Molecules and methods for modifying RNA with aryl groups

Abstract

Compositions and methods are provided for a class of electrophilic aromatic reagents that react with RNA in high yield to produce heteroaryl or aryl adducts at the 2′-OH positions of RNA. A number of structural variants of the reagent are described with applications for labeling, mapping, and profiling RNA interactions. The reagent and reactive group are easily synthesized, easily conjugated, function in pure water without organic solvents, are chemically stable for storage, and persist in water for a long time during reaction with RNA.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] Pursuant to 35 USC § 119(e), this application claims priority to the filing date of the following application:

[0003] U.S. provisional application serial number 63 / 603,981 filed on November 29, 2023, the disclosure of said application is incorporated herein by reference.

[0004] Government support

[0005] This invention was made with government support under contract GM145357 granted by the National Institutes of Health. The government holds specific rights to this invention. Background Technology

[0006] Molecules that react with RNA to form chemical bonds have proven useful in numerous applications in biology, biotechnology, and medicine. For example, RNA-reactive molecules are widely used to map the folded structure of RNA. In such applications, molecules such as dimethylsulfate (DMS) and nitro isatoic anhydride (1M7) preferentially react with unpaired nucleotides over nucleotides within the double-stranded structure. The application of reverse transcriptase enzymes and RNA-specific DNA primers induces polymerase stoppage at the chemical reaction site, and analyzing these stops yields information regarding RNA folding. In a second example, reagents that react with the 2′-OH groups of RNA can be used to introduce biotin or fluorescent labels into the RNA, allowing researchers to isolate, separate, image, and quantify the RNA of interest.

[0007] Some applications of RNA-reactive molecules add utility in living cellular environments. For example, groups that react with high yield at the 2′-OH group of RNA can be used to stabilize RNA from hydrolysis and enhance its protein expression. Additionally, RNA-reactive functional groups can be attached to drugs or drug candidates, allowing researchers to identify the cellular RNA to which the drug binds.

[0008] To date, the types of RNA-reactive chemical structures are limited. This is particularly true for compounds that react with 2′-OH groups. Reacting with the 2′-OH groups of RNA is potentially very useful because such groups appear at virtually every position on all RNA, potentially enabling a wide range of applications as mentioned above. However, to date, only acyl (carbonyl) groups and structurally similar sulfonyl groups are known to react with 2′-OH groups, providing limited structural diversity as adducts on RNA. In most cases, known reagents require a cosolvent, such as DMSO, to maintain solubility with RNA, which can be toxic to cells, interfere with RNA folding, or disrupt cell biology. Acyl reagents often have a short lifetime in water, limiting their usefulness as reactants. In some cases, adducts on RNA after reaction may be unstable to hydrolysis, causing them to detach prematurely from the RNA before their applications can be realized.

[0009] The present specification provides a composition and a method for modifying RNA.

[0010] Molecules that react with RNA are useful for modifying the properties of RNA and probing RNA structure and function. Compositions and methods are provided for a class of electrophilic aromatic reagents that react with RNA in high yield to produce heteroaryl or aryl adducts at the 2′-OH positions of RNA. A number of structural variants of the reagent are described along with applications for labeling, mapping, and profiling RNA interactions. The reagent and reactive group are easily synthesized, easily conjugated, function in pure water without organic solvents, are chemically stable for storage, and persist in water for a long time during reaction with RNA.

[0011] A reactant that selectively modifies RNA at the 2′-OH group is provided. In some embodiments, the reactant has structure (I).

[0012] (I)

[0013]

[0014] Here, LG is a leaving group and may include a halide, e.g., F, Cl, Br, I, etc.; ammonium salts, tertiary amines, e.g., trialkylamine, -N(CH3)3 + , may be N-methyl morpholine (NMM);

[0015] X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, provided that at least one of X and Y is nitrogen;

[0016] R1 and R2 are selected independently and may be absent or present. If present, R1 and R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, -NHR, and may include an azide or alkyne reactant group suitable for click chemistry, e.g., NHCH2CH2OCH2CH2N3.

[0017] In some embodiments, the reactants have structure (II), where LG, X, R1 and R2 are as defined above.

[0018] (II)

[0019]

[0020] Examples of reactants include, without limitation, those presented in FIG. 3, and include, without limitation, the following:

[0021] (DMTMM); (2PMM);

[0022] (2PN3); (AzTMM)

[0023] (DMTN3); (AzTN3)

[0024] In some embodiments, the aromatic compound of interest for the reaction in the method of the present disclosure is activated by a tertiary amine, for example, by substituting a halogen leaving group on a halogen-substituted heterocycle of Formula I or Formula II with N-methylmorpholine (NMM) or trimethylamine. In some embodiments, the reactants comprise an azide or alkyne functional group for click chemical conjugation as one or both of R1 and R2, for example, using Cu-mediated or Cu-free click chemistry.

[0025] In some embodiments, a composition comprising a compound of structure (I) is provided. In some embodiments, the compound is provided in powder form. In some embodiments, the compound is provided as a solution, e.g., an aqueous solution. In some embodiments, the compound is provided as a kit. The kit may further include instructions for use, click chemical reactants, e.g., detectable labels, therapeutic moiety, RNA control samples, etc.

[0026] The reactant of the present disclosure selectively reacts with RNA 2′-OH groups, for example, its reactivity may be at least 2 times greater than its reactivity with DNA, at least 5 times greater than its reactivity with DNA, and at least 10 times greater than its reactivity with DNA. The reaction may be carried out with high efficiency in an aqueous solution, for example, in the absence of an organic solvent such as DMSO, wherein the reaction may proceed to at least about 50% conversion of RNA, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more. The reactant is selective for ssRNA compared to dsRNA. The aryl adduct on RNA may block enzymatic reactions, for example, polymerase.

[0027] In some embodiments, a method for selectively modifying RNA at the 2′-OH group is provided, said method comprising contacting RNA with a reactant of structure (I). In some embodiments, said reaction is carried out in an aqueous solution. In some embodiments, said reaction is carried out in vitro. In some embodiments, said reaction is carried out in vivo. In some embodiments, said RNA is mRNA and is selectively modified at the polytail, for example, to reduce enzymatic degradation and increase stability.

[0028] In some embodiments, the reaction produces RNA having click chemoadditions, e.g., azide or alkyne groups, including, e.g., strained cyclooctyne. In some embodiments, the RNA is further modified by conjugation at the click chemoadditions and is conjugated, e.g., to detectable groups, therapeutic moiety, drugs for profiling, etc.

[0029] In some embodiments, a method for mapping RNA structures is provided in vivo, e.g., in living cells, or in vitro, wherein RNA is reacted according to the method disclosed herein so that the RNA is preferentially modified in unfolded (single-stranded) regions. The regions may be mapped by labeling the reacted sequence, by reaction with an enzyme that stops at the adduct site, etc., and by analysis by gel electrophoresis, sequencing, hybridization, etc., as known in the art.

[0030] In some embodiments, a method for reactivity-based RNA profiling (RBRP) is provided. To analyze the interaction between a drug and RNA across the cell, the RNA of interest is reacted with a reactant of structure (I) or (II), said reactant comprising a drug of interest that binds to the RNA as an R1 or R2 group. When said conjugate is exposed to a cell containing RNA, an adduct is introduced at the drug binding site, which can be analyzed as described above. In some embodiments, said LG group comprises the drug of interest, and after the drug binds and the aromatic group reacts, an aromatic adduct is left on the RNA as a tag. Brief explanation of the drawing

