule within the set, correlating the determined deaminase reactivity of the adenosine(s) and / or cytosine(s) of each RNA molecule with one or more of the candidate secondary structures from the initial set; (e) ranking at least two (and optionally up to all) of the candidate structures for each RNA molecule; and (f) predicting the most likely secondary structures (optionally, and / or tertiary structures) for each RNA molecule based on the correlation ranking, wherein the inputs comprise the primary structure of each RNA molecule, the deaminase reactivity, and (optionally) the calculated thermodynamic free energy of each RNA molecule and the secondary structure prediction system determines the thermodynamic free energy of each RNA molecule within the set based on its primary structure and utilizes the thermodynamic free energy and primary structure to generate the set of predicted candidate secondary structures for each RNA molecule; wherein the ranking further comprises ranking the at least two (and optionally up to all) of the candidate structures for each RNA molecule based on the correlation between secondary structure based on thermodynamic free energy and the deaminase reactivity. In some embodiments, generating candidate structures (e.g., secondary structures and / or tertiary structures) may include calculating a thermodynamic free energy for an RNA molecule (or a portion thereof) with or without accounting for deaminase reactivity of one or more (up to all) adenosines and / or cytosines in the RNA molecule or portion thereof. According to some embodiments, a method of interrogating RNA structure may include (a) contacting (i) a deaminase (e.g., an adenosine deaminase or a cytosine deaminase); and (ii) a nucleic acid (A) comprising one or more nitrogenous bases selected from adenine and cytosine, and (B) having a nucleotide sequence to produce a deamination product comprising one or more deaminated adenines and / or one or more deaminated cytosines; (optionally) (b) determining the position in the nucleotide sequence of (i) the one or more deaminated adenosines and / or (ii) the one or more deaminated cytosines; and (optionally) (c) determining the secondary structure context of one or more of the nitrogenous bases corresponding to the one or more deaminated adenines and / or the one or more deaminated cytosines; and (optionally) (d) (i) building a secondary structure and optionally tertiary structure model of of PATENT APPLICATION NEB-481-PCT the RNA using the generated secondary structure context data; or (ii) annotating or revising a pre-existing secondary and / or tertiary structure model of the RNA using the generated secondary structure context data; or (iii) annotating or revising a pre-existing secondary and / or tertiary structure model of the RNA using the generated secondary structure context data; or (iv) selecting the RNA for administration to a subject if the generated secondary structure meets a pre-determined criteria; or (v) selecting an interacting molecule and contacting the RNA with the interacting molecule to produce modify the structure of the RNA; or (vi) preparing a second RNA with a second nucleotide sequence, wherein differences between the nucleotide sequence and the second nucleotide sequence are selected in light of the generated secondary structure. BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 shows an example representation of RNA structure including primary sequence information (SEQ ID NO:4) and secondary structure information in the form of base-paired regions and single-stranded loop regions. Such two-dimensional representations of an RNA molecule may provide a simplified view, but such views may be oversimplified or errant where conditions in a solution or cell disrupt base pairing shown and / or allow additional interactions (e.g., base pairing, non-Watson-Crick base pairing, such as Hoogsteen base pairs, base-backbone interactions, backbone interactions, and / or pseudoknots). FIGURE 2 shows an example representation of RNA tertiary structure including three-dimensional spatial relationships of nucleotides. FIGURE 3 shows four categories of A’s based on their relative positions in predicted RNA local structure (SEQ ID NO:5). The regions marked with numbers in bold are as follows: L marks a loop; B marks a bulge; SS marks single-stranded RNA; and DS marks double-stranded RNA. FIGURE 4 shows example results of deamination with an adenosine deaminase in which deaminated A’s tended to locate near the stem at the 3’ side in the loop of a stem-loop structure (SEQ ID NO:6). FIGURE 5 shows an example representation of loop sizes of converted A’s versus non-converted A’s. FIGURE 6 shows example results of adenosine deaminase conversion of adenosine to inosine at 25°C and 42°C. FIGURE 7 shows example results of HPAMPD conversion of adenosine in AHP1 transcripts (both panels, SEQ ID NO:7) to inosine at 25°C and 42°C. PATENT APPLICATION NEB-481-PCT FIGURE 8 shows an example conversion of adenosine to inosine at genomic A positions across a 100 base pair region of the yeast RPS31 gene transcript catalyzed by AMP deaminase under each of the three indicated reaction conditions. FIGURE 9 shows example conversions of adenosine to inosine at genomic A positions across a 52 base pair region of the yeast AHP1 gene transcript catalyzed by AMP deaminase under each of the three indicated reaction conditions. FIGURE 10 shows example conversions of cytosine to uracil at genomic C positions across 52 base pair region of the yeast ERG3 gene transcript catalyzed by cytidine deaminase, RhDa01_extN10. FIGURE 11 shows example conversions of adenosine to inosine at genomic A positions that occurred on the RNA transcriptome across the 74 base pair region corresponding to yeast genomic positions of Chr XII:254478-254552 catalyzed by the adenosine deaminase TadA8.20 FIGURE 12 shows an example embodiment of a combination of methods for predicting, probing and experimentally determining the structure of RNA. FIGURE 13A illustrates secondary structure of E. coli 5S rRNA (SEQ ID NO:9) with example normalized reactivity of adenosines after HPAMPD deamination as described in EXAMPLE 10. The secondary structure of E. coli 5S RNA is depicted and the normalized reactivity of the adenosines after HPAMPD deamination is shown at adenosine (“A”) nucleotide positions that have low reactivity (white) , medium activity (gray) or high reactivity (black). FIGURE 13B illustrates secondary structure of E. coli 5S RNA with example reactivity of adenosines after dimethyl sulfate (DMS) from published data (P. Cordero, W. Kladwang, C. C. VanLang, R. Das, Biochemistry.51, 7037–7039 (2012)). FIGURE 14 illustrates an example arrangement of system 1400 that may be networked and may dynamically generate / predict structure of one or more RNA molecules. FIGURE 15A illustrates secondary structure of an in vitro transcribed RNA Turnip yellow mosaic virus tRNA-like structure (TYMV-TLS; SEQ ID NO:10) with select adenosines deaminated by HPAMPD. Deamination rates were potted on the secondary structure with positions above background annotated with a deamination % in box. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. PATENT APPLICATION NEB-481-PCT FIGURE 15B illustrates the secondary structure of an in vitro transcribed Zika virus xrRNA1 (Zkv-xrRNA; SEQ ID NO:11) with select adenosines deaminated by HPAMPD. Deamination rates were plotted on the secondary structure with positions above background (deamination rate > 0.1%) annotated with a deamination % in box. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. FIGURE 15C illustrates the secondary structure of an in vitro transcribed Adenovirus viral associated RNA1 (VA-I; SEQ ID NO:12) with select adenosines deaminated by HPAMPD. Deamination rates were plotted on the secondary structure with positions above background (deamination rate > 0.1%) annotated with a deamination % in box. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. FIGURE 15D Illustrates the secondary and tertiary structure of an in vitro transcribed RNAseP (SEQ ID NO:13) with select adenosines deaminated by HPAMPD. Deamination rates were plotted on the secondary structure with positions above background (deamination rate > 0.1%) annotated with a deamination % in box. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. FIGURE 16 Illustrates the secondary structure of an in vitro transcribed RNAseP (SEQ ID NO:13) with cytosine deaminase MsddA. Deamination rates were plotted on the secondary structure with positions above background (a deamination rate > 0.1%) annotated with a deamination % in box. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. FIGURE 17A illustrates example effects of magnesium on HPAMPD deamination and DMS reactivity across the RNAseP RNA (SEQ ID NO:13) mapped onto the proposed secondary structure under example reaction conditions. Regions of interest are highlighted in box 1 (with all panels showing nucleotides 102-113 of SEQ ID NO:13), box 2 (with all panels showing nucleotides 123-139 of SEQ ID NO:13), box 3 (with all panels showing nucleotides 222-232 of SEQ ID NO:13), and box 4 (with all panels showing nucleotides 79- 93 and 150-165 of SEQ ID NO:13). PATENT APPLICATION NEB-481-PCT FIGURE 17B illustrates data shown in FIGURE 17A as a histogram with the RNAseP RNA forming the x-axis in a 5’-3’ direction. Boxes 1-4 are indicated in the grey fields. Box 4 appears twice because the structure comprises two discontiguous portions of the linear sequence. FIGURE 18A illustrates an example deamination profile of TadA8r on RNAseP RNA transcribed in vitro that was deaminated in 0mM MgCl2. The secondary structure was depicted in a way to reflect the domain architecture present in the crystal structure PDB ID: 3dhs. Deamination rates were plotted on the secondary structure with positions above background (deamination rate > 0.1%) annotated with a deamination % in box. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. For this FIGURE 18A and similar figures disclosed herein, bases are shown in circles. The phosphate backbone, in places, is generally represented by a line that touches the linked bases (circles). In other places, the phosphate backbone is not specifically shown and the bases (circles) simply appear in close proximity to adjacent bases. Generally, lines that do not touch either base (circle) represent base pairing (Watson-Crick or otherwise). Each of these figures may be viewed in light of the sequence listing, which clearly sest forth the 5’-3’ arrangement of the nucleotides. FIGURE 18B illustrates an example deamination profile of TadA8r on RNAseP RNA transcribed in vitro that was deaminated in 5mM MgCl2. Deamination rates were plotted on the secondary structure with positions above background (deamination rate > 0.1%) annotated with a deamination % in box. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. FIGURE 19A is an example secondary structure rendering of non-Watson-Crick pairing present in RNAseP (represented with small circles positioned between bases). FIGURE 19B is an enlargement of the portion of the molecule shown in FIGURE 19A. This helix contains three non-Watson-Crick base pairs involving 215A, 245A, and 246A. The three A positions are highly reactive to DMS (right diagonal lines) but not reactive to HPAMPD (white). A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. PATENT APPLICATION NEB-481-PCT FIGURE 19C is an example rendering of non-Watson-Crick pairing of the same portion of RNase P shown in FIGURE 19B visualized in the three-dimensional structure of RNAse P (PDBID: 3DHS). FIGURE 19D is an example secondary structure rendering of non-Watson-Crick pairing present in E.coli 5s rRNA E-loop. FIGURE 19E is an enlargement of the portion of the molecule shown in FIGURE 19D. Within this region there are five A’s that are participating in non-Watson-Crick pairing interactions (89A,94A,115A,117A, and 120A) that have medium to high reactivity to DMS, and are not reactive to HPAMPD. A coloring scheme of adenosines is provided reflecting background (white), low (grey circle with black text), medium (dark grey circle with black text), and high (black circle with white text) percentages of deamination. FIGURE 19F is an example rendering of non-Watson-Crick pairing of the same portion of E.coli 5s rRNA E-loop shown in FIGURE 19E visualized in the three-dimensional structure of E.coli 5s rRNA E-loop (PDBID: 1A4D). FIGURE 20A illustrates an example method of assessing archaeal endonuclease V cleavage activity on various RNA substrates comprising inosine. FIGURE 20B illustrates capillary electrophoresis traces from example kinetic analysis of archaeal endonuclease V cleaving ssRNA, dsRNA, and RNA:DNA hybrid constructs containing a site specific inosine. FIGURE 20C provides illustratative kinetic graphs of an archaeal endonuclease V cleaving ssRNA, dsRNA and RNA:DNA hybrid constructs with a site-specific inosine. Data were fit to a single exponential equation ((y = m1*(1-exp(-m2*m0)) where m2 equals rate of cleavage (displayed in TABLE 4). FIGURE 21A illustrates an embodiment of a method to site-specifically detect adenosine to inosine deamination. As shown, contacting a substrate RNA (left) comprising an adenosine and an adenosine deaminase may form a deaminated RNA comprising an inosine (middle left). As further shown in FIGURE 21A, a method may also include contacting a deaminated RNA and an archaeal endonuclease V to produce a cleavage product (middle right). A cleavage product may be analyzed, for example, to assess what positions of the original substrate RNA are adenosines and which, if any, were converted to inosine. FIGURE 21B illustrates idealized results expected from analysis of an RNA with adenosine deaminase & archaeal Endonuclease V using UHPLC-MS / MS according to a method shown in FIGURE 21A, where the RNA comprises a single adenosine as shown. PATENT APPLICATION NEB-481-PCT FIGURE 22A illustrates an example of archaeal Endonuclease V cleavage of the Hepatitis C virus internal ribosomal entry site (HCV IRES) in vitro transcribed RNA (SEQ ID NO:19). Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis using a 6% TBE- Urea polyacrylamide gel visualized with SYBR Gold on a UV imager. FIGURE 22B illustrates the position of the highly deaminated A’s at position 72A and 329A. These highly reactive positions are depicted with solid black circles and white text in the context of the Hepatitis C virus internal ribosomal entry site (HCV IRES) in vitro transcribed RNA proposed secondary structure. Position 72A was deaminated to 20.5% and position 329A was deaminated to 51.0% by HPAMPD respectively. FIGURE 23 shows an example screening method for identification and / or purification of an RNA of interest that is included in an RNA pool. As illustrated, a method may comprise contacting the RNA pool with a 3’ hydroxyl blocking reagent to form a 3’ blocked RNA pool, contacting the 3’ blocked RNA pool with an endonuclease V, the guide having a sequence complementary to at least a portion (e.g., a unique portion) of the RNA of interest to form a cleaved RNA of interest comprising a 3’-OH, contacting the cleaved RNA of interest with a 3’-OH labelling agent (here, biotin-ATP and poly(A) polymerase) to form a labeled RNA of interest, and enriching (here, with streptavidin) and / or detecting the labeled RNA of interest. FIGURE 24 illustrates an example of HPAMPD deamination to detect differences in a dynamic RNA, thiamine pyrophosphate-specific riboswitch (TPP Riboswitch; SEQ ID NO:20), in the presence or absence of its ligand thiamine pyrophosphate (TPP). Three positions 40A, 75A, and 85A annotated by a black circle with white text decreased in reactivity between absence and presence of TPP. BRIEF DESCRIPTION OF THE SEQUENCES Some embodiments of this disclosure relate to the following provided sequences of example polynucleotides and / or example polypeptides. SEQ ID NO:1 is an example RNA deaminase, Helix pomatia AMP deaminase (HPAMPD) of which aa1-27 may be signal sequence and residues H121, H123, D124, W212, F215, D216, F219, A301, V333, A334, H366, E369, H394, L425, D451, and D452 may define and / or be included in the catalytic site. MSQVGAVTMVSIICVVVLGAVGGPVAGLAVRFPTMDEYTNAREELIGSEQYLRVGGSINLNNK EKKLNQFILREKRAIIENSRLNKTQYIPAVSFFLSKSQMESTPIFKIIKDMPKGAALHLHDTA SARIDWIVSNATYRDHVYMCMDQDNFVRLTVSGTGPPANSGCEWKLVETERANSGDIAAFDHW PATENT APPLICATION NEB-481-PCT LKSNISLLTTDPLVTYPSLDKVWGRFDKHFSQLRGIIYHTPIRRDYYRQILEEFRSDNVQYVE VRSSLSGYYDLDGTVHDPEYGLQLYKAVTEEFVRTYPDFSGAKIIKSTARVKPNTDIFNDVKL SMDLYKRYPGFFLGFDLVAQEDPNTSLLGYIDSLLYPSRQNPPVSLPYYFHAGETNWQGTEVD YNLVDALLLNATRIGHGFALIKHPRVIELVKSRGVAVEVNPVSNQLLGLVKDLRNHAAAPLLA QNVPVVISSDDPGVWEALPMSHDMYVAFMDLVGEDAGLDVLKQLVWNSIQYSSMNATEKKTAL KLLQAKWNNFINDSLIKWKLTNKKVIG SEQ ID NO:2 is an example RNA deaminase, a Clostridium hungatei deaminase designated RhDa01_extN10. MKPLEGPVNSVGGMIEQVTDQATGVVDNAEQSAMSSMGNGSKLLSGFAGSSGSQNRDAGKAGE SKGQEKNSGKSGQEGGKSQGGGDFLGLLKSGVHTRLMTFGLMNIKKLGAKVIAAGAAKVTGVI KKMLTPKVKFRLGAEEHELWVEKGKNGNVVMMASKKPGPIKRKIEEGEIPDNGEISNKRRKVE AEKDEQQVVQQNEAMASTIQAVTAGVGEAGKKEPWEAIDRFRKSNGLEPLGDRIPVRGDGLET VALMEVSGNKVFGVNSSLLSDELKNLGRDFFKVIKEKGLLGNAKHYGSGEAQVLTHAEAHALM KARKEAGGHLGDSVVLYVDRLTCPNCQKYLPEVRAAMGIKTLKVITKGGIELIL SEQ ID NO:3 is an example RNA deaminase, a synthetic deaminase designated TadA8.20. SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMN HRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD SEQ ID NO:4 is an example RNA sequence, which may hybridize to itself to form base-paired stems and single-stranded loops as shown in FIGURE 1. GGAGGGUUGGCCGAGUGGUCUAAGGCGGCAGACUUAAGAUCUGUUGGACGGUUGUCCGCGCGA GUUCGAACCUCGCAUCCUUCA SEQ ID NO:5 is an example RNA sequence, which may hybridize to itself to form base-paired stems and single-stranded loops as shown in FIGURE 3. AGGUCGGGCCGGCGAAAGUCGCCACAGUUUGGGGAAAGCUGUGCAGCCCGUAACCCCCCCACG AAAGUGGG SEQ ID NO:6 is an example RNA sequence, which may hybridize to itself to form a base-paired stem (nt3-10 and 17-24) and a single-stranded loop as shown in FIGURE 4. ACGUGCCACGAUUCAACGUGGCACAG SEQ ID NO:7 is an example RNA sequence read from DNA amplified from RNA specimens contacted with a deaminase as shown in FIGURE 7 (left and right panel). GUGCUUUCAC CAAAUCCAUU GGUUUCGAAU UAGCCGUCGG UGACGGUGUU UACUGGAGUG GUAGAUG SEQ ID NO:8 is an example RNA as shown in FIGURE 12 (left and right panels). GGAGGGUUGG CCGAGUGGUC UAAGGCGGCA GACUUAAGAU CUGUUGGACG GUUGUCCGCG CGAGUUCGAA CCUCGCAUCC UUCA PATENT APPLICATION NEB-481-PCT SEQ ID NO:9 is an example E. coli 5S rRNA as shown in FIGURE 13A and FIGURE 13B. GGGGCCTTCGGGCCAATGCCTGGCGGCCGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAAC TCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAAC TGCCAGGCATTCGATCAGGTTCGCCTGATCCAAATCGGGCTTCGGTCCGGTTCG SEQ ID NO:10 is an example in vitro transcribed RNA Turnip yellow mosaic virus tRNA-like structure as shown in FIGURE 15A. GGUAAGUUCUCGAUCUUUAAAAUCGUUAGCUCGCCAGUUAGCGAGGUCUGCGAAAGCAGAUAA UCGGGUGCAACUCCCGCCCUUUCUCCGAGGGUCAUCGGAAC SEQ ID NO:11 is an example in vitro transcribed Zika virus xrRNA1 as shown in FIGURE 15B. GGGGCCUUCGGGCCAAGGGUCAGGCCGGCGAAAGUCGCCACAGUUUGGGGAAAGCUGUGCAGC CUGUAACCCCCCCACGAAAGUGGGUCGAUCAGGUUCGCCUGAUCCAAAUCGGGCUUCGGUCCG GUUCGG SEQ ID NO:12 is an example in