[0031] The present invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. In accordance with common practice, it is emphasized that various features in the drawings are not drawn to scale. On the contrary, the dimensions of various features have been enlarged or reduced at will for clarity. The drawings include the following. Fig. 1: Initial set of reagents screened for reaction with RNA 2′-OH in water (all purchased from various commercial suppliers). Numbers (in %) represent the conversion yield estimated by MALDI-TOF MS after ethanol precipitation: the percentage of total tRF3 RNA in which at least one 2′-OH was covalently modified by an electrophilic reagent after the reaction. "nd" indicates no conversion was observed, and *: no RNA recovered after ethanol precipitation. All molecules described in this figure did not react with DNA oligonucleotides having the same sequence as tRF3. Fig. 2: (A) Reaction scheme for RNA 2′-OH modification using electrophilic aromatic reagents. (B) MALDI-TOF M / S analysis of the reaction between 200 mM Reagent 11 and tRF3 RNA. The number of aryl adducts on RNA is indicated by the red label. (C) MALDI-TOF M / S analysis of the reaction between 200 mM Reagent 11 and tRF3DNA (DNA oligonucleotide having the same sequence as tRF3 RNA). No DNA modification was detected. Note: Reaction performed with 18-mer tRF RNA (or tRF3DNA), reaction volume 10 μL, 20% DMSO, reaction time 24 h, temperature 37 °C, [MOPS] = [NaCl] = 100 mM, [MgCl2] = 6.06 mM, pH 7.5, MALDI-TOF M / S analysis after ethanol precipitation. Fig. 3: Structure and reactivity of the investigated cationic aryl reagent. Note: Reaction performed with 18-mer tRF RNA (or tRF3DNA), reaction volume 10 μL, reaction time 24 h, temperature 37 ℃, [MOPS] = [NaCl] = 100 mM, [MgCl2] = 6.06 mM, pH 7.5, MALDI-TOF M / S analysis after ethanol precipitation. Fig. 4: Measurement of the hydrolysis half-life of DMTMM. This reagent is estimated to have a half-life of >10 days by fitting the natural logarithm of the mole fraction of DMTMM to a straight line. NMR sample preparation: 15 mg of DMTMM is dissolved in 750 μL of D2O containing 5 μL of acetonitrile as an internal standard. Fig. 5: MALDI-TOF M / S analysis of tRF3 and tRF3DNA after reaction with 200 or 100 mM DMTMM under 0% or 20% DMSO conditions. Fig. 6. Mass peak. Specific details for implementing the invention

[0032] Before describing the method and composition, it should be understood that the present invention is not limited to the specific method or composition described and, of course, may vary. Furthermore, it should be understood that the terms used herein are merely for the purpose of describing specific embodiments and are not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0033] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range is specifically disclosed, up to one-tenth of the lower limit unit, unless the context clearly indicates otherwise. Any smaller range between any described value or intervening value within the described range and any other described value or intervening value within the described range is included in the present invention. The upper and lower limits of such smaller ranges may be independently included in or excluded from the range, and any range in which one limit, both limits, or neither limit is included in the smaller range is also included in the present invention, subject to any limit specifically excluded within the described range. Where the described range includes one or both limits, the range excluding one or both of the included limits is also included in the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. Any method and material similar or equivalent to that described herein may be used in the practice or testing of the present invention, but some potential and preferred methods and materials are described hereafter. All publications mentioned herein are referenced by reference in this specification to disclose and describe methods and / or materials related to those cited. It is understood that this disclosure takes precedence over any disclosure of the referenced publications to the extent of any inconsistencies.

[0035] It should be noted that the singular forms “a,” “an,” and “the” used in this specification and the appended claims include plural references unless the context clearly indicates otherwise. Accordingly, for example, a reference to “cell” includes a plurality of such cells, and a reference to “peptide” includes one or more peptides and their equivalents known to those skilled in the art, e.g., polypeptides, etc.

[0036] The publications discussed herein are provided only for their disclosures prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment that the present invention is not qualified to precede such publications by prior art. Furthermore, the provided disclosure dates may differ from actual disclosure dates that may need to be independently verified.

[0037] Oilseed Raw Materials Acros Organics(Pittsburgh PA), Aldrich Chemical(Milwaukee WI, Sigma Chemical 및 Fluka 포함), Apin Chemicals Ltd.(Milton Park UK), Avocado Research(Lancashire UK), BDH Inc.(Toronto, Canada), Bionet(Cornwall, UK), Chemservice Inc.(West Chester PA), Crescent Chemical Co.(Hauppauge NY). Company(Rochester NY), Fisher Scientific Co.(Pittsburgh PA), Fisons Chemicals(Leicestershire UK), Frontier Scientific(Logan UT), ICN Biomedicals, Inc.(Costa Mesa CA), Key Organics(Cornwall UK), Lancaster Synthesis(Windham NH), Maybridge Chemical Co. Ltd.(Cornwall UK), Parish Chemical Co.(Orem UT), Pfaltz & Bauer, Inc.(Waterbury CN), Polyorganix(Houston TX), Pierce Chemical Co.(Rockford IL), Riedel de Haen AG(Hannover, Germany), Spectrum Quality Product, Inc.(New Brunswick, NJ), TCI America(Portland OR), Trans World Chemicals, Inc.(Rockville MD), Wako Chemicals USA, Inc.It may be obtained from commercial suppliers including, but not limited to (Richmond VA), Novabiochem and Argonaut Technology.

[0038] As used herein, 'methods known to those skilled in the art' may be identified through various reference books and databases. Suitable reference books and papers that describe the synthesis of reactants useful for the preparation of the compounds of the present invention, or provide references to papers describing such preparation, include, for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; SR Sandler et al., "Organic Functional Group Preparations", 2nd Ed., Academic Press, New York, 1983; HO House, "Modern Synthetic Reactions", 2nd Ed., Benjamin, Inc., Menlo Park, Calif., WA, 1972; TL Gilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; Includes J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 4th Ed., Wiley-Interscience, New York, 1992. Specific and similar reactants can also be identified through the index of known chemicals compiled by the American Chemical Society’s Chemical Abstract Service, which is available at most public and university libraries, and through online databases (for further information, contact the American Chemical Society, Washington, DC). Chemicals known in the catalog but not commercially available can be manufactured by custom chemical synthesizers, and many of the standard chemical suppliers (e.g., those listed above) provide custom synthesis services.

[0039] The term "alkyl" refers to C1-C which can be linear, branched, or cyclic. 20 It refers to an alkyl group. "Lower alkyl" refers to C1-C, as in "lower alkyl" or "substituted lower alkyl." 10 It means alkyl. The terms "alkyl," "lower alkyl," or "cycloalkyl" refer to methyl, ethyl, isopropyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, C6 to C 12 Includes spirocycl, cyclopropylethyl, cyclobutylethyl, decalinyl, bicyclo-[1.1.1]-pentyl, norboranil, bicyclo-[2.2.2]-octyl, cubyl, adamantanil, and related cage hydrocarbon moiety. In certain embodiments, the alkyl is C1-C 20 It is an alkyl. In certain embodiments, the alkyl group is polydeuterated.

[0040] "Substituted alkyl" is typically heterocycloalkyl, aryl, substituted aryl, heteroaryl, nitro, cyano (also referred to herein as nitrile), azido, halo, -OR, -SR, -SF5, -CHO, -COR, -C(O)OR, -C(O)-NR2, -OC(O)R, -OC(O)NR2, -OC(O)OR, -P(O)(OR)2, -OP(O)(OR)2, -NR2, -N + R3 (counterion may exist), -CONR2, -NRCOR, -NHC(O)OR, -NHC(O)NR2, -NHC(NH)NR2, SO3 -alkyl that is mono-, di-, or tri-substituted with -SO2OR, -OSO2R, -SO2NR2, or -NRSO2R, wherein each R is independently hydrogen, lower alkyl, R′-substituted lower alkyl, aryl, R′-substituted aryl, heteroaryl, heteroaryl(alkyl), R′-substituted aryl(alkyl), or aryl(alkyl), and each R′ is independently hydroxy, halo, alkyloxy, cyano, thio, SF5, nitro, alkyl, halo-alkyl, or amino. Substituted alkyl that is substituted with one to three substituents selected from the group consisting of alkynyl, cyano, halo, alkyloxy, thio, nitro, amino, or hydroxy is of particular interest.

[0041] The term "Aryl" refers to an aromatic ring having (4n+2) pi electrons and may contain 6 to 20 cyclic carbon atoms, comprising a single ring (e.g., phenyl), or two or more condensed rings, such as 2 to 3 condensed rings (e.g., naphthyl), or two or more aromatic rings, such as 2 to 3 aromatic rings connected by a single bond (e.g., biphenylyl). In certain cases, the aryl is C6-C 16 or C6 to C 14 is. In a specific embodiment, one or more hydrogen atoms of the alkyl group are substituted with deuterium.

[0042] A heteroaryl comprises (4n+2) pi electrons and consists of 1 to 10 cyclic carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se, and refers to an aromatic ring system having a single ring (e.g., thiophene, pyridine, pyrazine, imidazole, oxazole, tetrazole, etc.), or two or more condensed rings, e.g., 2 to 3 condensed rings (e.g., indole, benzimidazole, quinolone, quinoxaline, phenothiazine, etc.), or two or more aromatic rings, e.g., 2 to 3 aromatic rings connected by a single bond (e.g., bipyridyl). In some cases, the heteroaryl is C1-C 16 It has 1 to 5 heteroatoms selected from the group consisting of S, Se, N and O.

[0043] The terms "heterocycloalkyl," "heterocycle," "heterocyclic group," or "heterocyclile" refer to a saturated or unsaturated non-aromatic ring system containing 1 to 10 cyclic carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se, and having a single ring (e.g., tetrahydrofuran, aziridine, azetidine, pyrrolidine, piperidine, tetrathiopyran, hexamethylene oxide, oxazepan, etc.) or two or more condensed rings, such as 2 to 3 condensed rings (e.g., indoline, tetrahydrobenzodiazine, etc., including fused, cross-linked, and spiro-cyclic systems having 3 to 15 cyclic atoms and containing 1 to 4 heteroatoms). In certain cases, the heterocycloalkyl is C1-C 16It has 1 to 5 heteroatoms selected from the group consisting of S, Se, N, and O. In the fused ring system, one or more rings may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the attachment point is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0044] Examples of heterocyclic and heteroaryl compounds include azetidine, pyrrole, imidazole, benzimidazole, pyrazole, benzopyrazole, tetrazole, 1,2,3-triazole, benzotriazole, 1,2,4-triazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolidine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazolin, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiaazole, benzisothiazole, phenazine, isoxazole, benzisooxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, Piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, benzothiazole, thiazolidine, furan, benzofuran, thiophene, benzothiophene, benzo[b]thiophene, morpholinyl, thiomophorinyl (also called thiamophorinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranil, benzotetrahydrofuranil, etc. are included but not limited thereto.

[0045] Substituted heterocycloalkyl, aryl, heteroaryl are optionally hydrogen, 1 to 3 alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, substituted aryl, aryl(alkyl), -SO2NR 5 R 5 , -PO3H2, -NR 5 SO2R 6 or -NR 5 C(=O)R6 It is replaced with, where R 5 and R 6 is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, optionally substituted heterocycloalkyl, aryloxy, heteroaryl, heteroaryl(alkyl), or R 5 and R 6 Together -(CH2) 3-6 - or -(CH2) 0-3 X(CH2) 0-3- And, where X= NR, O, S, SO2, substituted aryl(alkyl), halo(alkyl), SF5, NR 5 3 + , azido, cyano (also referred to as nitrile in this specification), -OR 5 , -SR 5 , -NR 5 R 6 , halogen, nitro, SCH3, OCF3, SO2CH3, SCF3, SO2CF3, CF3, -SO2OR 5 , -OSO2R 5 , CCl3, -C(=O)R 5 , -C(=O)OR 5 ; -C(=O)NR 5 R 6 , -OC(=O)R 5 am.

[0046] As implied in some of the definitions mentioned above, "substituted" in "substituted alkyl," "substituted aryl," etc. means that at least one hydrogen atom bonded to a carbon (or other) atom in a hydrocarbyl, alkyl, aryl, or other moiety is substituted with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups and hydrocarbyl moiety C1-C24 alkyl (including C1-C18 alkyl, further C1-C12 alkyl, further C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further C2-C12 alkenyl, further C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further C2-C12 alkynyl, further C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, further C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, further C6-C12 aralkyl). The aforementioned hydrocarbyl moiety may be further substituted with one or more functional groups or additional hydrocarbyl moiety such as those specifically listed. Unless otherwise indicated, any group described in this specification should be interpreted as comprising a substituted and / or heteroatom-containing moiety in addition to an unsubstituted group.

[0047] The term "water-soluble group" refers to a functional group that is well solvated in an aqueous environment and imparts improved water solubility to the compound to which it is attached. Water-soluble groups of interest include, but are not limited to, polyalcohols, straight-chain or cyclic sugars, primary, secondary, tertiary, or quaternary amines and polyamines, sulfate groups, sulfonate groups, sulfinate groups, carboxylate groups, phosphate groups, phosphonate groups, phosphinate groups, ascorbate groups, glycols including polyethylene glycol (PEG) and modified PEG, and polyethers. In some cases, the water-soluble group is a primary, secondary, tertiary, and quaternary amine, carboxylate, phosphonate, phosphate, sulfonate, sulfate, -N(H) 0-1 (CH2CH2OH) 1-2 , -NHCH2CH2N(CH3) 2-3 , -NHCH2CH2SO3H, -NHCH2CH2PO3H2, and -NHCH2CH2CO2H, --(CH2CH2O) yy CH2CH2XR yy , --(CH2CH2O) yy CH2CH2X--, --X(CH2CH2O) yy CH2CH2--, glycol, oligoethylene glycol, and polyethylene glycol, where yy is selected from 1 to 1000, and X is O, S, and NR ZZ Selected from, R ZZ and R YY H and C independently 1-3 Selected from alkyls.

[0048] The term "carboxy isostere" refers to a standard pharmaceutical bioisostere substitute for carboxylic acids, amides, and esters. These include acyl cyanamide, tetrazole, hydroxychrome, 3-hydroxy-1,2,4-triazole, 1-hydroxypyrazole, 2,4-dihydroxyimidazole, 1-hydroxyimidazole, 1-hydroxy 1,2,3-triazole, alkylsulfonyl carboxamide, hydroxyisoxazole, 5-hydroxy 1,2,4-oxadiazole, thiazole, 1,2,4-oxadiazole, 1,2,4-oxadiazolone, oxazole, triazole, thiazole, other hydroxyamic acids, sulfonimides, acylsulfonamides, sulfonylureas, oxadiazolone, thiazolidinediones, oxadiazole, thiadiazole, isothiazol, difluorophenol, tetramic acid, tetronic acid, squaric acid, Hydroxyquinoline-one, hydroxyquinoline-2-one, boronic acid and phosphate are included but not limited to these.

[0049] As used herein, the term “PEG” refers to polyethylene glycol or modified polyethylene glycol. Modified polyethylene glycol polymers include methoxypolyethylene glycol and polymers that are unsubstituted or have one end substituted with an alkyl, substituted alkyl, or substituent (e.g., as described herein).

[0050] The term "functional group" refers to halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X, where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), Carboxylato(-COO-), carbamoyl(-(CO)-NH2), mono-substituted C1-C24 alkylcarbamoyl(-(CO)-NH(C1-C24 alkyl)), di-substituted alkylcarbamoyl(-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl(-(CO)-NH-aryl), thiocarbamoyl(-(CS)-NH2), carbamido(-NH-(CO)-NH2), cyano(-C≡N), isocyano(-N+≡C-), cyanato(-OC≡N), isocyanato(-O-N+≡C-), isothiocyanato(-SC≡N), azido(-N=N+=N-), formyl(-(CO)-H), Thioformyl(-(CS)-H), amino(-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido(-NH-(CO)-alkyl), C5-C20 arylamido(-NH-(CO)-aryl), imino(-CR=NH, where R = hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino(-CR=N(alkyl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), arylimino(-CR=N(aryl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), nitro(-NO2), nitroso(-NO), sulfo(-SO2-OH), Sulfonato(-SO2-O-), C1-C24 alkylsulfanyl(-S-alkyl; also called "alkylthio"), arylsulfanyl(-S-aryl;It refers to chemical groups such as C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), and phosphino (-PH2), mono- and di-(C1-C24 alkyl)-substituted phosphino, and mono- and di-(C5-C20 aryl)-substituted phosphine. In addition, the aforementioned functional group may be further substituted with one or more additional functional groups or one or more hydrocarbyl moietyes as specifically listed above, if permitted by a specific group.