vitro transcribed Adenovirus viral associated RNA1 as shown in FIGURE 15C. GGACCUCGCAAGGGUAUCAUGGCGGACGACCGGGGUUCGAACCCCGGAUCCGGCCGUCCGCCG UGAUCCAUGCGGUUACCGCCCGCGUGUCGAACCCAGGUGUGCGAGGUCC SEQ ID NO:13 is an example in vitro transcribed RNAseP as shown in FIGURE 15D. GGCCTTCGGGCCAAGTTAATCATGCTCGGGTAATCGCTGCGGCCGGTTTCGGCCGTAGAGGAA AGTCCATGCTCGCACGGTGCTGAGATGCCCGTAGTGTTCGTGGAAACACGAGCGAGAAACCCA AATGATGGTAGGGGCACCTTCCCGAAGGAAATGAACGGAGGGAAGGACAGGCGGCGCATGCAG CCTGTAGATAGATGATTACCGCCGGAGTACGAGGCGCAAAGCCGCTTGCAGTACGAAGGTACA GAACATGGCTTATAGAGCATGATTAACGTCTCGATCCGGTTCGCCGGATCCAAATCGGGCTTC GGTCCGGTTC SEQ ID NO:14 is an example endonuclease V, namely a wild type Thermococcus kodakarensis endonuclease V. MSEGLFKKLEEVQRKLAERIVERPLEVSKIKTVGAVDVSYRDERARAAFVLCSFPDCELLKQR VVEVDVSFPYIPTFFFLRETRPVLIALGKERPDVLLVEGHGRAHPRGYGLASHIGLVLGIPTI GISKRLLRGTPEGSWVKVGKAYVSVGHLIDLPSAVEVVKTLNKNGYPLPLRIADRLSRGHTSW SGGVNDEH SEQ ID NO:15 is an example FAM-labeled ssRNA with a single central inosine further described in EXAMPLE 16 (FAM at nt1, inosine at nt30). FAM- rCrArCrArArCrArArCrArArCrArArCrArArCrCrGrUrArGrArGrCrUrArCrIrGr ArUrCrGrGrUrCrArCrCrGrCrArArCrArArCrA SEQ ID NO:16 is an example dsDNA complement further described in EXAMPLE 16. PATENT APPLICATION NEB-481-PCT TGTTGTTGCGGTGACCGATCCGTAGCTCTACGGTTGTTGTTGTTGTTGTG SEQ ID NO:17 is an example dsRNA complement further described in EXAMPLE 16. rUrGrUrUrGrUrUrGrCrGrGrUrGrArCrCrGrArUrCrCrGrUrArGrCrUrCrUrArCr GrGrUrUrGrUrUrGrUrUrGrUrUrGrUrUrGrUrG SEQ ID NO:18 is an example 32mer ssRNA with the only A at position 16. This RNA is further described in EXAMPLE 17. rCrUrUrCrUrUrCrUrUrCrUrUrCrUrUrArCrUrUrCrUrUrCrUrUrCrUrUrCrUrUr C SEQ ID NO:19 is an example in vitro transcribed hepatitis C virus internal ribosomal entry site (HCV IRES). GGGGCCUUCGGGCCAACCAUGAAUCACUCCCCUGUGAGGAACUACUGUCUUCACGCAGAAAGC GUCUAGCCAUGGCGUUAGUAUGAGUGUCGUGCAGCCUCCAGGACCCCCCCUCCCGGGAGAGCC AUAGUGGUCUGCGGAACCGGUGAGUACACCGGAAUUGCCAGGACGACCGGGUCCUUUCUUGGA UAAACCCGCUCAAUGCCUGGAGAUUUGGGCGUGCCCCCGCAAGACUGCUAGCCGAGUAGUGUU GGGUCGCGAAAGGCCUUGUGGUACUGCCUGAUAGGGUGCUUGCGAGUGCCCCGGGAGGUCUCG UAGACCGUGCACCAUGAGCACGAAUCCUAAACCUCAAUCGAUCAGGUUCGCCUGAUCCAAAUC GGGCUUCGGUCCGGUUCG SEQ ID NO:20 is an example in vitro transcribed thiamine pyrophosphate-specific riboswitch (TPP Riboswitch). GGCCTTCGGGCCAAGGACTCGGGGTGCCCTTCTGCGTGAAGGCTGAGAAATACCCGTATCACC TGATCTGGATAATGCCAGCGTAGGGAAGTTCTCGATCAGGTTCGCCTGATCCAAATCGGGCTT CGGTCCGGTTCG SEQ ID NO:21 is a FAM-labeled ssDNA with a single central deoxyinosine further described in EXAMPLE 16 (FAM at nt1, deoxyinosine at nt30). FAM-CACAACAACAACAACAACCGTAGAGCTACdIGATCGGTCACCGCAACAACA DETAILED DESCRIPTION The present disclosure relates to probing, determining, and / or predicting RNA structures. Obtained RNA structure data (e.g., by enzymatic probing) may be compared with and / or integrated into predicted and biophysically derived RNA structure. Structure probing may be used to inform machine learning algorithms for predicting RNA structure. The structure of an RNA molecule may influence its interaction with other molecules and / or its utility and function in vivo and / or in vitro. There is a strong correlation between a given RNA’s structure and function, and understanding an RNA’s structure may provide invaluable information especially in the context of therapeutic design (Cao, Xinang, et al. PATENT APPLICATION NEB-481-PCT "Identification of RNA structures and their roles in RNA functions." Nature Reviews Molecular Cell Biology (2024): 1-18.). The secondary and tertiary structure of a regulatory RNA molecule, for example, may inform a chemist on a design of a small molecule drug that could interact specifically with that RNA and thereby influence the RNA’s regulatory activity, controlling the accessibility of genetic information. Analogously disease related RNA molecules could be targets for small molecule drugs. The structure (primary, secondary, and / or tertiary) of an RNA molecule may impact its function in vivo and / or in vitro. For example, RNA structure may impact how an RNA (e.g., a coding RNA, a non-coding RNA, a synthetic RNA) interacts with cellular machinery, proteins, enzymes, and a host of other molecules. The efficacy, ease of manufacture, and / or other properties of a therapeutic RNA, for example, may be impacted by its structure. Accordingly, it may be desirable to produce RNAs having a preselected structure. The structure of synthetic therapeutic RNA molecules may be influenced by codon usage. For example, a therapeutic RNA having a first sequence encoding a protein may be observed to have a structure that is undesirable for some reason (e.g., is less readily taken up by cells, is more immunostimulatory). The sequence of the therapeutic RNA may be changed to improve or even optimize certain structural elements of the RNA. These elements can be predicted from the first sequence and variants may be created by altering that first sequence to effect one or more of these structural elements, such as replacing a base that participates base pairing for double-stranded regions to disfavor that base pairing. The multiple codons which encode the same amino acid allow for these changes of RNA sequence while not affecting the amino acid sequence of the protein which is encoded by the RNA. The obtained codon usage variants and the wild type mRNA may be compared to one another by probing. After probing results are examined the variant which most closely aligns with the expected (more desirable) RNA structure may be identified. Improving or optimizing structure of a coding RNA (e.g., a therapeutic RNA) can be extended further than the degeneracy of the genetic code. For example, if a region of a coding RNA has an important role in its structure and also encodes a portion of the amino acid sequence that tolerates amino acid substitutions (e.g., conservative substitutions), an even broader range of codons may be selected for their desirable impact on RNA structure than is available from degeneracy of the genetic code alone. These additional codon selections would be available without impacting function of the encoded protein. Even more flexibility may be possible in non-coding RNAs where the constraints of codon selection would not apply. PATENT APPLICATION NEB-481-PCT Replacing, adding, and / or removing bases may be possible with coding RNAs under some conditions, but would be more broadly available for non-coding sequences to permit RNA structure to be fine-tuned. RNA viruses having unique RNA tertiary structures may be targeted by antivirals, wherein the antivirals may be designed or selected for their ability to interact with the unique RNA tertiary structure. When modifying RNA, for example by treatment with enzymes in vitro, knowledge of the structure of the RNA to be modified before, during, and / or after modification may be desirable, beneficial or even required for evaluating and characterizing properties of the RNA modifying enzymes. The present disclosure relates, in some embodiments, biochemical probes for RNA structural analyses. Structural probes include, for example, nucleotide modifying enzymes that modify bases in RNA. Probing RNA structure with base modifying enzymes (e.g., AMP, adenosine, and cytosine deaminases) may be employed to determine which bases are sufficiently accessible to be enzymatically modified. RNA structure may be revealed by contacting an RNA molecule of interest with a structural probe and identifying modifications of the RNA molecule of interest. A resulting modified base would indicate that position is accessible to the specific deaminase used. For example, bases which are more susceptible to modification may be in surface and / or flexible regions of the molecule while less susceptible bases may be oriented toward the interior and / or less flexible regions of the molecule. RNA structure is typically studied using biophysical, biochemical, computational methods, and combined approaches. Biophysical methods including x-ray crystallography, NMR, cryo-electron microscopy, and small angle x-ray scattering (SAXS) may be used to study RNA structure and / or provide atomistic information of a given RNA molecule. However, these methods are time consuming, low throughput, require specialized instrumentation, and may produce artifacts. Additionally, some of these methods cannot be used unless the target RNA has certain prerequisite properties, such as the ability to be crystallized. Dynamic RNA molecules, which may adopt multiple conformations, or those that change conformation in a response to a drug, may not be compatible with some of these biophysical methods. RNA structure may be predicted computationally using a single sequence or multiple sequence alignments, in each case coupled with thermodynamic, machine learning, or deep learning methods. However, such predictions may be and often are inaccurate, especially for longer RNA species. More accurate structural models may be achievable with a combination PATENT APPLICATION NEB-481-PCT of biophysical, biochemical, and computational methods. Biochemical structure probing may be used to inform computational methods improving the accuracy of the structural predictions. Biochemical methods include small molecule or nuclease probing (footprinting) which provide per-nucleotide information identifying paired vs non-paired regions. Although these methods provide lower resolution information compared to biophysical methods, there are less experimental constraints and may be used to investigate dynamic RNA molecules. Detection of these modifications / cleavages and downstream analysis of the sequencing results are challenging. While RNA structure probing improves the accuracy of structural predictions, current methods require the use of highly reactive chemicals with short half-lives in solution. Furthermore, the analysis of the effect of the chemical probes requires the copying of the RNA strand with a reverse transcriptase into a cDNA. The cDNA copy should record the effect of the probing, however the resulting chemical probe modifications of the RNA have inconsistent and unpredictable effects on the reverse transcriptase as to which nucleotide is incorporated opposite the modification or if a deletion occurs or if the cDNA is truncated at that position. Therefore interpretation of the resulting changes in the cDNA sequence and its correspondence to which base in RNA was modified and at what frequency is challenging. Probing the structure of an RNA of interest with a small molecule and / or enzymatic probe may include modifying the RNA of interest and then detecting the modification. Small molecule based probing methods may be nucleotide specific or general and provide a means for interrogating aspects of structure such as accessibility of a Watson-crick face accessibility or nucleotide. For example, probing RNA dimethyl sulfate (DMS) provides information about the Watson-Crick face of A’s and C’s or under certain conditions all bases. As another example, selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) may be used to modify the free 2’ hydroxyl on the ribose backbone, providing information about the local flexibility of a given nucleotide ribose. In addition to small molecules, nucleases have been used to identify regions accessible to a given nuclease. For example, RNAse V1 has been used to identify regions of double stranded RNA while mung bean nuclease prefers single stranded regions. These nucleases provide valuable information, but detection of these cleavage events is challenging with many experimental caveats including multiple cleavage events and library prep with cleaved RNA. In some embodiments, biochemical structural probing (e.g., using one or more deaminases) may be used to inform computational folding algorithms, thereby improving their PATENT APPLICATION NEB-481-PCT predictive power. Deaminases are a class of enzymes that catalyze removal of an amino group from a nitrogenous base of a given nucleotide. Adenosine deaminases catalyze the conversion of adenosine to inosine while cytosine deaminases catalyze the conversion of cytidine to uracil, in each case changing the identity of the modified base without cleaving the phosphate backbone. These deamination events are easily read through reverse transcription coupled with next generation sequencing followed by mutation frequency calculation at a given position, reducing the complexity of downstream analysis. Each deaminase may have and often does have specificity for removing an amino group from a specific nucleobase. Adenosine deaminases acting on RNA (sometimes referred to as ADARs and ADATs) may act on a region of a polymeric RNA having certain required structural and / or sequence properties. Some deaminases do not interact with polymeric RNA. For example, Helix pomatia AMP deaminase (HPAMPD) is secreted by Helix pomatia cells where the enzyme converts extracellular AMP to IMP, such that, in vivo, it appears to have no role or need to bind to polymeric RNA. An RNA comprising adenosines contacted in vitro with HPAMPD may undergo deamination at only those adenosines, which by consequence of the structure of the RNA, are accessible to the HPAMPD catalytic site. The results of probing RNA with the HPAMPD support predicted structures where the adenosines that are susceptible to deamination by HPAMPD are flexible and not stabilized by hydrogen bonding networks. Without limiting any embodiment to any specific mechanism of action, when the base of an adenosine (i.e., adenine) or cytosine is Watson-Crick base paired or for some other reason the base is sequestered within the three-dimensional structure of RNA (e.g., by non-canonical base pairing / hydrogen bonding), it is inaccessible to deaminase (e.g., HPAMPD or MsddA), accordingly, will undergo little or no deamination. MsddA is a cytosine deaminase. Differing reactivity profiles between cytosine and adenosine deaminases and even differences among cytosine deaminases and among adenosine deaminases may be leveraged to get considerable information about the structure of a subject RNA. Multiple deaminases may be used individually, combined, or added sequentially to a deaminase probing reaction. Deaminases may be used before or after treatment with small molecule chemical probes. Deaminase probing may be used to assess the validity of RNA structures predicted by other means and / or may inform computational prediction of RNA structures, much as the chemical probing and nuclease probing have to date contributed to such computational methods. Deaminase probing may also support determining whether a given sequence modification (e.g., substitution, insertion, and / or deletion) is associated with a change in the PATENT APPLICATION NEB-481-PCT structure of the subject RNA by revealing differential deamination of the nucleosides in the vicinity of the modification or elsewhere in the molecule. Furthermore, where the activity of a deaminase on 5' AMP is not markedly affected by reaction conditions (e.g., presence of magnesium ions or pH or ionic strength) the differences in the conversion of adenosine in RNA to inosine may be a reflection (e.g., direct reflection) the impact of such conditions on RNA structure. RNA structures arising from X-ray, cryoEM, and / or NMR experimental analyses may be used to validate and define the three dimensional context for the accessibility of bases to a deaminase. Relative accessibility of each nucleoside position to deaminases may provide data-rich input for computational predictions of RNA structure and a supplemental method to query RNA structure, which may be used, in turn, to create higher confidence structure prediction algorithms. Accessibility information, according to some embodiments, may be used to support screening for reagents and / or conditions that have some effect of interest (or no effect) on the structure of a given RNA. In some embodiments, accessibility information may be used to support diagnostic methods, for example, where a change in accessibility is associated with health condition in a subject or an environmental condition. General Considerations Aspects of the present disclosure can be understood in light of the provided descriptions, figures, sequences, embodiments, section headings, and examples, none of which should be construed as limiting the entire scope of the present disclosure in any way. Accordingly, the innovations set forth herein should be construed in view of the full breadth and spirit of the disclosure. Each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the components and / or features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Lists of example species within a particular genus may vary in length at different places throughout the disclosure. Species lists shortened for convenience shall not be construed to exclude example species listed elsewhere in the specification. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. Unless otherwise expressly stated to be required herein, each component, feature, and method step disclosed herein is optional and the disclosure contemplates embodiments in which each optional element may be expressly excluded. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements PATENT APPLICATION NEB-481-PCT or use of a “negative” limitation. It is further intended to serve as antecedent basis for use of such elective terminology as “optionally” and the like in connection with the recitation of one or more claim elements. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Still, certain terms are defined herein with respect to embodiments of the disclosure and for the sake of clarity and ease of reference. Provided genomic positions in the yeast genome reference the yeast genome as disclosed in the NCBI database for Saccharomyces cerevisiae S288C (assembly R64). Sources of commonly understood terms and symbols may include: standard treatises and texts such as Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton, et al., Dictionary of Microbiology and Molecular biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, the Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) and the like. As used herein and in the appended claims, the singular forms “a” and “an” include plural referents unless the context clearly dictates otherwise. For example, the term “a protein” refers to one or more proteins, i.e., a single protein and multiple proteins. Numeric ranges are inclusive of the numbers defining the range. All numbers should be understood to encompass the midpoint of the integer above and below the integer i.e., the number 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55 etc. When sample numerical values are provided, each alone may represent an intermediate value in a range of values and together may represent the extremes of a range unless specified. Percent ranges with only one end point (e.g., ≥ 90% or ≤ 10%) optionally include a second endpoint at the maximum or minimum percentage (e.g., ≥ 90% includes a range of 90%-100% and ≤ 10% includes a range of 0%-10%). Ranges (including percent ranges) with only one end point (e.g., ≥ 90 or ≤ 10) optionally include a second endpoint 10% higher or 10% lower than the provided endpoint (e.g., ≥ 90 includes a range of 90-99 and ≤ 10 includes a range of 1-10). Concentration percentages are w / v percentages unless otherwise indicated. PATENT APPLICATION NEB-481-PCT The nomenclature of nucleobases, nucleosides, and nucleotides is well established. The present disclosure contemplates deamination in any context including deamination of nucleobases alone as well as deamination of nucleobases included in nucleosides, nucleotides, and polynucleotides. For example, identification of a specific deaminated nucleobase by name is illustrative of and includes corresponding deaminated nucleosides and deaminated nucleotides, for example, in a polynucleotide. Likewise, identification of a specific deaminated nucleoside by name is illustrative of and includes corresponding deaminated nucleobases and deaminated nucleotides, for example, in a polynucleotide. In addition, references to modified nucleobases include corresponding nucleosides, nucleotides, and polynucleotides comprising such modified nucleobases. In the context of the present disclosure, “adenosine deaminase” refers to enzymes that deaminate adenosine to inosine and deaminate deoxyadenosine to deoxyinosine (e.g., in RNA or DNA comprising adenosine and / or deoxyadenosine). Adenosine deaminases include adenine deaminases, AMP deaminases, TadAs, and adenosine deaminases that act on RNA (ADARs). In the context of the present disclosure, “AMP deaminase” refers to enzymes that convert adenosine 5’ monophosphate (AMP) to inosine 5’ monophosphate and ammonia. AMP deaminases may be found in organisms of the animal kingdom and fungi. Examples of AMP deaminases include Helix pomatia AMP deaminase (HPAMPD), Aspergillus melleu AMP deaminase, and Porphyra yezoensis Ueda AMP deaminase. HPAMPD is found in the foot muscle tissue of the snail and converts AMP to IMP. It also converts 5' ADP, 5' ATP, and NADH to their respective inosine derivatives. Wild type HPAMPD comprises an amino acid sequence according to SEQ ID NO:1. HPAMPD may also convert AppppA to IppppI. While these activities do not suggest that it converts adenosine in RNA to inosine, the present disclosure provides evidence that it does catalyze this conversion. In the context of the present disclosure, “buffer” and “buffering agent” refer to a chemical entity or composition that itself resists and, when present in a solution, allows such solution to resist changes in pH when such solution is contacted with a chemical entity or composition having a higher or lower pH (e.g., an acid or alkali). Examples of suitable non- naturally occurring buffering agents that may be used in disclosed compositions, kits, and methods include HEPES, MES, MOPS, TAPS, tricine, and Tris. Additional examples of suitable buffering agents that may be used in disclosed compositions, kits, and methods include ACES, ADA, BES, Bicine, CAPS, carbonic acid / bicarbonic acid, CHES, citric acid, DIPSO, PATENT APPLICATION NEB-481-PCT EPPS, histidine, MOPSO, phosphoric acid, PIPES, POPSO, TAPS, TAPSO, cacodylate (e.g., sodium cacodylate), and triethanolamine. In the context of the present disclosure, “contacting” refers to any act of bringing one item (e.g., a molecule or group of molecules) in contact with one or more other items (e.g., a molecule or group of molecules, like or unlike the first molecule(s)). Contacting includes, for example, adding, amalgamating, blending, combining, connecting, emulsifying, joining, mixing, precipitating, stirring, and / or touching one item with one or more other items. In the context of the present disclosure, “container” refers to a human-made container. A container may comprise one or more walls (e.g., defining an interior volume) and optionally one or more openings. Containers comprising one or more openings may further comprise one or more closures (e.g., a removable closures) for some or all such openings. A closure optionally may comprise an aperture or a septum, for example, to provide fluid communication with a volume of the container and a connected or inserted tube or syringe. Examples of containers include boxes, cartons, bottles, tubes (e.g., test tubes, microcentrifuge tubes), plates (e.g., 96-well, 384-well plates), vials, pipette tips, and ampules. Containers and / or closures may comprise any desired material including paper, plastics, glass, silicone, composites, metals, alloys, or combinations thereof. Containers and / or closures may comprise materials that are compostable, recyclable, and / or sustainable. In the context of the present disclosure and with respect to an amino acid residue or a nucleotide base position, “corresponding to” refers to positions that lie across from one another when sequences are aligned, e.g., by the BLAST algorithm. An amino acid position in a functional or structural motif in one polymerase may correspond to a position within a functionally equivalent functional or structural motif in another polymerase. In the context of the present disclosure, “cytosine deaminase” refers to enzymes that deaminate cytosine to uracil and enzymes that deaminate cytidine to uridine (the latter of which may be referred to as cytidine deaminases where a distinction is intended), for example, where such cytosine or cytidine is present in RNA or DNA. Cytosine deaminases include members of the apolipoprotein B mRNA editing enzyme complex (APOBEC) protein family (e.g., APOBEC1, APOBEC3A, and APOBEC3G), and activation-induced cytidine deaminase (AID). Further examples of cytosine deaminases are provided in International PCT Application No. PCT / US2022 / 080345 (Publication No. WO2023097226) and International PCT Application No. PCT / US2023 / 067416, each of which is incorporated herein in its entirety, by this reference. PATENT APPLICATION NEB-481-PCT In the context of the present disclosure, “deaminase” refers to an enzyme (e.g., a hydrolase) that catalyzes removal of an amino group from a nitrogenous base (e.g., adenine, cytosine, and modified versions thereof). Nitrogenous bases to be deaminated may be included in a nucleoside, a nucleotide, or polynucleotide (e.g., RNA and / or DNA). Examples of deaminases include RNA deaminases, adenosine deaminases and cytosine deaminases. Deaminases may be active on bases in single-stranded and / or duplex polynucleotides. Catalytic activity of a deaminase may be limited, in some embodiments, to bases that are on the surface of a folded polynucleotide molecule (e.g., RNA). A deaminase may lack the capacity to alter (e.g., denature, unwind, melt) the primary, secondary, and tertiary structure of a folded polynucleotide molecule (e.g., RNA) other than to deaminate surface bases (e.g., bases in contact with solvent). Examples of deaminases include TadA, AMP deaminases (e.g., HPAMPD), RhDa01_extN10, and TadA8.0. In the context of the present disclosure, “deaminase substrate” refers to any nucleobase comprising a cleavable amino group (e.g., —NH2) and includes any nucleoside, nucleotide, and polynucleotide (e.g., an RNA) molecule comprising one or more such nucleobases. Deaminase substrates include molecules that may have or adopt a conformation that makes inaccessible one or more otherwise cleavable amino groups from a deaminase that is otherwise capable of cleaving such amino group. A deaminase substrate may be exclusively double- stranded, partially double-stranded and partially single-stranded, or exclusively single- stranded. A deaminase substrate may comprise one or more cytosines, one or more modified cytosines, one or more adenines, one or more modified adenines, or combinations thereof. A deaminase substrate may comprise one or more polynucleotide strands (e.g., RNA strands). A deaminase substrate may comprise duplex portions and single-stranded portions and / or may comprise interior-facing bases and exterior-facing bases, wherein interior-facing bases have little or no contact with solvent and little or no ability to interact with molecules other than the polynucleotide of which it is a part, and wherein exterior-facing bases may contact solvent and may interact with other molecules (e.g., deaminases). A deaminase substrate may have any desired size. For example, a deaminase substrate may comprise or have a size of 1-25 nucleotides, ≥25 nucleotides, ≥50 nucleotides, ≥75 nucleotides, ≥100 nucleotides, ≥250 nucleotides, ≥500 nucleotides, ≥1,000 nucleotides, ≥2,000 nucleotides, ≥5,000 nucleotides, ≥10,000 nucleotides, ≥25,000 nucleotides, ≥50,000 nucleotides, ≥75,000 nucleotides, ≤100,000 nucleotides, ≥100,000 nucleotides, 25-1,000 nucleotides, 500-5,000 nucleotides, PATENT APPLICATION NEB-481-PCT 1,000-10,000 nucleotides, 1,000-100,000 nucleotides, 1,000-50,000 nucleotides, 10,000- 75,000 nucleotides, or 25,000-100,000 nucleotides. In the context of the present disclosure, “duplex” and “double stranded” refer to any conformation of a polynucleotide in which two polynucleotide strands (e.g., separate molecules or spatially separated portions of a single molecule) are arranged anti parallel to one another in a helix with complementary bases of each strand paired with one another (e.g., in Watson-Crick base pairs). Paired bases may be stacked relative to one another to permit pi electrons of the bases to be shared. Duplex stability, in part, may be related to the ratio of complementary bases to mismatches (if any) in the two strands, ratio of pairs with three hydrogen bonds (e.g., G:C) to pairs with two hydrogen bonds (e.g., A:T, A:U) in the duplex, and the length of the strands with higher ratios and longer strands generally associated with higher stability. Duplex stability, in part, may be related to ambient conditions including, for example, temperature, pH, salinity, and / or the presence, concentration and identity of any buffer(s), denaturant(s) (e.g., formamide), crowding agent(s) (e.g., PEG), detergent(s) (e.g., SDS), surfactant(s), polysaccharide(s) (e.g., dextran sulfate), chelator(s) (e.g., EDTA), and nucleic acid(s) (e.g., salmon sperm DNA). A duplex polynucleotide may comprise one or more unpaired bases including, for example, a mismatched base, a hairpin loop, a single-stranded (5’ and / or 3’) end. Duplex polynucleotides (including duplex portions of polynucleotides comprising other structures (e.g., single-stranded loops)) may have any desired length. For example, a duplex polynucleotide may have a length of ≤ 5 nucleotides, ≤ 10 nucleotides, ≤ 20 nucleotides, ≤ 50 nucleotides, 5-20 nucleotides, 5-50 nucleotides, 10-200 nucleotides, 80-400 nucleotides, 50- 500 nucleotides, ≤ 500 nucleotides, ≤ 1 kb, ≤ 2 kb, ≤ 5 kb or ≤ 10 kb. Optionally, duplex polynucleotides may be ≥ 5 kb. Duplex polynucleotides may have any desired number of mismatched or unpaired nucleotides, for example, ≤ 1 per 100 nucleotides, ≤ 2 per 100 nucleotides, ≤ 3 per 100 nucleotides, ≤ 5 per 100 nucleotides, ≤ 10 per 100 nucleotides, ≤ 25 per 100 nucleotides, ≤ 50 per 100 nucleotides, ≤ 75 per 100 nucleotides. In the context of the present disclosure, “endonuclease V” refers to archaeal (thermostable) enzymes that cleave the nucleic acid backbone of nucleic acids comprising inosine or deoxyinosine at the second phosphodiester bond 3’ to each inosine / deoxyinosine site, with each cleavage leaving a 5’ hydroxyl group and 3’ phosphate group. Examples of endonuclease V include Thermococcus kodakarensis endonuclease V, Pyrococcus furiosus endonuclease V, Pyrococcus abyssi endonuclease V, Sulfolobus solfataricus endonuclease V, PATENT APPLICATION NEB-481-PCT Sulfolobus acidocaldarius endonuclease V, Nitrososphaera gargensis endonuclease V, Nanoarchaeum equitans endonuclease V, Haloferax volcanii endonuclease V, Methanothermobacter thermautotrophicus endonuclease V, Archaeoglobus fulgidus endonuclease V, and Lokiarchaeum ossiferum endonuclease V. Wild type Thermococcus kodakarensis endonuclease V comprises an amino acid sequence according to SEQ ID NO:14. An endonuclease (e.g. endonuclease V) may have greater catalytic activity towards cleavage of RNA over DNA . For example, an endonuclease may have ≥2x, ≥5x, ≥10x, ≥20x, ≥50x, ≥100x, ≥150x, ≥200x, ≥250x, ≥275x, or ≤300x more activity toward RNA than DNA. An endonuclease (e.g., a thermostable endonuclease V) may achieve complete or near complete cleavage of RNA. For example, at a molar ratio (m / m) of enzyme to RNA substrate <1:1, ≤0.5:1, ≤0.25:1, ≤0.05:1, ≤0.025:1, ≤0.01:1, ≤0.005:1, ≤0.0025:1, or ≤0.001:1, an endonuclease (e.g., a thermostable endonuclease V) may cleave ≥95% of the RNA substrate at inosine. In the context of the present disclosure, “endonuclease V substrate” refers to any nucleic acid molecule that contains an inosine, deoxyinosine, or modified inosine. An endonuclease V substrate may be exclusively double stranded, partially double-stranded and partially single- stranded, or exclusively single stranded. An endonuclease V substrate may be exclusively DNA, exclusively RNA, or DNA / RNA hybrids. An endonuclease V substrate may be created by deaminating adenosine to inosine (e.g., by contacting a nucleic acid comprising adenosine with an adenosine deaminase). In the context of the present disclosure, “fusion protein” refers to a protein composed of two or more polypeptide components that are un-joined in their native state. Fusion proteins may be a combination of two, three or four or more different proteins. For example, a fusion protein may comprise two naturally occurring polypeptides that are not joined in their respective native states. A fusion protein may comprise two polypeptides, one of which is naturally occurring and the other of which is non-naturally occurring. The term polypeptide is not intended to be limited to a fusion of two heterologous amino acid sequences. A fusion protein may have one or more heterologous domains added to the N-terminus, C-terminus, and or the middle portion of the protein. If two parts of a fusion protein are “heterologous”, they are not part of the same protein in its natural state. Examples of fusion proteins include proteins comprising a deaminase fused to another enzyme (e.g., an endonuclease), an antibody, a binding domain suitable for immobilization such as maltose binding domain (MBP), a histidine tag (“His-tag”), a chitin binding domain, an alpha mating factor or a SNAP-Tag® (New PATENT APPLICATION NEB-481-PCT England Biolabs, Ipswich, MA (see for example US patents 7,939,284 and 7,888,090)), a DNA-binding domain, and / or albumin with the deaminase optionally positioned closer to the N-terminus or closer to the C-terminus than the other component(s). A binding peptide may be used to improve solubility or yield of the deaminase during the production of the protein reagent. Other examples of fusion proteins include fusions of a deaminase and a heterologous targeting sequence, a linker, an epitope tag, a detectable fusion partner, such as a fluorescent protein, β-galactosidase, luciferase and / or functionally similar peptides. Components of a fusion protein may be joined by one or more peptide bonds, disulfide linkages, and / or other covalent bonds. In the context of the present disclosure, “immobilized” refers to covalent attachment of an enzyme to a solid support with or without a linker. Examples of solid supports include beads (e.g., magnetic, agarose, polystyrene, polyacrylamide, chitin). Beads may include one or more surface modifications (e.g., O6-benzyleguanine, polyethylene glycol) that facilitate covalent attachment and / or activity of an enzyme of interest. For example, a support may comprise a ligand and an enzyme may have a receptor for such ligand or an enzyme may comprise a ligand and a support may comprise a receptor for such ligand. Receptor-ligand binding may be covalent or non-covalent. Non-covalent attachment (e.g., avidin:biotin, chitin:CBP) may be useful in some embodiments, for example, where the level of dissociation of the binding partner is deemed tolerable. A linker may be disposed between a support and an enzyme. For example, linker disposed between a support and an enzyme may have a first covalent bond to the support and a second covalent bond to the enzyme. An immobilized enzyme comprising a ligand- receptor attachment may have a linker disposed between the support and the ligand-receptor attachment, a linker disposed between the enzyme and the ligand-receptor attachment, or both. An immobilized enzyme comprising a linker may also comprise an optional covalent bond directly between the enzyme and the support. A linker may be of any desired length and have any desired range of motion. A peptide linker may comprise one or more repeats (e.g., 1-10 repeats) of glycine-serine. In the context of the present disclosure, “modified adenine” refers to any covalent modification of adenine including naturally occurring and non-naturally occurring modifications. Modified adenines include, for example, inosine (xi), methyladenine (mA), N6 methyl adenine (m6A), 1-methyladenine (m1A), 6-hydroxymethyladenine (6hmA), 6- hydroxymethylpurine (HAP), 2-aminoadenine, 2-amino-6-hydroxymethylpurine (AHAP), PATENT APPLICATION NEB-481-PCT N6,N6,aminodimethyladenine (6-DMA), and N6-carbamoylmethyladenine (ncm6A). Additional examples of modified nucleobases may be found at https: / / dnamod.hoffmanlab.org. In the context of the present disclosure, “modified cytosine” refers to any covalent modification of cytosine including naturally occurring and non-naturally occurring modifications. Modified cytosines include, for example, 1-methylcytosine (1mC), 2-O- methylcytosine (m2C), 3-ethylcytosine (e3C), 3,N4-ethylenocytosine (^C), 3-methylcytosine (3mC), 4-methylcytosine (4mC), 5-carboxylcytosine (5CaC), 5-formylcytosine (5fC), 5- hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), N4-methylcytosine (N4mC), and pyrrolo-cytosine (pyrrolo‐C). Additional examples of modified nucleobases may be found at https: / / dnamod.hoffmanlab.org. In the context of the present disclosure, “modified inosine” refers to any covalent modification of inosine or deoxyinosine including naturally occurring and non-naturally occurring modifications. Modified inosines may include, for example 2’-O-methyl-inosine (2OmeI), and 1-methyl-inosine (1mI). In the context of the present disclosure, “modified nucleotide” refers to nucleotides having a modification on the sugars (e.g., 2′‐fluororibose, ribose, 2′‐deoxyribose, arabinose, and hexose); and / or in the phosphate groups (e.g., phosphorothioates and 5′‐N‐ phosphoramidite linkages); and / or in the nucleotide base (e.g., as described in US 8,383,340; WO 2013 / 151666; US 9,428,535 B2; US 2016 / 0032316). Modified nucleotides include modified nucleobases (e.g., modified adenines and modified cytosines) as well as corresponding modified nucleosides and modified nucleotides. Examples of modified nucleotides include pseudouridine and N1-methyl-pseudouridine. In the context of the present disclosure, “non-naturally occurring” refers to a molecule (e.g., a polynucleotide, polypeptide, carbohydrate, or lipid) or composition that does not exist in nature. Such a molecule or composition may differ from naturally occurring molecules or compositions in one or more respects. For example, a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) may differ in the kind and arrangement of the component parts (e.g., nucleotide sequence, amino acid sequence, or sugar molecules). A polymer may differ from a naturally occurring