[0051] Where the term “substituted” appears before a list of possible substituted groups, the term is intended to apply to all members of that group. For example, the phrase “substituted alkyl and aryl” should be interpreted as “substituted alkyl and substituted aryl”.

[0052] In addition to the disclosure of this specification, the term “substituted” may also mean that when used to modify a particular group or radical, one or more hydrogen atoms of the particular group or radical are each, independently of one another, substituted with the same or different substituent groups as defined below.

[0053] In addition to the groups disclosed with respect to individual terms in this specification, for substituting one or more hydrogens on a saturated carbon atom within a specific group or radical (any two hydrogens on a single carbon are =O, =NR 70 , =N-OR 70 , can be substituted with =N2 or =S) Substituents are, unless otherwise specified, -R 60 , Halo, =O, -OR 70 , -SR 70 , -NR 80 R 80, 트리할로메틸, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O-M + , -SO2OR 70 , -OSO2R 70 , -OSO2O-M + , -OSO2OR 70 , -P(O)(O-)2(M + )2, -P(O)(OR 70 )O-M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O-M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O-M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO 2- M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 및 -NR 70 C(NR 70 )NR 80 R 80 이고, 여기서 R 60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, and each R 70 is independently hydrogen or R 60 and; each R 80 R independently 70 Either or, alternatively, 2 Rs 80 Silver, together with the nitrogen atoms to which they are bonded, forms a 5-, 6-, or 7-membered heterocycloalkyl group, which may optionally comprise 1 to 4 identical or different additional heteroatoms selected from the group consisting of O, N, and S, wherein N may have an -H or C1-C3 alkyl substitution; and each M + is a counterion with a net single positive charge. Each M + is independently, for example, K + , Na + , Li + Alkali ions such as; +N(R 60 Ammonium ions such as )4; or [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 It may be an alkaline earth ion such as ("subscript 0.5 means that one of the counterions for such a divalent alkaline earth ion may be an ionized form of the compound of the present invention and the other may be a typical counterion such as chloride, or two ionized compounds disclosed herein may act as counterions for such a divalent alkaline earth ion, or a double ionized compound of the present invention may act as a counterion for such a divalent alkaline earth ion"). As a specific example, -NR 80 R 80 -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazine-1-yl, N-morpholinyl, -N(H)0-1 (CH2CH2OH) 1-2 , -NHCH2CH2N(CH3) 2-3 It is intended to include -NHCH2CH2SO3H, -NHCH2CH2PO3H2 and -NHCH2CH2CO2H.

[0054] In addition to the disclosure of this specification, the substituent group for a hydrogen on an unsaturated carbon atom in a “substituted” alkene, alkyne, aryl, and heteroaryl group is, unless otherwise specified, -R 60 , Halo, -O - M + , -OR 70 , -SR 70 , -SM + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO 3- M + , -SO3R 70 , -OSO2R 70 , -OSO 3- M + , -OSO3R 70 , -PO 3- 2 (M + )2, -P(O)(OR 70 )OM + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO 2- M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO 2- M +, -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO 2- M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 and, here R 60 , R 70 , R 80 and M + is as previously defined, provided that in the case of substituted alkenes or alkynes, the substituent is -O - M + , -OR 70 , -SR 70 , or -SM + It is not.

[0055] In addition to the groups disclosed in relation to individual terms in this specification, the substituent group for the hydrogen on the nitrogen atom in the "substituted" heteroalkyl and cycloheteroalkyl groups is, unless otherwise specified, -R 60 , -OM + , -OR 70 , -SR 70 , -SM + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O-M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O-M +, -OS(O)2OR 70 , -P(O)(O-)2(M + )2, -P(O)(OR 70 )OM + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 and, here R 60 , R 70 , R 80 and M + is as previously defined.

[0056] Salts include, but are not limited to, Na, K, Ca, Mg, ammonium, tetraalkylammonium, aryl and alkyl sulfonates, phosphates, carboxylates, sulfates, Cl, Br, and guanidinium.

[0057] Unless otherwise specified, references to atoms are intended to include their isotopes. For example, references to H are 1 H, 2 H (i.e., D) and 3 It is intended to include H (i.e., T), and the reference to C is 12 C and all isotopes of carbon (e.g. 13 It is intended to include C).

[0058] In addition to the disclosure of this specification, in certain embodiments, the substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0059] Unless otherwise indicated, the nomenclature of substituents not explicitly defined in this specification is derived by naming the terminal portion of a functional and then naming the adjacent functional toward the attachment point. For example, the substituent "heterocycloalkyl(alkyl)" refers to the group (heterocycloalkyl)-(alkyl).

[0060] With respect to any group disclosed herein containing one or more substituents, of course, it is understood that such group does not contain any substitution or substitution pattern that is stereoscopically impractical and / or synthetically impossible to realize. Additionally, the target compounds include all stereochemical isomers arising from the substitution of these compounds.

[0061] In certain embodiments, substituents may contribute to the optical isomerism and / or stereoisomerism of the compound. Salt, solvate, hydrate, and prodrug forms of the compound are also of interest. Polymorphic, pseudo-polymorphic, amorphous, and cocrystalline forms of the compound are also of interest. All such forms are included in this disclosure. Accordingly, the compounds described herein include their salt, solvate, hydrate, prodrug, and isomer forms, and include their pharmaceutically acceptable salt, solvate, hydrate, prodrug, and isomer forms. In certain embodiments, the compound may be metabolized into a pharmaceutically active derivative.

[0062] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are commercially available. Additionally, pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, isotonic adjusters, stabilizers, and wetting agents, are commercially available. Any compound useful for the methods and compositions of the present invention may be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salt" refers to such a salt that retains the biological efficacy and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and salts of basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, ethylenediamine, glucosamine, methylglucarmin, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0063] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; and amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or TWEEN TM , PLURONICS TM Or it contains nonionic surfactants such as polyethylene glycol (PEG). The formulation to be used for in vivo administration must be sterile. This is easily achieved by filtration through a sterile filter membrane.

[0064] The term “sample” in relation to a patient includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens, or tissue cultures or cells and their offspring derived therefrom. The term also includes samples that have been manipulated in any manner after acquisition, such as treatment with reagents; washing; or concentration for specific cell populations such as disease cells. The definition also includes samples concentrated for specific types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” includes clinical samples and also includes tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, etc. “Biological sample” includes samples obtained from a patient’s disease cells, e.g., samples containing polynucleotides and / or polypeptides obtained from a patient’s disease cells (e.g., cell lysates or other cell extracts containing polynucleotides and / or polypeptides); and samples containing disease cells from a patient. Biological samples containing disease cells from a patient may also contain non-disease cells.

[0065] RNA.The methods and reactants of the present disclosure may be used to modify any form of single-stranded RNA, or a single-stranded region of RNA, and include, without limitation, mRNA, tRNA, rRNA, viral RNA, siRNA, RNA aptamers, synthetic RNA such as chemically synthesized or in vitro transcribed forms, or any other form of RNA such as hnRNA and viroid RNA. The RNA may be a mixture of different types of RNA. The RNA may be a synthetic product or a natural product. In some embodiments, the RNA is mRNA of eukaryotic or prokaryotic origin. The mRNA may or may not have a cap and / or a poly-A tail. The RNA may or may not contain a non-natural modified nucleobase. The RNA may be at least 12 nt, at least about 15, at least about 20, at least about 25 in length, and may be about 100 nt, 500 nt, 750 nt, 1 kb, 1.5 kb, greater than 2 kb, or larger. RNA can be linear or circular.

[0066] Selective modification of a region of RNA, e.g., a poly-A tail, can be achieved by hybridizing the RNA to a sequence other than the region selected for modification, e.g., the mRNA's 5'-UTR, open reading frame, and 3'-UTR, specifically to complementary DNA. In some embodiments, the mRNA's 5'-UTR, open reading frame, and 3'-UTR are hybridized to a complementary DNA oligo having a length ranging from about 18 nt to about 120 nt. In some embodiments, substantially the entire mRNA sequence, excluding the poly-A tail, is hybridized. In alternative embodiments, a single strand of complementary DNA hybridized to the mRNA's 5'-UTR, open reading frame, and 3'-UTR is synthesized, e.g., by reverse transcriptase. Subsequent removal of the DNA strand with DNase produces mRNA having modifications in the target region.