polymer with respect to the molecule(s) to which it is linked. For example, a “non-naturally occurring” polypeptide (e.g., protein) may differ from naturally occurring polypeptides in its secondary, tertiary, or quaternary structure, by having (or lacking) a chemical bond (e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others) to a lipid, a carbohydrate, a second polypeptide PATENT APPLICATION NEB-481-PCT (e.g., a fusion protein), or any other molecule. Similarly, a “non-naturally occurring” polynucleotide or nucleic acid may comprise (or lack) one or more other modifications (e.g., an added label or other moiety) to the 5’- end, the 3’ end, and / or between the 5’- and 3’-ends (e.g., methylation) of the nucleic acid. A “non-naturally occurring” molecule or composition may differ from naturally occurring compositions in one or more of the following respects: (a) having components that are not combined in nature, (b) having components in ratios and / or concentrations not found in nature, (c) lacking one or more components otherwise found in naturally occurring molecules or compositions (e.g., a cell-free composition, a chromosome- free composition, a histone-free composition, a polymerase-free composition, a cell membrane- free composition), (d) having a form not found in nature (e.g., dried, freeze dried, lyophilized, crystalline, aqueous, immobilized), and (e) having one or more additional components beyond those found in nature (e.g., a buffering agent, a detergent, a dye, a solvent or a preservative). With reference to an amino acid, “position” refers to the place such amino acid occupies in the primary sequence of a peptide or polypeptide numbered from its amino terminus to its carboxy terminus. In the context of the present disclosure, “primary structure” refers to the linear, ordered arrangement of nucleotides joined one to the next by phosphodiester bonds in a polynucleotide (e.g., RNA), for example, from a (more) 5’ position to a (more) 3’ position. For clarity, circular and linear polynucleotides have a primary structure. In the context of the present disclosure, “RNA structure” refers to any spatial arrangement of nucleotides in a single stranded molecule or a polynucleotide duplex including, for example, primary structure, secondary structure, and tertiary structure. Secondary and / or tertiary RNA structure may be static or may be transient, changing over time with a time scale of microseconds, seconds, and / or minutes. A transient RNA structure may convert from one conformation into one or more different conformations. A given nucleobase in one RNA structure may or may not have the same degree of accessibility to solvent, enzymes (e.g., deaminases), and / or other molecules as that RNA molecule takes on other RNA structures. For example, a given nucleobase of an RNA molecule may only be accessible to a deaminase in one RNA structure that such RNA molecule adopts. It may be inaccessible to (and therefore, unreactive with) the deaminase in other structures of such RNA molecule. In the context of the present disclosure, “salt” refers to an ionic compound comprising a cation of a base and an anion of an acid. Salts include monovalent salts (e.g., NaCl, KCl), PATENT APPLICATION NEB-481-PCT divalent salts (e.g., MgCl2, CaCl2), organic salts (NaCH3COO), and inorganic salts (e.g., NaHCO3, Na2SO4, CaCO3, ). In the context of the present disclosure, “secondary structure” refers to the base pairing state of nucleotides in a polynucleotide including unpaired nucleotides in which the base does not form hydrogen bonds with any other base and paired nucleotides that form at least two hydrogen bond with another base. Base pairs may be Watson-Crick base pairs or Hoogsteen base pairs. The polynucleotide backbone of the first nucleotide of a base pair may be arrange antiparallel to the polynucleotide backbone of the second nucleotide of the base pair. A secondary structure of a polynucleotide may include base-paired stems (e.g., one or more nucleotides in length) and / or single-stranded loops (e.g., one or more nucleotides in length). In the context of the present disclosure, “tertiary structure” refers to the three- dimensional arrangement of nucleotides in a polynucleotide and any higher order structures in which they are included (e.g., base-pairs, stems, helices, and single-stranded loops). In the context of the present disclosure, “tRNA-specific adenosine deaminases” and “TadA” each refer to enzymes that deaminate adenosine to inosine in tRNAs and the TadA orthologs that deaminate cytidine and adenosines in RNA. In the context of the present disclosure, “substitution” refers to an amino acid residue at a position in a comparator amino acid sequence that differs with respect to a corresponding position of a reference amino acid sequence, where the comparator and reference sequences are at least 60% identical to each other or at least 70% identical to each other or at least 80% identical to each other. A substitute amino acid residue at a position, in addition to differing from the corresponding position of a reference amino acid sequence, may differ from the amino acid at the corresponding position of all naturally-occurring sequences that are at least 60% identical to each other or at least 70% identical to each other or at least 80% identical to the reference sequence. A reference sequence and comparator sequence may have the same length or similar lengths (e.g., differing by ≤ 12%, ≤ 5%, ≤ 1%). A substitute amino acid residue at a position, in addition to differing from the corresponding position of a reference amino acid sequence, may differ from the amino acid at the corresponding position of all naturally- occurring sequences that are at least 60% identical to each other or at least 70% identical to each other or at least 80% identical to the reference sequence. Optionally, a substitute amino acid may have different properties than the amino acid in the corresponding position of the reference sequence. Optionally, a substitute amino acid may have similar properties to the amino acid in the corresponding position of the reference sequence (a “conservative” PATENT APPLICATION NEB-481-PCT substitution). For example, a non-polar amino acid (e.g., A, V, L, I, M, W, and F (and optionally C, G, and P) may substitute for another non-polar amino acid, a polar amino acid (e.g., N, Q, S, T, and Y) may substitute for another polar amino acid (e.g., C, D, E, H, K, N, P, Q, R, S, and T), a positively charged amino acid (H, K, and R) may substitute for another positively charged amino acid, and a negatively charged amino acid (e.g., D and E) may substitute for another negatively charged amino acid. A substitute amino acid may be a natural amino acid (e.g., replacing another natural amino acid or a non-natural amino acid). A substitute amino acid may be a non-natural amino acid (e.g., replacing a natural amino acid or another non-natural amino acid). All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Reagents referenced in this disclosure may be made using available materials and techniques, obtained from the indicated source, and / or obtained from New England Biolabs, Inc. (Ipswich, MA). Molecule / Chemical Entity The present disclosure relates to naturally occurring and non-naturally occurring deaminases (e.g., RNA deaminases). Examples of naturally occurring deaminases include HPAMPD. A non-naturally occurring deaminase (e.g., an RNA deaminase) may relate to, but differ from, a naturally occurring protein. Naturally-occurring proteins often include a deaminase as a single domain of a larger, multi-domain structure with the deaminase domain positioned at the most C-terminal end. Non-naturally occurring deaminases (e.g., RNA deaminases) may constitute truncated versions of a naturally-occurring protein, in which cases, the non-naturally occurring deaminases (e.g., RNA deaminases) may have a high degree of identity to a portion of a naturally-occurring sequence, but lack, for example, structural and / or functional domains or sub-units of the corresponding naturally-occurring proteins. A non-naturally occurring deaminase (e.g., RNA deaminase) may have any number of insertions, deletions, or substitutions relative to a naturally occurring enzyme. For example, a non-naturally occurring deaminase (e.g., RNA deaminase) may have less than 100% identity, less than 99% identity, less than 98% identity, less than 90% identity, less than 85% identity, less than 80% identity, less than 70% identity, less than 60% identity, less than 50% identity, less than 40% identity, less than 30% identity, or less than 20% identity to a naturally occurring enzyme. Non-naturally occurring deaminases (e.g., RNA deaminases) PATENT APPLICATION NEB-481-PCT may include expression and / or purification tags. Non-naturally occurring deaminase (e.g., RNA deaminase) disclosed herein may have an amino acid sequence that is at least 80% identical (e.g., at least 90% identical, at least 95% identical or at least 98% identical or at least 99% identical to) the C-terminal deaminase domain of a naturally-occurring protein, wherein the deaminase (e.g., RNA deaminase) possesses a deaminase (e.g., RNA deaminase) activity and does not comprise the N-terminus of the corresponding naturally-occurring protein (if any). In some embodiments, a non-naturally occurring deaminase (e.g., RNA deaminase) lacks at least 10, at least 20, at least 50 or at least 100 of the N-terminal amino acids of the corresponding naturally-occurring protein. In some embodiments, a deaminase (e.g., RNA deaminase) is no more than 300 amino acids in length, e.g., no more than 200 amino acids in length or no more than 150 amino acids in length. According to some embodiments, a deaminase (e.g., an RNA deaminase) may comprise an amino acid sequence having at least 80%, at least 85%, at least 88% identical, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1. In some embodiments, a deaminase (e.g., an RNA deaminase) may be encoded by a nucleic acid sequence that, when transcribed, translated, and / or processed, results in an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:1. In some embodiments, a non-naturally occurring deaminase (e.g., an RNA deaminase) lacks the N-terminus of its corresponding naturally-occurring protein, for example, lacking at least 1, at least 10, at least 20, at least 50 or at least 100 of the N-terminal amino acids. Variants may be designed using sequence alignments and structural information. In some embodiments, a deaminase (e.g., RNA deaminase) may contain a fragment of a wild type protein, where the fragment contains a deaminase domain, but lacks other domains of the wild type protein that may be C-terminal and / or N-terminal to the deaminase domain. In some embodiments, a deaminase (e.g., an RNA deaminase) may be included in a fusion protein, for example, a fusion comprising the deaminase and a linker. A deaminase (e.g., alone or included a fusion) and / or a deaminase substrate may be immobilized, according to some embodiments. A deaminase (e.g., an RNA deaminase) optionally may deaminate one or more nucleobases in a nucleoside, nucleotide, and / or polynucleotide. For example, a deaminase may deaminate adenine, but not cytosine, may deaminate cytosine, but not adenine, or may deaminate both adenine and cytosine (appreciating that one may be a better substrate than the PATENT APPLICATION NEB-481-PCT other under otherwise equivalent conditions). A deaminase (e.g., an RNA deaminase) may be modification sensitive. For example, a deaminase (e.g., RNA deaminase) may deaminate cytosine, but not deaminate one or more modified cytosines in double stranded DNA. For example, a deaminase (e.g., RNA deaminase) may deaminate adenine, but not deaminate xi, mA, 6hmA, HAP, AHAP, 6-DMA, and / or ncm6A. Compositions The present disclosure provides deaminase (e.g., RNA deaminase) compositions including, for example, reaction mixtures. According to some embodiments, deaminase compositions may comprise (a) a deaminase (e.g., RNA deaminase) and (b) a polynucleotide (e.g., an RNA). A deaminase composition may comprise, for example, a deaminase (e.g., having an amino acid sequence at least 80% identical to SEQ ID NO:1). A deaminase (e.g., RNA deaminase) composition may be free of one or more other catalytic activities. For example, a deaminase (e.g., RNA deaminase) composition may be free of nucleases that cleave dsRNA, free of nucleases that cleave ssRNA, free of polymerase activity, free of RNA modification activity, and / or free of protease activity, in each case, under desired test conditions (e.g., conditions of time, temperature, pH, salinity, model substrate and / or others), for example, conditions intended to replicate conditions of a specific use of the deaminase (e.g., RNA deaminase) composition or intended to represent conditions for a range of uses. In some embodiments, deaminase (e.g., RNA deaminase)s and compositions comprising one or more deaminase (e.g., RNA deaminase) may have any desirable form including, for example, a liquid, a gel, a film, a powder, a cake, and / or any dried or lyophilized form. A deaminase (e.g., RNA deaminase) composition may comprise a deaminase (e.g., RNA deaminase) and a support or matrix, for example, a film, gel, fabric, or bead comprising, for example, a magnetic material, agarose, polystyrene, polyacrylamide, and / or chitin. In some embodiments, a reaction mix may comprise: an RNA substrate that comprises adenines and a deaminase (e.g., RNA deaminase). An RNA substrate may comprise adenines and at least one modified adenine, e.g., xi, mA, 6hmA, HAP, AHAP, 6- DMA, and / or ncm6A. An RNA substrate may be eukaryotic RNA (e.g., plant, virus or animal) or bacterial or bacteriophage. In some embodiments, an RNA substrate may be mammalian, e.g., from a human. In some embodiments, an RNA substrate may be total RNA or mRNA fraction of total RNA. In some embodiments, an RNA substrate may be an RNA therapeutic molecule (e.g., a vaccine). In some embodiments, an RNA substrate may be an in PATENT APPLICATION NEB-481-PCT vitro transcribed RNA molecule. An RNA substrate may have any desired length. For example, an RNA substrate may have a length of ≥ 10 nucleotides, ≥ 25 nucleotides, ≥ 50 nucleotides, ≥ 75 nucleotides, ≥ 100 nucleotides, or ≥ 250 nucleotides to ≤ 1,000 nucleotides, ≤ 5,000 nucleotides, ≤ 10,000 nucleotides, ≤ 25,000 nucleotides, ≤ 50,000 nucleotides, ≤ 75,000 nucleotides, or ≤ 100,000 nucleotides. A reaction mix may additionally comprise a buffer and / or a salt. A reaction mix may be free of chemical denaturants (e.g., detergents, formamide) and / or free of RNA binding proteins (e.g., RNA chaperones, RNA helicases, ssRNA binding proteins). A reaction mix may have a temperature ≥ 15°C, ≥ 20°C, ≥ 22°C, ≥ 25°C, ≥ 30°C, ≤ 35°C, ≤ 40°C, ≤ 45°C, ≤ 50°C, ≤ 55°C, and / or ≤ 60°C (e.g., 15°C-60°C, 20°C-50°C, 20°C-35°C, 30°C-50°C). According to some embodiments, a deaminase composition may comprise a deaminase (e.g., an RNA deaminase) and, optionally, any of (including one or more of) a buffering agent (e.g., a storage buffer, a reaction buffer), an excipient, a salt (e.g., NaCl, MgCl2, CaCl2), a protein (e.g., albumin, an enzyme), a stabilizer, a detergent (for example, ionic, non-ionic, and / or zwitterionic detergents (e.g., octoxinol, polysorbate 20)), a deaminase inhibitor (for example, pentostatin or pentostatin phosphate), a polynucleotide, a cell (e.g., intact, digested, or any cell-free extract), a biological fluid or secretion (e.g., mucus, pus), an aptamer, a crowding agent, a sugar (e.g., a mono, di, tri, tetra, or higher saccharide), a starch, cellulose, a glass-forming agent (e.g., for lyophilization), a lipid, an oil, aqueous media, a support (e.g., a bead) and / or (non-naturally occurring) combinations thereof. Combinations may include for example, two or more of the additional components (e.g., a salt and a buffer) or a plurality of a class of components (e.g., two different salts or two different sugars). Examples of proteins that may be included in a double-stranded DNA deaminase composition include one or more enzymes that alter the deamination susceptibility of one or more bases. Deamination Methods The present disclosure provides methods for assessing the sensitivity of adenines and / or cytosines in a polynucleotide (e.g., an RNA) to deamination using, for example, a deaminase (e.g., an RNA deaminase). In some embodiments, a method may comprise providing a deaminase substrate of any desired length. For example, a deaminase substrate may have a length of ≤ 50 nucleotides, 10-200 nucleotides, 80-400 nucleotides, 50-500 nucleotides, ≤ 500 nucleotides, ≤ 1 kb, ≤ 2 kb, ≤ 5 kb or ≤ 10 kb or ≤ 100 kb. A deaminase substrate, in some embodiments, may be or comprise a messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small RNA (sRNA), microRNA (miRNA), long PATENT APPLICATION NEB-481-PCT noncoding RNA (lncRNA), circular RNA (circRNA), transfer RNA (tRNA), aptamer RNA, antisense RNA, silencing RNA (siRNA), guide RNA (gRNA), therapeutic RNA, other RNAs of interest or any combination thereof and may be or arise from any desired source (e.g., human, non-human mammal, plants, insects, microbial, viral, or synthetic DNA). A deaminase substrate may be prepared, in some embodiments by extracting RNA (e.g., total RNA, mRNA, or other ) from a biological sample. In some embodiments, methods may include forming or revising a representation of a structure of a deaminase substrate (e.g., RNA or comprising RNA), wherein deamination sensitive bases are positioned at or near the surface of the substrate and deamination insensitive bases are positioned away from the surface of the substrate. In some embodiments, methods may include forming or revising a structural representation of a structure of a deaminase substrate (e.g., RNA or comprising RNA), wherein deamination sensitive bases are positioned at loops or bulges of the substrate and deamination insensitive bases are positioned at based paired regions of the substrate. In some embodiments, methods may include developing a structural representation (e.g., de novo or from a prior representation) of a structure of a deaminase substrate (e.g., RNA or comprising RNA), wherein deamination sensitive bases are not a member of a Watson-Crick base pair. Methods may include, according to some embodiments, ligating one or more adapters to polynucleotide (e.g., RNA) ends. Adapters may comprise one or more sample tags, unique molecular identifiers (UMIs), modified nucleotides, primer sequences (e.g., for sequencing). In some embodiments, adapters may comprise adenines (or cytosines) that are not substrates for the deaminase to be used (e.g., modified adenines and / or spatially inaccessible adenines). If desired, ligation products may be cleaned up, for example, to separate ligation products from enzymes, unreacted nucleotides and / or unligated adapters. A method may comprise contacting a deaminase substrate that comprises adenosines and a deaminase (e.g., an RNA deaminase) to produce a deamination product that may (depending on the structure of the deaminase substrate) comprise one or more deaminated adenosines (e.g., inosines). A deaminase substrate may further comprise one or more