[0067] Click Chemistry Click is a concept in organic chemistry that describes a series of highly efficient, selective chemical reactions that can be used to rapidly and reliably generate molecular assemblies or conjugates. The best-known example of a click reaction is copper-catalyzed azide-alkyne cycloaddition (CuAAC), where an azide group reacts with an alkyne group to form a stable triazole link. Other click reactions include strain-promoted azide-alkyne cycloaddition (SPAAC), which eliminates the need for a copper catalyst, and the Diels-Alder reaction.

[0068] The easy scalability, modularity, and biocompatibility of click reactions enable applications in the field of nucleic acids, for example, labeling of oligonucleotides (ODNs) using small molecule probes; linking of oligonucleotide sequences (e.g., single strands, double strands including complementary strands); cyclization of oligonucleotides to form circular DNA or RNA constructs; metabolic labeling of RNA; and the like.

[0069] To modify nucleic acids, click chemical reactions provide bioorthogonal and highly specific options compatible with DNA and RNA. In some embodiments, the reactants of Formula (I) or Formula (II) comprise functional groups for click chemical conjugation as one or both of R1 and R2, for example, azides, alkynes, for example, copper-catalyzed azide-alkyne cyclization addition (CuAAC); strained alkynes, for example, DBCO for SPAAC, tetrazine and trans-cyclooctene (TCO), thiols and alkenes for UV-initiated thiol-ene reactions; oximes and hydrazones; aminooxy or hydrazines; isothiocyanates; etc. as known in the art. For example, see Fantoni et al. (2021) Chem Rev. 121:7122-7154, which is specifically incorporated herein by reference.

[0070] In some embodiments, click chemistry reactants provide metal-free chemistry, e.g., SPAAC, conjugation between an oxanorbornadiene derivative and an azide; reaction between a strained alkene and a tetrazine; and alkene-tetrazole photoclick reaction. The SPAAC reaction involves cyclization addition between a strained cyclooctane and an organic azide, wherein the RNA modified by the method of the present disclosure may contain a strained cyclooctane or an azide group, typically an azide.

[0071] Click chemical reactions can be used to label RNA with detectable groups, radioisotopes, therapeutic moiety, etc. In some embodiments, the detectable label is a fluorescent dye or fluorophore. Examples include, without limitation, cyanine dyes such as fluorescein, rhodamine, Cy3 and Cy5, the Alexa fluor series, e.g., Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647, green fluorescent protein (GFP) and its variants, boron-dipyrromethene (BODIPY) dye, Texas Red, Dylight fluorophore; Atto dye; tetramethylrhodamine, etc.

[0072] Therapeutic moiety refers to pharmacologically active molecules or substances capable of inducing specific biological responses in the body to treat or alleviate specific medical conditions. These moiety can vary and include small organic molecules, peptides, nucleic acids, or larger bioagents such as proteins and antibodies.

[0073] composition

[0074] A reactant that selectively modifies RNA at the 2′-OH group is provided. In some embodiments, the reactant has structure (I).

[0075] (I)

[0076]

[0077] Here, LG is a leaving group and may include halides, e.g., F, Cl, Br, I, etc.; ammonium salts, tertiary amines, e.g., trialkylamines, -N(CH3)3 + , may be N-methylmorpholine (NMM);

[0078] X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, provided that at least one of X and Y is nitrogen;

[0079] R1 and R2 are selected independently and may be absent or present. If present, R1 and R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, -NHR, and may include groups suitable for click chemistry, e.g., NHCH2CH2OCH2CH2N3.

[0080] In some embodiments, the reactants have structure (II), where LG, X, R1 and R2 are as defined above.

[0081] (II)

[0082]

[0083] Examples of reactants include, without limitation, the following:

[0084] (DMTMM); (2PMM);

[0085] (2PN3); (AzTMM)

[0086] (DMTN3); (AzTN3)

[0087] In some embodiments, the aromatic compound of interest for the reaction is activated by a tertiary amine, for example, by substituting a halogen on a halogen-substituted heterocycle with NMM or trimethylamine. In some embodiments, the reactant comprises an azide or alkyne functional group for RNA conjugation using Cu-free click chemistry as one or both of R1 and R2.

[0088] In some embodiments, a composition comprising a compound of structure (I) or (II) is provided. In some embodiments, the compound is purified, for example, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or more of the composition is the compound of interest. In some embodiments, the compound is provided in powder form. In some embodiments, the compound is provided as a solution, for example, an aqueous solution. In some embodiments, the compound is provided as a kit. The kit may further include instructions for use, click chemical reactants, for example, detectable labels, RNA control samples, etc.

[0089] Modified RNA comprising an aryl adduct, for example, an adduct of structure (I) without a leaving group, is also provided. As discussed above, any RNA comprising at least one single-stranded region is suitable for this purpose.

[0090] reaction

[0091] In some embodiments, a method for selectively modifying RNA at the 2′-OH group is provided, said method comprising contacting the RNA with a reactant of structure (I) or (II). In some embodiments, said reaction is carried out in an aqueous solution. In some embodiments, said reaction is carried out in a test tube. In some embodiments, said reaction is carried out in vivo. In some embodiments, said RNA is mRNA and is selectively modified at the poly-A tail, for example, to reduce enzymatic degradation and increase stability.

[0092] In some embodiments, the reaction produces RNA having a click chemoaddition, e.g., an azide or alkyne group. In some embodiments, the RNA is further modified by conjugation at the click chemoaddition and is conjugated, e.g., to a detectable group, a therapeutic moiety, a drug for profiling, etc.

[0093] The reactant of the present disclosure selectively reacts with RNA 2′-OH groups, for example, its reactivity may be at least 2 times greater than its reactivity with DNA, at least 5 times greater than its reactivity with DNA, and at least 10 times greater than its reactivity with DNA. The reaction may be carried out with high efficiency in an aqueous solution, for example, in the absence of an organic solvent such as DMSO, wherein the reaction may proceed to at least about 50% conversion of RNA, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more. The reactant is selective for ssRNA compared to dsRNA. The aryl adduct on RNA may block enzymatic reactions, for example, polymerase.

[0094] Kit

[0095] A kit may be provided. The kit may contain reactants suitable for modifying RNA. The kit may further include, for example, click chemical reactants, for example, detectable labels, RNA control samples, etc. Components may be individually packaged in two or more containers suitable for use in the method disclosed herein. The kit may also include tubes, buffers, etc., and instructions for use.

[0096] experiment

[0097] The following examples are presented to provide a complete disclosure and description of the methods for manufacturing and using the invention to those skilled in the art, and are not intended to limit the scope of what the inventor considers to be his invention, nor are they intended to indicate that the experiments below are the only or all experiments performed. While efforts have been made to ensure accuracy with respect to the figures used (e.g., amounts, temperatures, etc.), some experimental error and deviation must be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near it.

[0098] Example 1

[0099] We first tested whether classical electrophilic aromatic species could react with RNA in water. An initial set of 33 commercially available reagents, 1-32 and CDMT (Fig. 1), was screened for the 18-mer RNA oligonucleotide tRF3. All RNA reactions were performed over 24 hours at 37 °C in pH 7.5 SHAPE buffer ([MOPS] = [NaCl] = 100 mM, [MgCl2] = 6.06 mM). In these 10 μL reactions, the RNA concentration was fixed at 10 μM, and the electrophilic reagent concentration was 200 mM. All RNA screening reactions for this set contained 20% DMSO by volume, and parallel control reactions using DNA (under the same conditions) were performed to determine the RNA selectivity of these reagents.

[0100] Screening of this set revealed that several reagents selectively react with RNA 2′-OH in water with low to medium yields. Reagents 11–21 are halogen-substituted pyrimidines possessing electron-withdrawing groups at various positions on the heterocycle. In contrast to similar pyrimidine motifs in reagents 1–6, which lack electron-withdrawing substituents on the aromatic ring and do not react with RNA under screening conditions, reagents 11–21 (excluding 19) exhibit significant RNA conversion. The presence of strong electron-withdrawing groups at the para position (relative to the halogen substituents) significantly improves the RNA reactivity of these reagents, which is likely attributed to a decrease in electron density (via resonance) at the electrophilic carbon. Molecules 7–10 demonstrate that pyridine and benzene motifs do not result in RNA modification, which is likely due to a combination of poor solubility and lower reactivity compared to the pyrimidine reagents. Surprisingly, reagents 22–24 do not exhibit any detectable RNA modifications, despite being pyrimidine reagents containing strong electron-withdrawing substituents with highly electrophilic carbon centers. This is likely due to steric hindrance caused by large, electron-rich substituents at ortho positions relative to the electrophilic center. Finally, triazine and tetrazine reagents 27–32 exhibit poor solubility and result in little to no detectable RNA modifications. Interestingly, CDMT, reported as a peptide coupling reagent, resulted in RNA 2′-OH selective modification (13% conversion). CDMT has not previously been reported to react with alcoholic motifs and is considered selective for carboxylic acid modifications.

[0101] We noted that N-methylmorpholine was previously used to convert the chloro-triazine species CDMT into the more water-soluble peptide coupling reagent DMTMM. The cationic reagent DMTMM (Fig. 3) is commercially available for this purpose; it reacts with a carboxylic acid to produce an active ester intermediate that reacts well with a primary amine. We tested DMTMM and examined whether it reacts with RNA. Surprisingly, the reagent provided >98% conversion of tRF3 RNA and <6% modification of tRF3 DNA under the same reaction conditions (20% DMSO, total reaction volume = 10 μL, [RNA] = [DNA] = 10 μM, [DMTMM] = 200 mM, [MOPS] = [NaCl] = 100 mM, [MgCl2] = 6.06 mM, pH 7.5, reaction at 37 °C for 24 h). However, DMTMM is reported to be much more soluble and stable in water compared to DMSO. Therefore, we decided to remove DMSO from the reaction workflow and prepared a DMTMM stock solution in RNase-free water for use in RNA reactions. Upon optimizing reaction conditions, we obtained >99% tRF3 RNA conversion with 100 mM DMTMM and <5% tRF3 DNA modification under the same conditions (0% DMSO, total reaction volume = 10 μL, [RNA] = [DNA] = 10 μM, [MOPS] = [NaCl] = 100 mM, [MgCl2] = 6.06 mM, pH 7.5, reaction at 37 °C for 24 h).

[0102] Next, we tested whether other halogen-substituted heterocycles could also react with NMM to form RNA-reactive species. Pyrimidine reagent 17 was selected as a test substrate because it contains an ester group that can inspire the design and easy synthesis of analogs with useful functional handles. Advantageously, the NMM-activated reagent 2PMM (Fig. 3) was synthesized by simply adding 2 molar equivalents of N-methylmorpholine to a solution of 17 in anhydrous THF (on a scale of 50–100 mg, initial

[17] = 0.5 M). After stirring for 30 min at ambient temperature (25 °C), pure 2PMM (characterized by NMR and LCMS) was obtained in quantitative yield by filtering the reaction mixture and washing the solid residue (product) with >5 mL THF to remove excess N-methylmorpholine. Using the DMSO-free RNA reaction conditions employed in the DMTMM experiment, we observed >98% RNA conversion and <1% DNA conversion with 200 mM 2PMM. Thus, we demonstrate that the activation of triazine and pyrimidine heterocycles using N-methylmorpholine is an effective strategy for designing molecules that selectively react with RNA 2′-OH in an aqueous environment. Importantly, these molecules can perform RNA modification in the complete absence of DMSO, a characteristic useful for future biological applications.

[0103] Encouraged by these results, we designed and synthesized an NMM-activated triazine reagent containing an azide functional group that can be used for RNA conjugation using Cu-free click chemistry. We designed the reagent AzTMM (Fig. 3), which was synthesized in two steps: (1) conjugating an N3-(PEG)2-NH2 linker to molecule 32 using a reported procedure, 11and (2) activated an azide-containing chloro-triazine intermediate with N-methylmorpholine to synthesize AzTMM using a standard procedure for NMM activation of triazines. AzTMM was successfully synthesized and characterized by LCMS, but this reagent has poor solubility in both water and DMSO. Regardless of whether the co-solvent is DMSO or water, the reaction of AzTMM with RNA results in only <60% RNA conversion, even when [AzTMM] = 200 mM under the previously described RNA reaction conditions. Fortunately, the reagent AzTN3 (Fig. 3), synthesized by activating the azide-containing chloro-triazine intermediate with trimethylamine in THF, was found to be well soluble in water. Using the DMSO-free RNA reaction conditions used in the DMTMM experiment, we observed >93% RNA conversion and <5% DNA conversion with 200 mM AzTN3.

[0104] Encouraged by the favorable solubility characteristics of trimethylamine-activated aryl species, reagents DMTN3 and 2PN3 were also synthesized using a similar procedure. Interestingly, these highly water-soluble reagents maintain RNA 2′-OH reactivity in the complete absence of DMSO, demonstrating >99% RNA conversion in water. Thus, we demonstrate that N-methylmorpholine is not the only activation motif for high RNA reactivity in these cationic molecules. In general, activation by any tertiary amine can lead to high RNA 2′-OH reactivity, provided that water solubility can be maintained.

[0105] Stability in water is an important and desirable characteristic for RNA-reactive molecules to maintain high effective reagent concentrations for significant levels of RNA modification. Generally, useful 2′-OH acylation reagents are highly reactive and have aqueous half-lives ranging from seconds to minutes. Recently reported sulfonylation reagents are relatively more stable, and the RNA-reactive sulfonyl reagent P3S has an aqueous half-life of approximately 90 minutes. Since aryl molecules for RNA 2′-OH modification represent a new class of RNA-reactive reagents, we measured the aqueous half-life of the reagent DMTMM in D2O. Using acetonitrile as an internal standard, we 1 The mole fraction of DMTMM was measured at various time points using H NMR. This analysis revealed that DMTMM is remarkably stable in water with an aqueous half-life of >10 days (Fig. 4). Thus, this reagent is >66,000 times more stable in water compared to 1M7, >450 times more stable than NAI, and >170 times more stable than P3S (1M7, NAI: common acylating agent, P3S: sulfonylating agent).

[0106] In summary, we demonstrated that tertiary amine-activated aryl groups can react with RNA at the 2′-OH group in very high yields. The resulting aryl ether adduct is stable on RNA and is the first reactive species that does not react as a carbonyl or sulfonyl structure. We showed that the reagent can be synthesized and purified very easily and that the NMM-activated species is exceptionally stable in water. We also report that these cationic reagents are sufficiently water-soluble, eliminating the need for organic solvents when reacting with RNA. Finally, we demonstrated numerous applications of these groups, including RNA labeling and RNA folded structure mapping.

[0107] Example 2. Preparation of NMM reagent

[0108]

[0109] 50 mg (0.29 mmol, commercial source: AA Blocks) of compound 17 was dissolved in 580 μL of anhydrous THF in a 20 mL glass scintillation vial equipped with a magnetic stirrer. 58.7 mg (0.58 mmol, commercial source: TCI America) of N-methylmorpholine was added to the solution at ambient temperature (25 °C), and the mixture was stirred for 30 min. The reaction mixture was then filtered, and the solid residue was washed with >5 mL of THF to remove excess N-methylmorpholine. The residue was dried under vacuum to obtain 79 mg of 2 PMM (quantitative yield).