modified adenines, e.g., one or more modified adenines selected from m1A, m6A, xi, mA, 6hmA, HAP, AHAP, 6-DMA, and / or ncm6A. While a deamination substrate may be exposed to cell extracts or RNA binding proteins prior to or during deamination, a deaminase substrate may be free of RNA binding proteins during deamination (e.g., for structural assessments). While a deamination substrate PATENT APPLICATION NEB-481-PCT may be denatured prior to deamination (e.g., as a control), a deaminase substrate does not need to be denatured before or during deamination (e.g., for structural assessments). As such, methods may be practiced in the absence of a denaturation step. In some embodiments, deamination methods may comprise contacting a deaminase substrate comprising adenines and a deaminase (e.g., an RNA deaminase) to produce a deamination product comprising one or more deaminated adenines (e.g., hypoxanthines). Deamination methods may further comprise reverse transcribing the deamination product to produce a cDNA product, thereby copying any deaminated As in the original RNA strand to inosines in the cDNA product. The cDNA may further comprise amplifying the cDNA by PCR. Deamination methods may further comprise ligating an asymmetric (or "Y") adapter, e.g., an Illumina P5 / P7 adapter, onto the deamination product and amplifying the deaminated product using primers complementary to sequences in the adapter. In some embodiments, a method may comprise sequencing a deamination product, or amplifying a deamination product to produce amplification products and sequencing the amplification products, in each case, to produce sequence reads. Deamination products and / or amplification products may be sequenced using any suitable system including Illumina’s reversible terminator method (see, e.g., Shendure et al, Science 2005309: 1728). In some embodiments, a deaminated product may be sequenced directly, without amplification, for example, by nanopore or a deaminated product may be reversed transcribed to a cDNA product and sequenced directly by PacBio sequencing (Pacific Biosciences of California, Inc.). A sequencing step may result in at least 10,000, at least 100,000, at least 500,000, at least 1M, at least 10M, at least 100M, at least 1B or at least 10B sequence reads per reaction. In some cases, the reads may be paired-end reads. A method may comprise analyzing sequence reads to identify adenines and deaminated adenines in the deaminase substrate. Analyzing sequence reads may include mapping the read back to a reference sequence (e.g., a verified sequence of the deaminase substrate). Methods may include identifying adenines in the reads that match adenines at corresponding positions of the reference sequence and optionally may further include designating such adenines as having a position away from the surface of the deaminase substrate molecule. Methods may include identifying guanosines in the reads that match adenines at corresponding positions of the reference sequence and optionally may further include designating such adenines as having a position at the surface of the deaminase substrate molecule. PATENT APPLICATION NEB-481-PCT Workflows for example incorporating deaminase results into predictions of secondary and three dimensional structures are shown in FIGURE 12. Deaminase results may be incorporated into secondary and three-dimensional predictions. The deaminase results may assist in validating the secondary structure. For example, the image on the left and right of FIGURE 12 are the same illustrating a scenario in which deamination results were consistent with the initial structure prediction. Where an adenosine in the predicted structure is present in a Watson-Crick hydrogen bond base pair as in a RNA stem structure, a very low level of deamination of that adenosine would be consistent with the structure prediction. If there were a high level of deamination of that adenosine in the predicted stem structure, the deamination result would indicate that the predicted structure was incorrect and that the adenosine was not in a Watson-Crick base pair stem. Where an adenosine in the predicted structure is present in loop such as at the end of a RNA stem / loop structure, a high level of deamination of that adenosine would be consistent with the structure prediction. If there were no observed deaminations of that adenosine in the predicted loop structure, the deamination result would provide experimental evidence inconsistent with the description decreasing confidence in the prediction and optionally generating a modified predicted structure in which the adenosine is not in a loop structure. More complex arrangements of nucleotides in three dimensional structures, other than Watson-Crick base pairing and loops, may be predicted from comparisons of deaminase accessible bases to experimentally (e.g. X-ray, cryoEM and NMR) derived three dimensional RNA structures. Any method disclosed herein for enzymatically interrogating RNA structure (e.g., with a deaminase) may optionally further include actions using and / or acting on structural data obtained. For example, a method may include building a new RNA structure model and / or modifying an existing RNA structure model. In some embodiments, modifying an existing RNA structure model may comprise modifying one or more secondary and / or tertiary structure elements. In some embodiments, existing RNA structure model may comprise increasing a confidence metric for one or more structure elements and / or the overall model. A method may include, in some embodiments, enzymatically interrogating (e.g., with a deaminase) the structure of a plurality of RNA molecules to obtain structural information for each and using the obtained structural information to select RNA molecules that satisfy a predetermined metric (e.g., have a structure or structure element associated with a desired function) and / or deselect molecules that do not satisfy a predetermined metric (e.g., have a structure or structure element associated with an undesired property). For example, it may be PATENT APPLICATION NEB-481-PCT that a therapeutic RNA of interest has a plurality of possible structures, only one or a subset of which are associated with activity and / or minimal adverse events. A method may comprise enzymatically interrogating (e.g., with a deaminase) the structure of a plurality of these therapeutic candidate RNA molecules to obtain structural information for each and using the obtained structural information to select the RNA molecules having the desired confirmation and / or removing those lacking the desired conformation. A method as described may be configured for use in screening (large) numbers of candidates and / or quality control testing production lots. In some embodiments, a method may include enzymatically interrogating (e.g., with a deaminase) the structure of an RNA and rationally designing one or more nucleotide substitutions at one or more specific sites in the primary sequence to produce a modified RNA. A method may further include enzymatically interrogating (e.g., with a deaminase) the structure of the modified RNA to produce structure data and analyzing the produced structure data to assess whether the modified RNA has the rationally designed / predicted structure. In some embodiments, a method may comprise enzymatically interrogating (e.g., with a deaminase) the structure of a cellular RNA to produce cellular RNA structure data and selecting a therapy and / or therapeutic agent in light of the viral structure data. Selecting may include, for example, selecting a biologic and / or a small molecule pharmaceutical agent capable of interacting with (e.g., cleaving or otherwise modifying the structure of) the cellular RNA and / or capable of blocking or preventing replication, transcription, and / or translation of the cellular RNA. In some embodiments, a method may comprise enzymatically interrogating (e.g., with a deaminase) the structure of an RNA virus to produce viral structure data and selecting an antiviral in light of the viral structure data, for example, an antiviral capable of interacting with (e.g., cleaving) the viral RNA and / or otherwise capable of blocking or preventing replication, transcription, and / or translation of the viral RNA. The present disclosure further provides methods for screening proteins on the basis of their impact on RNA structure. For example, a method may comprise enzymatically interrogating (e.g., with a deaminase) the structure of an RNA (e.g., contacting an RNA and a deaminase and evaluating the reactivity of one or more nucleobases of the RNA) to produce RNA structure data. This may be performed on the RNA before contacting the protein of interest, while contacting the protein of interest, and / or after contacting the protein of interest. In some embodiments, a method may comprise comparing, RNA structure data from, for example, before and after contact with the protein of interest to characterize impact the PATENT APPLICATION NEB-481-PCT protein had on structure of the RNA, if any. For example, characterizing may include a binary operation of whether or not the structure of the RNA was changed. Characterizing may include identifying the nucleotides (adenosines and / or cytosines) within the RNA where changes in reactivity / accessibility were seen. A method may further comprise selecting or deselecting the protein for further work. For example, a protein (e.g., an antibody) that disrupts the structure of an undesirable RNA (e.g., an RNA involved in disease progression, a viral RNA) may be selected for further evaluation as a therapeutic and / or may be injected in a subject to slow or arrest disease progression. A protein that induces an RNA (e.g., an undesirable RNA) to move from a less desired (e.g., inactive and / or immunostimulatory) conformation to a more desirable conformation (e.g., active and / or less or non-immunogenic) may be selected for further evaluation as a therapeutic and / or may be injected in a subject to treat a condition associated with one or both conformations. Cleaving RNA comprising Inosine The present disclosure relates to compositions and methods for cleaving single- stranded RNA with an endonuclease. For example, a method may include contacting an RNA (e.g., a deaminated RNA produced from contacting a deaminase and an RNA) and an endonuclease V (e.g., an archaeal endonuclease V) to produce an RNA cleavage product. According to some embodiments, a endonuclease may have a preference for ssRNA over dsRNA. For example, a method may include (a) contacting a deaminase and (b) a nucleic acid (e.g., an RNA) comprising one or more nitrogenous bases selected from adenine and cytosine, and (B) having a nucleotide sequence to produce a deamination product comprising one or more deaminated adenines and / or one or more deaminated cytosines; (b) determining the position in the nucleotide sequence of (i) the one or more deaminated adenines and / or (ii) the one or more deaminated cytosines; (c) generating secondary structure context data for one or more of the nitrogenous bases corresponding to the one or more deaminated adenines and / or the one or more deaminated cytosines; and (d) contacting the deamination product with an archaeal endonuclease V (e.g., HPAMPD) to form a nucleic acid cleavage product (e.g., an RNA cleavage product). To enrich specific RNA species comprising inosine in a mixture of heterogenous RNA molecules, the 3’-OH ends of all RNA species in the mixture may be enzymatically modified or extended to alter the 3’ end to remove the 3’-OH (FIGURE 23). This may be accomplished, for example, by non-templated extension of the RNA 3’-OH with dNTPs using a polymerase. A mixture of heterogenous RNA molecules having such 3’ modifications PATENT APPLICATION NEB-481-PCT may be contacted with an archaeal endonuclease V to cleave adjacent to the inosine of the RNA of interest, generating cleavage product with 3’-OH ends. The 3’ phosphate fragments may be removed, if desired, by a T4 kinase. The 3’-OH ends may be leveraged to identify, enrich and / or isolate the RNA species of interest. For example, only the new 3’-OH ends may further be extended / biotinylated with Biotin-ATP by poly(A) polymerase since poly(A) polymerase requires a 3’-OH end for its polymerization activity. This will selectively biotinylate the RNA molecules of interest, which may subsequently be enriched for by affinity binding to a Streptavidin-coated matrix. Optionally, a precipitation step may be used to remove unincorporated biotein ATP after the polyA polymerase reaction. After elution of the RNA of interest from the Streptavidin matrix, the RNA may be sequenced (e.g., using the ONT Nanopore direct RNA sequencing protocol or Illumina sequencing using an oligo(dT) primer in a standard RNA library preparation). All other unwanted / uncleaved RNA species will not be biotinylated and thus not be sequenced. In some embodiments, a Biotin enrichment step may be omitted. Since the ONT nanopore adapter requires a free 3’-OH to be ligated to RNA, RNAs with blocked 3’ ends cannot be ligated and thus will not be sequenced. Only RNAs with endonuclease generated new 3’-OH ends will successfully ligate and be sequenced by ONT nanopore. It may be desirable to nick or cleave nucleic acids (e.g., RNA) comprising inosine, in some embodiments. For example, it may be desirable to release a nucleic acid comprising insosine from linked to a solid support. A method of releasing such a nucleic acid may comprise contacting a support-bound substrate nucleic acid comprising a single inosine with an archaeal endonuclease V under conditions that permit cleavage of the nucleic acid (e.g., cleavage of the second phosphodiester bond 3’ to the inosine), thereby releasing the nucleic acid. For example, if the substrate nucleic acid is tethered to the support at the 5’ end and the inosine is positioned near the 5’ end of the substrate nucleic acid, cleavage releases substantially the entire substrate RNA, less a few bases at the 5’ end (including the inosine) that remain tethered to the support. On the other hand, if the substate nucleic acid is tethered to the support at the 3’ end and the inosine is positioned near the 3’ end, cleavage releases substantially the entire substrate RNA (including the inosine), less a few bases at the 3’ end that remain tethered to the support. RNA Structure Predictions, Models, and Systems RNA secondary structure may be predicted computationally with thermodynamic, machine learning, or hybrid models (Nat Commun 12, 941, doi:10.1038 / s41467-021-21194-4 PATENT APPLICATION NEB-481-PCT (2021)). Many of the computational tools available to researchers, such as, RNAFold, Mfold, and RNAstructure (Algorithms Mol Biol 6, 26, doi:10.1186 / 1748-7188-6-26 (2011); Nucleic Acids Res 31, 3406-3415, doi:10.1093 / nar / gkg595 (2003); Nucleic Acids Res 41, W471-474, doi:10.1093 / nar / gkt290 (2013)) rely on the calculation and minimization of a thermodynamic free energy to predict the appropriate secondary structure. From a single nucleotide sequence, an ensemble of possible folded conformations is generated that frequently have similar predicted free energy. This makes deconvolution and selection of the appropriate predicted secondary structure challenging. According to some embodiments, experimental data (e.g., deaminase reactivity) may be used to select, from among the possible folded conformations generated by a computational tool, one or more candidates that are consistent with and / or best match such experimental data. In some embodiments, incorporating experimental data from structure probing or biophysical methods as a set of constraints in a computational tool itself may improve the accuracy of one or more of the ensemble of possible folded conformations and / or the predictive power of the tool, according to some embodiments. For example, reactivities from structure probing experiments, such as the accessibility or flexibility of a given nucleotide, may be used to reduce the number of predicted structures, improving the overall accuracy of the model. In some embodiments, deaminase reactivity provides a supplemental set of data constraints that may be used (optionally, in tandem with other methods) to improve the predictive power of secondary structure (and, optionally, tertiary) prediction methods. The present disclosure relates, in some embodiments, to systems for producing a structure (e.g., secondary structure) of an RNA molecule. An example system, system 1400, may comprise one or more deaminase reactivity analyzers 1410, modeling modules 1420, central processing units 1430, input / output interfaces 1440, mass storage devices 1450, and / or memory 1460 (FIGURE 14), wherein two or more of the foregoing may be integrated and / or may be in operable communication with one another. Deaminase reactivity analyzer 1410 may comprise instruments and containers for combining an RNA molecule of interest and a deaminase to produce a deamination product. A deaminase reactivity analyzer 1410 may comprise instruments for determining the position of deaminated nucleotides in the deamination product including, for example, a thermocycler (e.g., configured for reverse transcription reactions) and / or a sequencing instrument (e.g., configured to sequence RNA and / or DNA). Sequencing instruments may include instruments PATENT APPLICATION NEB-481-PCT for LCMS sequencing, Sanger sequencing, , ion semiconductor sequencing (e.g., Ion Torrent), sequencing by synthesis, next generation sequencing, long read sequencing, isoform sequencing, single molecule real time sequencing, and nanopore sequencing with instrumentation available from suppliers including, for example, Agilent, Waters Corporation, Perkin Elmer, Thermo Fisher, Inc., Illumina, Pacific Biosciences, Oxford Nanopore, and 10x Genomics. Deaminase reactivity data analyzer 1410 may be configured to produce deamination data from one or more RNA molecules comprising deaminase reactivity of one or more (up to all) adenosines and / or one or more (up to all) cytosines of the one or more RNA molecules. Modeling module 1420 may produce (e.g., dynamically produce) one or more candidate RNA structures (e.g., secondary structures, tertiary structures) of a given RNA molecule from the nucleotide sequence of the RNA molecule. Modeling module 1420 may comprise a predictive model process that is adapted to produce / predict one or more candidate structures of an RNA molecule from information including user information, nucleotide information (e.g., nucleotide position information, nucleotide sequence information, nucleobase information), thermodynamic free energy information, deaminase information, deaminase reactivity information, and structure information (e.g., experimental and / or predicted RNA secondary and / or tertiary structure information of the RNA molecule or similar RNA molecules, for example, in the same or similar conditions (e.g., temperature, salt, buffer, solvent)). A predictive model process may apply natural language processing and / or a deep learning process to characterize data and classify how much each class influences RNA structure. Central processing unit 1430 may perform operations (e.g., logical and arithmetic