[0110] 1 H NMR (300 MHz, DMSO-d6): δ 9.55 (s, 2H), 4.46 (d, J = 11.2 Hz, 2H), 4.11-4.05 (m, 4H), 3.99 (s, 3H), 3.74-3.66 (m, 2H), 3.58 (s, 3H) ppm

[0111] ESI-MS [M-Cl] + : Calculated: 238.12; Observed: 238.14

[0112] Example 2. Synthesis of Trimethylamine Triazine Conjugate

[0113]

[0114] AzTC.N,N-diisopropylethylamine (4.3 g, 33.3 mmol) was added to a solution of 32 (2 g, 11.1 mmol) in anhydrous THF (80 mL) in a 500 mL round-bottom flask equipped with a magnetic stirrer. After stirring for 10 min, 2-(2-azidoethoxy)ethane-1-amine (1.44 g, 11.1 mmol) was added. After stirring for 48 h, the solvent was removed under vacuum, and the resulting crude product was dissolved in ethyl acetate and washed with 100 mM HCl and water (once each). The organic phase was dried with Na2SO4 and concentrated under vacuum. AzTC (2.43 g, 80%) was obtained by silica gel column chromatography (hexane:EtOAc 1:1).

[0115] 1 ¹H NMR (400 MHz, CDCl3): [Mixture of conformational isomers] δ 6.05 (s, NH of the main isomer), 5.94 (s, NH of the minor isomer), 3.99 (s, OCH3 of the main isomer), 3.94 (s, OCH3 of the minor isomer), 3.70-3.63 (m, 6H), 3.41-3.37 (m, 2H) ppm

[0116] ESI-MS [M+H] + : Calculated: 274.08; Observed: 274.11

[0117] AzTN3. 50 mg (0.18 mmol) of AzTC was dissolved in 364 μL of anhydrous THF in a 20 mL glass scintillation vial equipped with a magnetic stirrer. 180 μL (0.36 mmol, commercial source: TCI America) of 2 M trimethylamine in dry THF was added to the solution at ambient temperature (25 °C), and the mixture was stirred for 30 min. The reaction mixture was then filtered, and the solid residue was washed with >5 mL THF to remove excess trimethylamine. The residue was dried under vacuum to obtain 60 mg of AzTN3 (quantitative yield).

[0118] 1 ¹H NMR (400 MHz, D2O): [Mixture of conformational isomers] δ 4.07 (s, OCH3 of the main isomer), 4.02 (s, OCH3 of the minor isomer), 3.79-3.72 (m, 6H), 3.54 (s, N(CH3)3 of the minor isomer), 3.50 (s, N(CH3)3 of the main isomer), 3.50-3.45 (m, 2H) ppm

[0119] ESI-MS [M-Cl] + : Calculated: 297.18; Observed: 297.26

[0120] Example 4. Reaction of RNA and NMM-activated aryl groups in pure water or DMSO

[0121] In a sterile 200 μL PCR tube, 3.3 μL of SHAPE 3.3X buffer (333 mM MOPS pH 7.5, 333 mM NaCl, 20 mM MgCl2 in water) was mixed with 4.7 μL of 21.3 μM tRF3 RNA stock solution. Fresh stock of DMTMM reagent was prepared in DMSO or water; 2 μL of 1 M or 500 mM stock was added to the 200 mM or 100 mM reactions, respectively. These reactions were incubated at 37 °C for 24 h, and the RNA was subsequently purified by ethanol precipitation. RNA modification levels were measured using MALDI-TOF M / S and analyzed using MestReNova™ software. Note: For all RNA reactions, parallel reactions using tRF3DNA were performed under identical conditions.

[0122] Ethanol Precipitation of RNA Reaction: For a total volume of X μL of RNA reaction, 9X μL of RNA precipitation solution (0.33 M NaOAc in water containing 0.2 mg / mL glycogen (pH 5.2)) was added and mixed thoroughly. Subsequently, 30X μL of ice-cold anhydrous ethanol was added, and the mixture was mixed by vortexing for at least 30 seconds. After incubation overnight at -80 °C, the mixture was centrifuged at 14.8 kJ RPM for 60 minutes at 4 °C. The supernatant was discarded to obtain the pellet, which was washed with 70% ethanol. The obtained pellet was air-dried for 15 minutes and then stored at -80 °C for future use or dissolved in water / PBS for direct use in further experiments.

[0123] MALDI-TOF MS: All MALDI-TOF spectra were recorded at the Stanford University Mass Spectrometry Facility using a Bruker Daltonik Microflex MALDI-TOF spectrometer equipped with an N2 laser. All spectra were recorded in linear anion mode, and samples were spotted onto MSP Anchorchip 96 target plates. 0.3 M trihydroxyacetophenone (matrix) and 0.1 M aqueous ammonium citrate (co-matrix) in EtOH were mixed in a 2:1 volume ratio and used as the matrix mixture for MALDI. This mixture was always prepared fresh prior to analysis. After RNA precipitation, the RNA pellet was redissolved in RNase-free water to prepare a 10 μM sample solution. 1 μL of this solution was transferred to the target plate and dried under an Ar stream. Subsequently, 1 μL of the matrix mixture was added directly onto the dried sample and completely dried under an Ar stream. Next, spectrum data was recorded using Flex Control software (Bruker) and analyzed using MNova (Mestrenova).

[0124] Example 5

[0125] In vitro and in vivo RNA structure mapping

[0126] In folding-supporting buffers, RNA is shown to react preferentially with DMTMM and other ammonium triazine and pyrimidine reagents in the unfolded regions of RNA compared to the double-stranded regions. When human 5S RNA (in vitro or in living cells) is exposed to one of these reagents at 50–200 mM, it is used to map the folded structure. The reaction time is adjusted to yield approximately one aryl group per RNA strand. Fluorescent-labeled DNA primers that specifically bind to 5S RNA are hybridized to the reacted RNA after this reaction, and a reverse transcriptase enzyme (e.g., Superscript 3) is added along with nucleoside triphosphate to support cDNA synthesis. The enzyme stops at the arylation site, generating a series of cDNAs of varying lengths. Analysis by gel electrophoresis reveals banding patterns consistent with the known folded structure of 5S RNA. This demonstrates that arylating agents can be used to map the folded structure of RNA.

[0127] Example 6

[0128] RNA labeling with triazine conjugates

[0129] In a sterile 200 μL PCR tube, 10 μM tRF3 RNA was treated with 2 μL of a 1 M AzTN3 stock solution in water (or water only for the control reaction) according to the RNA reaction protocol for 0% DMSO RNA reactions. Subsequently, 50 picomoles of the treated tRF3 RNA were dissolved in 40 μL of 1X PBS (pH 7.4) and incubated at room temperature for 10 minutes. Next, 10 μL of a 250 μM TAMRA-DBCO stock solution in DMSO was added to the RNA solution and mixed thoroughly. After incubating at 37°C for 2 hours, the sample was subjected to ethanol precipitation to isolate the RNA and remove unreacted TAMRA reagent. The obtained pellet was then dissolved in water for MALDI-TOF and PAGE analysis, or in 1X PBS (pH 7.4) for fluorescence experiments.

[0130] Mass peak index:

[0131] 5662 → Naked RNA

[0132] 6422 → RNA + 3 AzTN3 adducts

[0133] 6838 → RNA + 1 (AzTN3+TAMRA) Click Add-ons

[0134] 7601 → RNA + 3 AzTN3 adducts + 1 (AzTN3+TAMRA) click adduct

[0135] 8013 → RNA + 2 (AzTN3+TAMRA) Click Add-ons

[0136] 8772 → RNA + 3 AzTN3 adducts + 2 (AzTN3+TAMRA) click adducts

[0137] 9190 → RNA + 3 (AzTN3+TAMRA) Click Add-ons

[0138] Example 7

[0139] Drug Conjugate Profiling

[0140] Reactivity-based RNA profiling (RBRP) can be used to analyze drug-RNA interactions across the cell. Previously, acylimidazole reactive groups were used to react with RNA; however, these reactive groups have short half-lives, which destabilize the reagents and reduce the yield in the reaction with RNA. In a new experiment, we select a drug to be profiled that possesses an amine side chain and react it with the reagent AzTC to generate an RNA-reactive drug conjugate. When this conjugate is exposed to human cells, the drug enters the cell, binds to intracellular RNA, and reacts with the RNA it binds to. Analysis by isolation and sequencing of the reacted RNA (according to the published RBRP methodology) allows us to determine the location within the human transcriptome where the drug binds. A control experiment using an unmodified drug as an excess amount of competitor confirms drug-specific interactions.

[0141] Example 8

[0142] RNA modification for enhanced biological properties

[0143] An arylation reagent, such as DMTMM, is reacted with messenger RNA, such as eGFP mRNA, at 100–200 mM in the presence of DNA hybridized to the whole mRNA excluding the poly-A tail. This leaves the poly-A region open for selective arylation. The DNA is then digested according to the exposed TRAIL method. The resulting mRNA is transfected into cells using a cationic lipid preparation. Intracellular eGFP fluorescence is analyzed to determine whether arylation enhances the total expression level of this protein.

[0144] References

[0145] Zubradt, M. et al. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat Methods 14, 75-82 (2017).

[0146] Mortimer, S. A. & Weeks, K. M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. Journal of the American Chemical Society 129, 4144-4145 (2007).

[0147] Spitale, R. C. et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature 2015 519:7544 519, 486-490 (2015).

[0148] Chatterjee, S., Shioi, R. & Kool, E. T. Sulfonylation of RNA 2′-OH groups. ACS Cent. Sci. 9, 531-539 (2023).

[0149] Xiao, L., Habibian, M. & Kool, E. T. Site-Selective RNA Functionalization via DNA-Induced Structure. Journal of the American Chemical Society 142, 16357-16363 (2020).

[0150] Fang, L., Xiao, L., Jun, Y. W., Onishi, Y. & Kool, E. T. Reversible 2'-OH acylation enhances RNA stability. Nat Chem 15, 1296-1305 (2023).

[0151] Fang, L. et al. Pervasive transcriptome interactions of protein-targeted drugs. Nat. Chem. 15, 1374-1383 (2023).

[0152] Kami㎖ski, Z. J. 2-Chloro-4,6-dimethoxy-1,3,5-triazine. A New Coupling Reagent for Peptide Synthesis. Synthesis 1987, 917-920 (1987).

[0153] Kunishima, M. et al. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride: an efficient condensing agent leading to the formation of amides and esters. Tetrahedron 55, 13159-13170 (1999).

[0154] Kitamura, M. & Kunishima, M. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium Chloride. in Encyclopedia of Reagents for Organic Synthesis (ed. John Wiley & Sons, Ltd) rn01530 (John Wiley & Sons, Ltd, 2013). doi:10.1002 / 047084289X.rn01530.

[0155] Kunishima, M. et al. Development of chlorotriazine polymer dehydrocondensing reagents (Poly-Trzs). Tetrahedron 63, 2604-2612 (2007).

[0156] Spitale, R. C. et al. RNA SHAPE analysis in living cells. Nature Chemical Biology 2012 9:1 9, 18-20 (2012).

[0157] Jahn, K. et al. Site-Specific Chemical Labeling of Long RNA Molecules. Bioconjugate Chem. 22, 95-100 (2011).

[0158] D'Este, M., Eglin, D. & Alini, M. A systematic analysis of DMTMM vs EDC / NHS for ligation of amines to Hyaluronan in water. Carbohydrate Polymers 108, 239-246 (2014).

[0159] Todoroki, K. et al. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as an enantioseparation enhancer for fluorescence chiral derivatization-liquid chromatographic analysis of dl-lactic acid. Journal of Chromatography A 1360, 188-195 (2014).

[0160] The foregoing merely illustrates the principles of the present invention. Those skilled in the art will understand that various arrangements embodying the principles of the present invention and falling within the spirit and scope thereof, even if not explicitly described or illustrated in this specification, may be devised. Furthermore, all examples and conditional expressions described in this specification are intended primarily to help the reader understand the principles of the present invention and the concepts to which the inventor has contributed to the development of the technology, and such specifically described examples and conditions should be interpreted without limitation. Moreover, all statements in this specification describing the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to include both their structural and functional equivalents. Additionally, such equivalents are intended to include currently known equivalents and equivalents to be developed in the future, that is, any elements developed to perform the same function regardless of structure. Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments illustrated and described in this specification. Rather, the scope and spirit of the present invention are defined by the appended claims.

Claims

Claim 1 A method for selectively modifying RNA at the 2′-OH group comprises the step of reacting the RNA of interest with a reactant having structure (I): (I) where the leaving group (LG) is selected from F, Cl, Br, I; ammonium salt; or tertiary amine; X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, provided that at least one of X and Y is nitrogen; R1 and R2 are independently selected and may be absent or present, and if present, R1 and R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, or -NHR; a method for producing aryl modified RNA. Claim 2 In claim 1, the method wherein the reactant has structure (II): (II) Claim 3 A method according to claim 1 or 2, wherein the tertiary amine is a trialkylamine. Claim 4 In paragraph 3, the trialkylamine is -N(CH3)3 + Person, method. Claim 5 A method according to claim 1 or 2, wherein the tertiary amine is N-methylmorpholine (NMM). Claim 6 A method in which, in any one of the preceding claims, one or both of R1 and R2 comprise click chemical reactants. Claim 7 In claim 6, the method wherein the click chemical reactant comprises an azide or an alkyne. Claim 8 A method according to claim 1 or 2, wherein the reactant is selected from DMTMM, 2PMM, DMTN3, 2PN3, AzTMM, and AzTN3. Claim 9 A method according to claim 6 or 7, further comprising the step of reacting the aryl modified RNA with a click reactant. Claim 10 A method according to claim 9, wherein the aryl modified RNA comprises an azide group and the click reactant comprises a constrained alkyne. Claim 11 A method according to claim 9 or 10, wherein the click reactant conjugates a detectable label to the aryl modified RNA. Claim 12 A method according to claim 9 or 10, wherein the click reactant conjugates a drug to the aryl modified RNA. Claim 13 A method according to claim 9 or 10, wherein the click reactant conjugates a therapeutic moiety to the aryl modified RNA. Claim 14 A method in which, in any one of the preceding claims, the reaction step is performed in a test tube. Claim 15 A method in which, in any one of the preceding claims, the reaction step is performed in a living cell. Claim 16 A method in which, in any one of the preceding claims, the reacting step is performed in an aqueous solution. Claim 17 As a compound, it includes structure (I): (I) where the leaving group (LG) is selected from F, Cl, Br, I; ammonium salt; or tertiary amine; X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, provided that at least one of X and Y is nitrogen; R1 and R2 are independently selected and may be absent or present, and where present, R1 and R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, or -NHR, a compound. Claim 18 In paragraph 17, a compound having structure (II): (II) Claim 19 A compound according to claim 17 or 18, wherein the tertiary amine is a trialkylamine. Claim 20 In claim 19, the trialkylamine is -N(CH3)3 + Phosphorus, compound. Claim 21 A compound according to claim 17 or 18, wherein the tertiary amine is N-methylmorpholine (NMM). Claim 22 A compound according to any one of claims 17 to 21, wherein one or both of R1 and R2 comprise a click chemical reactant. Claim 23 In paragraph 17, the above-mentioned click chemical reactant comprises an azide or an alkyne. Claim 24 A compound according to claim 17 or 18, wherein the reactant is selected from DMTMM, 2PMM, DMTN3, 2PN3, AzTMM, and AzTN3. Claim 25 A compound conjugated to a drug in any one of R1, R2, or LG in any one of paragraphs 17 to 24. Claim 26 A kit comprising a compound of any one of claims 17 to 25 and instructions for use in modifying RNA. Claim 27 In Clause 26, a kit further comprising a click chemical reactant. Claim 28 RNA modified by the method of any one of paragraphs 1 to 16. Claim 29 In paragraph 28, the RNA is selectively modified in the region of interest. Claim 30 In paragraph 29, RNA, wherein the region of interest is a poly-A tail of mRNA. Claim 31 RNA according to any one of claims 28 to 30, wherein the RNA has increased stability, expression, and / or reduced immunogenicity compared to non-modified RNA. Claim 32 In any one of claims 28 to 30, the RNA wherein the aryl modification comprises a drug conjugate. Claim 33 A method for mapping an RNA structure comprising: modifying RNA by the method of any one of claims 1 to 16, wherein the RNA is selectively aryl-modified in a single-stranded region; binding a primer to the RNA for polymerization initiation, wherein the polymerase is stopped at an aryl adduct to produce a population of polymerization products; and analyzing the polymerization products for size and / or sequence.

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