operations) based on instructions received through an input of system 1400 (e.g., input / output interface 1440) and / or stored in memory 1460, for example, to implement one or more methods disclosed herein. CPU 1430 may comprise any desired processor or processors (e.g., microprocessors, controllers, microcontrollers, digital signal processors, field programmable gate arrays, and programmable logic devices) suitable for performing the desired data manipulations and / or desired logical and / or arithmetic operations. Input / output interface 1440 may comprise any desired element and / or component that (a) receives information (e.g., into system 1400), for example, user information, nucleotide information (e.g., nucleotide position information, nucleotide sequence information, nucleobase information), thermodynamic free energy information, deaminase information, PATENT APPLICATION NEB-481-PCT deaminase reactivity information, and structure information (e.g., experimental and / or predicted RNA secondary and / or tertiary structure information of the RNA molecule or similar RNA molecules, for example, in the same or similar conditions (e.g., temperature, salt, buffer, solvent)) and / or (b) conveys information (e.g., out of system 1400), for example, to a user of system 1400, to one or more external devices, and / or to a network (e.g., LAN, WAN, Internet). Input / output interface 1440 may be configured to receive and / or convey information via wireless and / or wired communication links. Input / output interface 1440 may be or comprise a keyboard, a touchpad, a display, a graphical user interface, a speaker, and / or a network. Input / output interface 1440 may comprise a user interface. Mass storage device 1450 may comprise hardware (e.g., one or more magnetic drives, solid state drives, optical media), firmware, and / or software. Mass storage device 1450 may serve as a repository for software (e.g., any type of machine-executable instructions including firmware, middleware, hardware description language, and microcode in any format including source code format, binary code format, and executable code format) and / or information (e.g., candidate RNA structures, RNA structure models, deaminase reactivity data). Memory 1460 may comprise RAM and / or ROM and may be in communication with and provide instructions and / or data to central processing unit 1430. For example, inputs received at input / output interface 1440 may be stored in memory 1460. A method (e.g., a computer implemented method) of predicting an RNA structure may include receiving structural information about a subject RNA molecule (e.g., a nucleotide sequence, a candidate structure, a simulation, deamination information), for example, from an input / output interface, memory, and / or a mass storage device. A method may further include extracting one or more structural and / or thermodynamic features of the subject RNA molecule from the received information. For example, a method may include generating an ensemble (collection) of candidate structures of the RNA molecule (or a portion thereof) by executing (e.g., in a processor) a molecular simulation, obtaining a feature set (e.g., by characterizing the hydrogen bonding of base pairs, the thermodynamic free energy), and / or executing a principal component analysis to the feature set. A method may include weighting or selecting one or more candidate structures from the ensemble to produce one or more predicted structures of the RNA molecule, which may be conveyed (e.g., displayed) to a user through an input / output device (e.g., a GUI). Weighting and / or selecting one or more candidate structures from the ensemble may include assessing the likelihood a PATENT APPLICATION NEB-481-PCT given candidate structure accounts for deamination reactivity of one or more adenosines and / or cytosines of the RNA molecule. In some embodiments, deamination reactivity may be used both to inform the generation of the ensemble and the candidate selection. In some embodiments, generating an ensemble of candidate structures (e.g., secondary structures and / or tertiary structures) may include calculating a thermodynamic free energy for an RNA molecule (or a portion thereof) with or without accounting for deaminase reactivity of one or more (up to all) adenosines and / or cytosines in the RNA molecule or portion thereof. For example, generating candidate structures may include determining whether deaminase reactivity impacts the thermodynamic calculation for one or more (up to all) of the adenosines and / or cytosines in a subject RNA molecule. This may include calculating a thermodynamic free energy with and without deaminase reactivity data. Kits The present disclosure relates, in some embodiments, to a deaminase kit comprising a deaminase (e.g., an RNA deaminase). A kit may comprise any of the components described herein. A deaminase composition or kit may include, for example, a deaminase (e.g., an RNA deaminase) and, optionally, a storage buffer (e.g., comprising a buffering agent and comprising or lacking glycerol), and / or a reaction buffer. A reaction buffer for a deaminase composition or a deaminase kit may be in concentrated form, and the buffer may include one or more additives (e.g., glycerol), one or more salts (e.g., KCl), one or more reducing agents, EDTA, one or more detergents, one or more non-ionic surfactants, one or more ionic (e.g., anionic or zwitterionic) surfactants, and / or crowding agents. In some embodiments, a kit may comprise one or more NTPs and / or one or more dNTPs. A kit may comprise one or more modified NTPs in addition to or instead of a corresponding unmodified NTP. One or more components of a kit may be included in one container for a single step reaction, or one or more components may be contained in one container, but separated from other components for sequential use or parallel use. For example, a kit may comprise two components in a single tube (e.g., a deaminase and a storage buffer) and all other components in separate, individual tubes, in each case, with the contents provided in any desired form (e.g., liquid, dried, lyophilized). One tube in a kit may contain a mastermix, for example, for receiving and amplifying an RNA (e.g., a deaminated RNA). For example, a deaminase (e.g., an RNA deaminase) may be deposited in the cap of a tube while components for transcribing a template nucleic acid are deposited in the body of the tube. As desired, for example, upon completion of the deamination reaction, the tube may be tapped, shaken, turned, spun, or PATENT APPLICATION NEB-481-PCT otherwise moved to contact the deposited deaminase (e.g., an RNA deaminase) with the deamination reaction mixture. A kit may include a deaminase (e.g., an RNA deaminase) and the reaction buffer in a single tube or in different tubes and, if included in a single tube, the deaminase (e.g., an RNA deaminase) and the buffer may be present in the same or separate locations in the tube. For example, a kit may comprise a deaminase (e.g., an RNA deaminase), as described above, and a reaction buffer (e.g., a 5x or 10x buffer). The contents of a kit may be formulated for use in a desired method or process. In some embodiments, a kit may additionally comprise a ligase, a polymerase, a proteinase K, and / or a thermolabile proteinase K. A deaminase (e.g., an RNA deaminase) may be lyophilized or in a buffered storage solution that contains glycerol. As would be apparent to those having the benefit of the present disclosure, a deaminase (e.g., an RNA deaminase) may be used in a variety of polynucleotide structural analysis methods, particularly methods whose goal is to identify the position and / or identity of one or more deamination sensitive bases. A subject kit may further include instructions for using the components of the kit to practice a desired method. The instructions may be recorded on a suitable recording medium. For example, instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. Instructions may be present as an electronic storage data file residing on a suitable computer readable storage medium (e.g., a CD-ROM, a flash drive). Instructions may be provided remotely using, for example, cloud or internet resources with a link or other access instructions provided in or with a kit. EXAMPLES Some specific example embodiments may be illustrated by one or more of the examples provided herein. Unless otherwise expressly stated, disclosures within the examples are applicable to and compatible with the disclosures of the other examples and the broader disclosures herein. EXAMPLE 1: HPAMPD Activity with Human RNA HPAMPD converts adenosines to inosines when the adenosine is located at the 5' or 3' terminus of the RNA molecule. Internal adenosines found in RNA vary greatly in their PATENT APPLICATION NEB-481-PCT reactivity with HPAMPD. This reactivity correlates with the structural context in which the adenosine is located. Total universal human reference RNA (5µg) was fragmented with 10 mM ZnCl2 at 94 C° for 1 min. Fragmented RNA was then purified with a clean and concentrator column (5µg, Zymo). RNA ends were repaired with T4 PNK (no ATP) and further column purified; 90 ng / µL RNA was incubated with 2.75 µM HPAMPD in buffer containing 50 mM Tris-HCl pH 7.5 and 1 mM EDTA, at 25 C° for 30 min. Treated RNA was then column purified, 5' phosphorylated with T4 PNK (+ ATP), and again column purified. Sequencing libraries were prepared using NEBNext® Small RNA Library Prep Set for Illumina (New England Biolabs, Inc., E7330). When inosines, due to deamination of adenosine, are present in RNA, the reverse transcriptase utilized for synthesis of cDNA for Illumina sequencing, incorporates cytosines across from the template RNA strand where inosines are located. Therefore, when the sequencing reads are mapped back to the genome, mismatches occur where adenosines were converted to inosines and cytosines were consequently incorporated during reverse transcription. Upon amplification of the DNA for sequencing, guanosines are now present in place of deaminated adenosines. HPAMPD converts adenosines to inosines when the adenosine is located at the 5' or 3' terminus of the RNA molecule. Internal adenosines were found in RNA to vary greatly in their reactivity with HPAMPD. This reactivity correlates with the structural context in which the adenosine is located. Illumina sequencing reads were aligned to the human reference genome. In the sample that was treated with HPAMPD greater than 95% of terminal 5’ and 3’ adenosines were converted to inosines (detected as guanosines upon sequencing amplified cDNAs), while adenosines that are not located at the termini are converted to guanosines to varying degrees. When displaying the sequencing reads that mapped to ribosomal RNA on the IGV genome browser, it was observed there were internal positions in sequencing reads that mapped to the genomic sequence of 18S and 28S where the conversion of adenosine to inosine (detected as guanosines upon sequencing amplified cDNAs) ranged from 1% to 70%. EXAMPLE 2: HPAMPD Activity Analysis PolyA enriched RNA from a human cancer SUM149 cell-line were treated as in EXAMPLE 1. Sequenced libraries were analyzed with respect to the frequency of occurrence of HPAMPD internal deamination events. These deamination events were called where high quality A to G mismatches were specific to HPAMPD treated samples (i.e., PATENT APPLICATION NEB-481-PCT mismatches that were not present in the control sample). Deamination rate was calculated for each covered A site as the number of A to G mismatches divided by the total number of reads that covered that site (coverage). Sites that have high deamination rates (^50% or ^90%) were selected and the 50 bp flanking sequences of these deaminated sites were extracted for local RNA structure prediction using the CONTRAFold program (Do CB et al., Bioinformatics.22, e90–e98 (2006)). Deaminated A sites were classified into 7 categories based on their relative positions in the predicted RNA local structures (FIGURE 3), and their frequencies in each category was calculated (TABLE 1). As a control, the frequencies of all the non-converted sites in these structure categories were also calculated (TABLE 1). Internal deaminated sites are highly enriched in the “loop” of the stem-loop structure (category 1) compared to non-converted sites. In addition, deaminated A's tend to locate near the stem at the 3’ side in the loop and 58% of the deaminated A's that are in a loop structure are located next to the stem (FIGURE 4). Also, deaminated sites tend to have smaller loop sizes (mean=5bp) than unconverted sites (mean=10bp) (FIGURE 5). TABLE 1: Frequencies of deaminated A's in 7 different local structure categories code Structure type 90% 50% non converted converted converted EXAMPLE 3: HPAMPD Activity with Yeast RNA Saccharomyces cerevisiae (S288C) RNA was subjected to adenosine deamination with HPAMPD. Total Yeast RNA (at 100 ng / µL) was incubated for 60 minutes in 20 mM Tris-HCl pH 7.5, 50 mM NaCl with 2.75 µM HPAMPD or without HPAMPD (control) at either 25°C or 42°C. RNA was purified from the reaction products with Monarch Spin RNA Cleanup Kit (10µg) (New England Biolabs, Inc., T2030). An aliquot of the RNA was then treated with NEB Nucleoside Digestion Mix (New England Biolabs, Inc., M0649) to convert RNA into nucleosides. The resulting relative quantity of inosines and adenosines was determined by LC-MS. The percent conversion of adenosine to inosine was calculated where PATENT APPLICATION NEB-481-PCT the quantity of inosines was divided by the sum of the quantity of inosines and adenosines (FIGURE 6). Furthermore, the initial rate of deamination of the nucleotide 5' AMP in 20 mM Tris 7.5, 50 mM NaCl was determined to be 1.5 times higher at 42°C than the rate at 25°C. EXAMPLE 4: Deamination at different temperatures Aliquots of the treated RNAs after the clean-up step from EXAMPLE 3 were reverse transcribed and sequenced by using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760). The sequencing reads were aligned to the yeast reference genome. Mismatches occur where adenosines were converted to inosines and cytosines were consequently incorporated during reverse transcription. Upon amplification of the DNA for sequencing, guanosines are present in place of deaminated adenosines. Data shown in Table 2 demonstrates the relatively higher conversion of adenosines to inosines at 42°C in the FLO1, HSC82 and RPS31 gene transcripts. TABLE 2: Conversion of adenosine to inosine by HPAMPD Genome position Chr I: 207455 Chr XIII: 632938 Chr XII: 499134 Transcript FLO1 HSC82 RPS31 e ra e o eam na on o e ree nuc eo e y s . mes grea er a 42°C than at 25°C. In contrast, a relatively higher conversion of adenosine to inosine was seen to occur (FIGURE 7) at 25°C (8% conversion) rather than 42°C (1% conversion) for the AHP1 transcript (S. cerevisiae genomic position Chr XII:369167). The higher conversion at the lower temperature correlated with the two different structures predicted for AHP1 at the two different temperatures. Specifically, RNAfold (available at http: / / rna.tbi.univie.ac.at) predicts that the affected adenosine (FIGURE 7, right arrow) occurs in a loop structure at 25°C but the same adenosine (FIGURE 7, left arrow) participates in a Watson-Crick base pair at 42°C. Analysis of the entire transcriptome indicates 6% of A to I conversions are higher at 25°C than 42°C. EXAMPLE 5: Deamination of 5’ AMP with HPAMPD under various reaction conditions Relative rates of deamination of 5’ AMP were determined in different reaction conditions. Results are shown in TABLE 3. PATENT APPLICATION NEB-481-PCT TABLE 3: 5’AMP conversion under different reaction conditions Reaction conditions Relative rate of deamination of 5’ AMP in various reaction conditions 20 M T i H 75 50 M N Cl 1 HPAMPD Saccharomyces cerevisiae (S288C) total RNA was subjected to adenosine deamination with 2.75 µM HPAMPD at 42° C for 60 minutes under three different reaction conditions, namely (a) 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2; (b) 20 mM Na Acetate pH 6.0, 50 mM NaCl; and (c) 20 mM Tris-HCl, pH 7.5, 50 mM NaCl. Aliquots of the treated RNAs after the clean-up step from EXAMPLE 3 were reverse transcribed and sequenced by using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760). The sequencing reads were aligned to the yeast reference genome. Mismatches occur where adenosines were converted to inosines and cytosines were consequently incorporated during reverse transcription. Upon amplification of the DNA for sequencing, guanosines are present in place of deaminated adenosines. FIGURE 8 shows the percent conversion of A to G at all the A positions and the three different reaction conditions across 100 base region of RPS31 gene RNA transcript between genomic positions Chr XII:499100-499200. EXAMPLE 7: Deamination of AHP1 gene RNA at 3 different reaction conditions with HPAMPD Total yeast RNA was treated as described in EXAMPLE 6 under the indicated three reaction conditions with HPAMPD and analyzed at all the A positions across the 52 base region of AHP1gene RNA transcript between genomic positions XII:368893-368945. Results are shown in FIGURE 9. EXAMPLE 8: Deamination of ERG3 gene RNA with RhDa01_extN10 PolyA enriched Yeast RNA (100 ng) was subjected to deamination with a cytidine deaminase, namely RhDa01_extN10, at 37° C for 60 minutes, specifically, 0.3 µg of RhDa01 in 30 µL of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2. Treated RNA molecules PATENT APPLICATION NEB-481-PCT were sequenced using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760). Sequencing reads were aligned to the yeast reference genome. Mismatches occur where cytidines were converted to uracils and adenosines were consequently incorporated during reverse transcription. Upon amplification of the DNA for sequencing, thymidines are present in place of deaminated cytidines. FIGURE 10 shows the percent conversion C to T at all the C positions across a 86 base region of ERG3 gene RNA transcript between genomic positions Chr XII:254476-254562. EXAMPLE 9: TADA8.20 deamination of yeast RNA PolyA enriched yeast RNA (100 ng) was subjected to adenosine deamination with 0.8 µM TadA8.20 at 37° C for 60 minutes in 30 µL of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2. The treated RNA were sequenced using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760). The sequencing reads were aligned to the yeast reference genome. Mismatches occur where adenosines were converted to inosines and cytidines were consequently incorporated during reverse transcription. Upon amplification of the DNA for sequencing, guanosines are present in place of deaminated adenosines. FIGURE 11 shows the percent conversion A to G at all the A positions across a 74 base region of ERG3 gene RNA transcript between genomic positions Chr XII:254478-254552. EXAMPLE 10: HPAMPD and DMS reactivities of adenosines of in vitro transcribed E. coli 5S RNA In vitro transcribed E. coli 5S RNA was incubated with 2.75 µM HPAMPD in 30 µL of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2at 37° C for 60 minutes. Treated RNA molecules were sequenced using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760). Sequencing reads were aligned to the reference sequence of the E.coli 5s rRNA (SEQ ID NO:9). In FIGURE 13A, the secondary structure of E. coli 5S RNA is depicted and the normalized reactivity of the adenosines after HPAMPD deamination is shown at adenosine (“A”) nucleotide positions that have low reactivity (white), medium activity (gray) or high reactivity (black). The relative reactivity of dimethyl sulfate (DMS) with E. coli 5S RNA adenosines (right image) is from P. Cordero, W. Kladwang, C. C. VanLang, R. Das, Biochemistry.51, 7037–7039 (2012). The reactivity of the adenosines shown in the HPAMPD and DMS reactions generally correlate, however there are differences, for example the different reactivities at PATENT APPLICATION NEB-481-PCT positions A55, A75, A94, and A117. These differences indicate there is different informational content on the flexibility and accessibility from the two methods of probing RNA structure. The information from the deamination experiments provides a complimentary set of data constraints that may be used in tandem with other methods to improve the predictive power for researchers using secondary structure prediction methods. EXAMPLE 11: HPAMPD deamination of in vitro transcribed RNA Four in vitro transcribed RNAs of known structure were deaminated with HPAMPD in triplicate in accordance with EXAMPLE 10. The four constructs were, the Turnip yellow mosaic virus tRNA-like structure (TYMV-TLS; Hartwick, E. W. et al. Ribosome-induced RNA conformational changes in a viral 3'-UTR sense and regulate translation levels. Nat Commun 9, 5074 (2018)) (FIGURE 15A), the exonuclease resistant RNA 1 (xrRNA1) from Zika Virus (ZkV-xrRNA; Akiyama, B. M. et al. Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a cellular exonuclease. Science 354, 1148-1152 (2016)) (FIGURE 15B), the Adenovirus virus associated RNA1 (VA-1; Hood, I. V. et al. Crystal structure of an adenovirus virus-associated RNA. Nat Commun 10, 2871 (2019)) (FIGURE 15C), and the RNAseP catalytic domain minimal crystal construct (RNAseP; Kazantsev, A. V., Krivenko, A. A. & Pace, N. R. Mapping metal-binding sites in the catalytic domain of bacterial RNase P RNA. RNA 15, 266-276 (2009)) (FIGURE 15D). All four RNAs were deaminated under identical conditions in a 20 µL reaction consisting of 0.53 µM HPAMPD (or no enzyme control), 20 mM Tris-HCl, pH 7.5, 50 mM NaCl at 37° C for 60 minutes. Treated RNA molecules were sequenced using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760). Sequencing reads were aligned to a fasta files containing SEQ 10, SEQ 11, SEQ 12, SEQ 13 and the A->I conversion (deamination rate) was calculated at each position. Deamination levels were binned into 4 categories based on percent deamination at each of the 130 A sites present in all four in vitro RNA constructs as follows: background (0.0-0.1%), low (0.10-0.3%), medium (0.3%-6.5%), and high (6.5-24%). The low, medium, and high classification was determined by the distribution of deamination rate (%) of a concatenated data frame of the 130 A’s present in each of the four RNAs with 0-25% quartile (low), 25- 75% quartile (medium), and 75%+ (high). Average HPAMPD reactivities were overlaid onto the RNA secondary structure diagrams that only depict canonical trans Watson-Crick base pairs and G-U wobble base pairing without consideration of any non-Watson-Crick base pairs or tertiary interactions. PATENT APPLICATION NEB-481-PCT Across all the RNA constructs, deamination sites were observed in predicted unpaired regions of the RNA’s secondary structure (loops, bulges, linker regions) and less so in helices. This indicates the adenosines in these regions are sufficiently flexible and accessible to the deaminase, while base paired double-stranded regions are not accessible for deamination. The deamination levels may be used as a proxy for detecting unpaired regions in vitro analogous to other structure probing methods. EXAMPLE 12: Deamination of RNAseP RNA with a cytosine deaminase, MsddA MsddA is a non-specific, modification-sensitive dsDNA deaminase that deaminates cytosines to uracils in both DNA and RNA molecules (Vaisvila et al., Molecular Cell 84, 854–866 (2024)). The RNAseP catalytic domain minimal crystal construct (RNAseP) described by Kazantsev et al. (RNA 15, 266-276 (2009)) was deaminated in duplicate with MsddA in accordance with EXAMPLE 10. Average MsddA reactivities were overlaid onto the predicted RNA secondary structure diagram (FIGURE 16). MsddA deamination percentages were binned into 4 categories: background (0.0-0.1%), low (0.10-0.25%), medium (0.25%-2.5%), and high (2.5-15%). The low, medium, and high bins were determined from by the 0-25% quartile (low), 25-75% quartile (medium), and 75%+ (high) of the 60 cytosines present in the RNase P molecule. Under the conditions tested, MsddA deaminated cytosines to uracils present in both paired and unpaired regions of RNAseP. Out of the 60 C’s present in the molecule 76.6% of the C’s are deaminated by MsddA above the background level. It appears that MsddA, under the conditions tested, may deaminate C’s that are present both in both paired and unpaired regions of the molecule. Many (8 / 10) of the highly deaminated C’s occur in predicted paired region that are close to junctions or helical termini. MsddA’s differential base specificity for deamination (relative to adenosine deaminases) may be used alone or combined with an adenosine deaminase for even more information about RNA structure. In a simple example, an RNA of interest comprising approximately even proportions of canonical ribonucleotides contacted with an adenosine deaminase or a cytosine deaminase might provide structural information at up to approximately one quarter of the positions within the RNA. This information, however, might not allow the position or even the presence of small bubbles or loops (e.g., 1-6 nucleotides) in the structure of the RNA to be found or confirmed. Separately contacting the same RNA of interest with each of an adenosine deaminase and a cytosine deaminase might provide structural information at up to approximately one half of the PATENT APPLICATION NEB-481-PCT positions within the RNA and reduce or eliminate the risk of missing or misapprehending the position of small bubbles or loops. EXAMPLE 13: Effects of magnesium on HPAMPD deamination and DMS reactivity on the RNAseP RNA RNAseP was both deaminated with HPAMPD and modified with DMS. Specifically, 200ng of RNAseP was modified with 105mM DMS (or DMSO control) in 100uL total volume of 20mM Tris-HCl, pH 7.5, 50mM NaCl, and either 0 mM or 5 mM MgCl2 for 5 minutes at 37°C. The reaction was quenched with 60% BME and cleaned up using the Zymo RNA Clean & Concentrator-5 spin columns (Zymo, R1013). The modified RNA was reverse transcribed to cDNA with 5.0 uM 3’-specific RNAseP single stranded DNA primers with 200U of Induro® Reverse Transcriptase (New England Biolabs, M0681) following the manufacturers protocol. The resultant cDNA was PCR amplified to double stranded DNA using 5’ and 3’ specific DNA primers, and sequencing libraries were prepared using the NEBNext UltraExpress® DNA Library Prep Kit (New England Biolabs, E3325). The final libraries were sequenced with Illumina Next generation sequencing. Sequenced data was processed using RNA Framework (Incarnato et al., Nucleic Acids Res 46, e97 (2018)) in combination within house python scripts. For deamination 200ng of in vitro transcribed RNAseP was subjected to deamination with 0.53 µM HPAMPD at 37° C for 60 minutes in 20 µL of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and either 0 mM or 5 mM MgCl2. The treated RNA was prepped for sequencing using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760) followed by Illumina next generation sequencing. Deamination levels were binned into 4 categories based on percent deamination at each of the 130 A sites present in all four in vitro RNA constructs as outlined in EXAMPLE 11. Regions of high level HPAMPD deamination are shown in FIGURE 17A, outlined in boxes, which occur in in predicted unpaired regions in the 0mM MgCl2conditions. When assayed with 5mM MgCl2, there is an overall reduction of HPAMPD deaminase activity seen in all regions of interest (FIGURE 17A and FIGURE 17B). This is due to the adoption of the divalent cation dependent tertiary fold, both stabilizing and burying residues within the molecule reducing accessibility to the enzyme. Compared to the deaminase treatment, only small reductions in reactivity are observed with DMS in the regions of interest. PATENT APPLICATION NEB-481-PCT EXAMPLE 14: Deamination of in vitro transcribed RNAseP with TadA8r In vitro transcribed RNAseP was deaminated with TadA8r (Xiao et al., Nat Biotechnol 42, 1442-1453 (2024)) in 0mM or 5mM MgCl2 in accordance with EXAMPLE 10. Briefly, 200ng of in vitro transcribed RNAseP was subjected to deamination with 0.25 µM TadA8r in 30 µL of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and either 0 mM or 5 mM MgCl2at 37° C for 60 minutes. Deaminated RNA was sequenced using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760) coupled with Illumina next generation sequencing. TadA8r reactivities were overlaid onto the predicted RNA secondary structure diagram for each MgCl2 condition (FIGURE 18A, FIGURE 18B). The deamination rate (%) of adenosines were binned into 4 categories: background (0.0-0.1%), low (0.10-0.52%), medium (0.52%-3.6%), and high (2.5- 15.4%) based on the 72 A sites in the RNAseP molecule. The low, medium, and high bins were determined from by the 0-25% quartile (low), 25-75% quartile (medium), and 75%+ (high) of the 72 A’s present in RNAseP. TadA8r appears to deaminate across the entire RNA molecule with positions of high deamination occurring predominantly in unpaired regions (FIGURE 18A & FIGURE18B). In the 0mM MgCl2condition the overall deamination rates are higher (FIGURE 18A) than in the 5mM MgCl2 condition (FIGURE 18B), due to the adoption of the divalent cation dependent tertiary structure. The frequency and rate of deamination differs from other adenosine deaminases such as HPAMPD. This example demonstrates the unique activity and preference by other adenosine deaminases. EXAMPLE 15: Response of non-Watson-Crick base pairs to deamination and chemical probing RNA secondary structure depictions are usually drawn where some RNA regions consist of bases that are unpaired such as loops and bulges and in other regions where bases are paired and form a helical stem with Watson-Crick hydrogen bonds in a configuration that form the minimum free energy structure overall. These minimum free energies are predicted from calculations with various optional parameters. Furthermore, some depictions in addition to Watson-Crick hydrogen bonding consider pairing which involve a multitude of other hydrogen bonds (Leontis, Stombaugh, Westhof 2002) five of which of which are shown in FIGURE 19A, FIGURE 19B, FIGURE 19C,and FIGURE 19D. The various parameters and the application of non-Watson-Crick base pairs may alter the secondary structure predictions. In addition, computational predictions may also include PATENT APPLICATION NEB-481-PCT experimental probing data which improve the accuracy of secondary structure predictions. Probing with deaminases may supplement experimental probing data and determine potential non-Watson-Crick base pairs and lead to a more accurate secondary structure prediction than that obtained by solely using chemical probing data. In these examples the secondary structure prediction in the boxed portion of both FIGURE 19A and FIGURE 19C depicts unpaired regions, however, these regions are stabilized by non-Watson-Crick base pair interactions which were observed from biophysical studies. When examining the RNAseP crystal structure (PDB ID 3DHS) (FIGURE 19B) the region boxed in the secondary structure consists of three non-Watson Crick base pair interactions depicted in the Leonitis-Westhof notation. These nucleotides are stabilized through hydrogen bonding constraining, the nucleotides within a helix. These nucleotides are not accessible to the deaminase HPAMPD, but are highly reactive to the small molecule DMS which is still able to methylate the Watson-Crick face of the A’s which are still solvent accessible. FIGURE 19C shows the E loop of the E.coli 5s rRNA which is drawn with a large bulged out single stranded region. When investigating the NMR structure of this region (PDB ID: 1A4D) this region is helical and stabilized by multiple non-Watson Crick base pair interactions. Similar to RNAseP in FIGURE 19A&B, these A’s (A89,A94,A115,A117, and A120) are not accessible to the deaminase but are still accessible by the small molecule DMS. The difference in reactivity of this region to deaminase and chemical probing may inform the secondary structure prediction and indicate there may be alternative hydrogen bonds that would abrogate the secondary structure prediction based only on Watson-Crick base pairs. EXAMPLE 16: Cleavage of inosine containing RNA with Thermococcus kodakarensis endonuclease V The ability of archaeal endonuclease V to cleave various single stranded and double stranded RNA and DNA nucleic acids containing a single site-specific inosine (in RNA) or deoxyinosine (in DNA) was determined (schematic depicted in FIG 20A). FAM-labeled ssRNA (SEQ ID NO:15), dsRNA(SEQ ID NO:17), RNA:DNA, ssDNA (SEQ ID NO:21), dsDNA, or DNA:RNA hybrid substrates (20nM final) containing a single inosine / deoxyinosine in 1X Thermopol buffer (New England Biolabs, Ipswich MA) (20 mM Tris-HCl,10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton® X-100, pH 8.8@25°C) was incubated with Thermococcus kodakarensis endonuclease V @ 60 °C for various time points, and quenched with 10 uL of 50 mM EDTA to halt the reaction. Reaction PATENT APPLICATION NEB-481-PCT products were separated and analyzed by capillary electrophoresis (FIG.20B). The fraction of cleaved product was calculated, plotted and fit to a single-exponential equation ((y = m1*(-exp(-m2*m0)) (FIG.20C and TABLE 4). TABLE 4 TkobKchem(m-1) ssRNAaa24^3.4dsRNA:RNA 37^0.7 dsRNA:DNAa37^2 ssDNAaa0.1^0.003dsDNA:DNAa0.25^0.01dsDNA:RNA0.20^0.01a Reactions were performed in triplicate and ^ denotes standard deviation b Reactions were performed at 60º C EXAMPLE 17: Site-specific detection of adenosine to inosine deamination in ssRNA using an adenosine deaminase and an archaeal endonuclease V FIGURE 21A illustrates an example method of determining the location(s) of adenosine deamination (A to I) comprising (a) contacting and RNA comprising adenosine and an adenosine deaminase to form a deamination product, (b) contacting the deamination product with an archaeal endonuclease V to form RNA cleavage products, wherein cleavage occurs at the second phosphodiester bond 3’ to each inosine, and (c) optionally (i) sequencing the RNA cleavage products (e.g., by high-throughput sequencing methods of Illumina, ONT, or Pacific Biosciences) or (ii) (1) fractionating and fragmenting the cleavage product to produce cleavage product fragments (e.g., LC-MS / MS, UHPLC-MS / MS) and (2) characterizing the cleavage product fragments. A specific example reaction may comprise: (a) contacting a 32mer ssRNA (30 pmol; SEQ ID NO:18) and TadA8.20 (40 µM final) for 90 min in 1X TadA8.20 reaction buffer (50 mM Tris / Cl pH 7.5, 25 mM KCl, 2.5 mM MgCl2, 2 mM DTT, 10% glycerol) at 37 °C to produce a deamination product; (b) contacting the deamination product and Thermococcus kodakarensis endonuclease V (1 µM final) @ 65° C for 15 min to produce cleavage products; and (c) analyzing the cleavage products by UHPLC-MS / MS (e.g., as described by Wolf et al., ACS Pharmacol. & Transl. Sci.6(11):1692-1702. Idealized spectra representing expected results of these conditions are shown in FIG.21B. EXAMPLE 18: Deamination of adenosines revealed by endonuclease V cleavage PATENT APPLICATION NEB-481-PCT The Hepatitis C IRES (HCV) in vitro transcript (330ng) in 10 µL of 20 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2 was incubated with 250 nM HPAMPD (A lanes) or 25 nM HPAMPD (B lanes) or no HPAMPD (C lanes) for 60 minutes at 37° C.1 µL of CutSmart Buffer (NEB) was added to each 10 µL reaction and split into 5.5 µL reactions. Five units (0.5 µL) of Thermococcus kodakarensis endonuclease V was added to one of the 250 nM HPAMPD reactions ( A + lane), and to one of the 25 nM HPAMPD reactions (B + lane) and to one of the no HPAMPD reactions (C + lane). No EndoV was added to the A-, B- and C- lanes. All 6 reactions were then incubated at 65 degrees for 10 minutes. The reactions were terminated by the addition of 6 µL of RNA Loading Dye heated for 2 minutes at 85 degrees and electrophoresed on a 6% TBE- Urea polyacrylamide gel (FIGURE 22A). The resulting fragment sizes are in agreement with cleavage by endonuclease V occurring at the most highly deaminated adenosines located within the HCV molecule by sequencing. (FIGURE 22B). The HCV RNA is 396 nucleotides in length and there are 2 reactive adenosines in the HCV RNA. One is located at nucleotide # 72 (FIGURE 22A) and one at 320 (FIGURE 22B). Cleavage at these positions would result in fragment lengths of 67, 72, 257 and 327 nucleotides, which are the observed sizes of fragments in FIGURE 22A. EXAMPLE 19: HPAMPD deamination of a thiamine pyrophosphate-specific riboswitch with or without thiamine pyrophosphate Riboswitches are a class of structured, highly conserved RNA molecules that may influence post-transcriptional processes through a conformational shift upon binding a specific small molecule or nucleic acid (Kavita, Kumari, and Ronald R. Breaker. "Discovering riboswitches: the past and the future." Trends in biochemical sciences 48.2 (2023): 119-141). The thiamine pyrophosphate-specific riboswitch (TPP Riboswitch) is a dynamic, well-studied riboswitch which undergoes a structural rearrangement upon binding its ligand TPP. This example outlines the ability of deaminase probing to detect conformational between the TPP riboswitch in the presence or absence of TPP. Briefly, 5 pmol of in vitro transcribed TPP riboswitch (SEQ ID NO:20) was denatured and then renatured / folded in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5 mM MgCl2 at 37°C for 10 minutes. After folding, TPP (+TPP) or water (-TPP) was added to the reaction at a final concentration of 5 µM, and then incubated at 37°C for 20 minutes. Next 0.53 µM HPAMPD (or no enzyme control) was added to the reaction (30 µL total volume) and incubated at 37°C for 60 minutes. The deaminated RNA was prepped for sequencing PATENT APPLICATION NEB-481-PCT using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760) and sequenced via Illumina next generation sequencing. Deamination rates (%) were compared between the -TPP and +TPP conditions (FIGURE 24). Three positions A40, A75, and A85 were reduced to background levels of reactivity in the +TPP condition, indicative of the conformational switch in the presence of TPP. These results demonstrate that deaminase probing may identify conformational changes of an RNA molecule of interest upon binding a small molecule. This is particularly useful in the context of RNA therapeutic design and identifying how a specific small molecule or other drug affects a given RNAs conformation upon binding.

Claims

PATENT APPLICATION NEB-481-PCT CLAIMS What is claimed is:

1. A method for deaminating a nucleic acid comprising: (a) contacting (i) a deaminase; and (ii) a nucleic acid acid (A) comprising one or more nitrogenous bases selected from adenine and cytosine, and (B) having a nucleotide sequence to produce a deamination product comprising one or more deaminated adenines and / or one or more deaminated cytosines; (b) determining the position in the nucleotide sequence of (i) the one or more deaminated adenines and / or (ii) the one or more deaminated cytosines; and (c) generating secondary structure context data for one or more of the nitrogenous bases corresponding to the one or more deaminated adenines and / or the one or more deaminated cytosines.

2. A method according to Claim 1, wherein the deaminase is an adenosine deaminase or a cytosine deaminase.

3. A method according to Claim 1 or 2, wherein the nucleic acid has a predicted secondary structure and the method further comprises (d) annotating the predicted secondary structure with the generated secondary structure context data for the one or more deaminated adenines and / or the one or more deaminated cytosines.

4. A method according to any of Claims 1-3, wherein the deaminated adenines are inosines and / or the deaminated cytosines are uracils.

5. A method according to any of Claims 1-4, wherein the nucleotide has a size of ≤ 20,000 nucleotides.

6. A method according to any of Claims 1-5, wherein the nucleotide has a size of ≤ 2,000 nucleotides.

7. A method according to any of Claims 1-6, wherein the nucleotide has a size of ≤ 200 nucleotides.PATENT APPLICATION NEB-481-PCT 8. A method according to any of Claims 1-7, wherein the nucleic acid consists of RNA.

9. A method according to Claim 8, wherein the deaminase is an adenosine deaminase that acts on RNA.

10. A method according to Claim 8 or Claim 9, wherein the RNA comprises adenine and the deaminase is an AMP deaminase.

11. A method according to any of Claims 8-10, wherein the the RNA comprises adenine and deaminase comprises an amino acid sequence at least 98% identical to any of SEQ ID NOS:1-3 12. A method according to any of Claims 8-11, wherein the RNA comprises adenosine and the deaminase is a Helix pomatia AMP deaminase.

13. A method according to Claim 8, wherein the RNA comprises cytosine and the deaminase is an APOBEC cytosine deaminase.

14. A method according to any of Claims 1-13, wherein the contacting occurs at a temperature of 20°C-50°C.

15. A method according to any of Claims 8-14 further comprising contacting the deamination product with an archaeal endonuclease V to form a nucleic acid cleavage product.

16. A method according to any of Claims 1-15 further comprising ligating an adapter to the 5’ end, to the 3’ end, or to both the 5’ end and the 3’ end of the deamination product or the cleavage product, in each case to form an adapter ligation product.

17. A method according to Claim 16, wherein the method comprises ligating an adapter to the 5’ end of the deamination product and ligating an adapter to the 3’ end of the deamination product and wherein the adapter ligated to the 5’ end is different from the adapter ligated to the 3’ end of the deamination product.

18. A method according to Claim 16 or Claim 17, wherein the determining further comprises sequencing the adapter ligation product.

19. A method according to Claim 16 or Claim 17 further comprising contacting the deamination product with a reverse transcriptase to copy the deamination product into a cDNA,PATENT APPLICATION NEB-481-PCT wherein the cDNA comprises cytosines at positions corresponding to the deaminated adenosines of the deamination product.

20. A method according to Claim 19, wherein the reverse transcriptase is Gka reverse transcriptase, Tbr reverse transcriptase, M-MuLV reverse transcriptase, Induro® reverse transcriptase, ProtoScript® reverse transcriptase, WarmStart® RTx reverse transcriptase, AMV reverse transcriptase, or combinations thereof.

21. A method according to Claim 19 or Claim 20 further comprising amplifying the cDNA to produce an amplified cDNA.

22. A method according to any of Claims 18-21, wherein the determining further comprises sequencing the cDNA or the amplified cDNA to produce one or more sequence reads.

23. A method according to Claim 22 further comprising comparing the sequence read to a reference sequence or the nucleotide sequence of the ribonucleic acid (x) to identify guanosines in the sequence read that match adenosines in (i) the reference sequence and / or (ii) the nucleotide sequence of the ribonucleic acid, and / or (y) to identify thymidines in the sequence read that match cytosines (i) in the reference sequence and / or (ii) the nucleotide sequence of the ribonucleic acid.

24. A method according to Claim 23 further comprising assigning a reactivity metric to each determined position of (i) the one or more deaminated adenosines and / or (ii) the one or more deaminated cytosines.

25. A method according to Claim 24 further comprising combining the assigned reactivity metric with a predicted structure of the ribonucleic acid to produce a modified predicted structure of the ribonucleic acid.

26. A method according to Claim 25, wherein the predicted structure is a covariation analysis structure, a DMS structure, or a selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) structure.

27. A method for producing at least one structural representation of an RNA comprising at least one adenosine and / or at least one cytosine, the method comprising:PATENT APPLICATION NEB-481-PCT (a) identifying a set of RNA molecules, the set comprising a plurality of individual RNA molecules, each with a different primary structure, and each comprising at least one adenine and / or at least one cytosine; (b) generating a set of candidate secondary structures (optionally, tertiary structures in addition to or instead of secondary structures) for each RNA molecule of the set with a secondary structure prediction system (optionally, a tertiary structure prediction system in addition to or instead of a secondary structure prediction system) and inputs to the secondary structure prediction system; (c) determining for each RNA molecule within the set a deamination reactivity of at least one (and optionally up to all) of the adenines in the RNA molecule and / or at least one (and optionally up to all) of the cytosines in the RNA molecule (e.g., by (I) contacting (x) a deaminase; and (y) the RNA molecule to produce a deamination product comprising one or more deaminated adenosines and / or one or more deaminated cytosines; and (II) determining the position in the nucleotide sequence of (x) the one or more deaminated adenines and / or (y) the one or more deaminated cytosines); (d) for each RNA molecule within the set, correlating the determined deaminase reactivity of the adenine(s) and / or cytosine(s) of each RNA molecule with one or more of the candidate secondary structures from the initial set; (e) ranking at least two (and optionally up to all) of the candidate structures for each RNA molecule; and (f) predicting the most likely secondary structures for each RNA molecule based on the correlation ranking, wherein the inputs comprise the primary structure of each RNA molecule, the deaminase reactivity, and the calculated thermodynamic free energy of each RNA molecule and the secondary structure prediction system determines the thermodynamic free energy of each RNA molecule within the set based on its primary structure and utilizes the thermodynamic free energy and primary structure to generate the set of predicted candidate secondary structures for each RNA molecule; wherein the ranking further comprises ranking the at least two of the candidate structures for each RNA molecule based on the correlation between secondary structure based on thermodynamic free energy and the deaminase reactivity.PATENT APPLICATION NEB-481-PCT 28. A method according to Claim 27, wherein generating a set of candidate secondary structures further comprises calculating a thermodynamic free energy for the RNA molecule (or a portion thereof) with or without accounting for deaminase reactivity of one or more (up to all) adenosines and / or cytosines in the RNA molecule or portion thereof.

29. A method enzymatically interrogating RNA structure comprising: (a) contacting (i) a deaminase; and (ii) an RNA (A) comprising one or more nitrogenous bases selected from adenine and cytosine, and (B) having a nucleotide sequence to produce a deamination product comprising one or more deaminated adenines and / or one or more deaminated cytosines; (b) determining the position in the nucleotide sequence of (i) the one or more deaminated adenines and / or (ii) the one or more deaminated cytosines; (c) generating secondary structure context data for one or more of the nitrogenous bases corresponding to the one or more deaminated adenines and / or the one or more deaminated cytosines; and (d) (i) building a secondary structure and optionally tertiary structure model of of the RNA using the generated secondary structure context data; or (ii) annotating or revising a pre-existing secondary and / or tertiary structure model of the RNA using the generated secondary structure context data; or (iii) annotating or revising a pre-existing secondary and / or tertiary structure model of the RNA using the generated secondary structure context data; or (iv) selecting the RNA for administration to a subject if the generated secondary structure meets a pre-determined criteria; or (v) selecting an interacting molecule and contacting the RNA with the interacting molecule to produce modify the structure of the RNA; or (vi) preparing a second RNA with a second nucleotide sequence, wherein differences between the nucleotide sequence and the second nucleotide sequence are selected in light of the generated secondary structure. .

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