Molecules and methods for modifying RNA with ARYL groups

Abstract

Compositions and methods are provided relating to a class of electrophilic aromatic reagents that react in high yields with RNA, resulting in heteroaryl or aryl adducts at 2¢-OH position of RNA. Multiple structural variants of reagents are described, along with applications in labeling, mapping, and profiling interactions of RNA. The reagents and reactive groups are easily synthesized, easily conjugated, function in pure water without organic solvents, are chemically stable for storage, and survive for extended times in water during reaction with RNA.

Description

MOLECULES AND METHODS FOR MODIFYING RNA WITH ARYL GROUPSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 603,981 , filed November 29, 2023, the disclosure of which application is herein incorporated by reference.GOVERNMENT SUPPORT

[0002] This invention was made with Government support under contract GM 145357 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Molecules that react with RNA to form chemical bonds have proven to be useful for a number of applications in biology, biotechnology, and medicine. For example, RNA-reactive molecules are used widely to map the folded structure of RNA. In that application, molecules such as dimethylsulfate (DMS) and nitro isatoic anhydride (1 M7) preferentially react with unpaired nucleotides over those in double-stranded structure. Applying a reverse transcriptase enzyme and an RNA-specific DNA primer results in polymerase stops at the site of chemical reaction, and analyzing these stops gives information about the folding of the RNA. In a second example, reagents that react with 2'-OH groups of RNA can be used to introduce biotin or fluorescent labels to the RNA, enabling researchers to isolate, separate, image, and quantify the RNA of interest.

[0004] Some applications of RNA-reactive molecules add utility in the setting of living cells. For example, groups that react in high yields at 2'-OH groups of RNA can be employed to stabilize RNA from hydrolysis and enhance their protein expression. In addition, RNA-reactive functional groups can be attached to drugs or drug candidates, enabling researchers to identify cellular RNAs that the drugs bind to.

[0005] To date, the types of RNA-reactive chemical structures are limited. This is especially true for compounds that react with 2’-OH groups. Reacting at the 2'-OH groups of RNA is potentially highly useful, because such groups appear at essentially every position of all RNAs, potentially enabling a wide range of applications such as those mentioned above. However, to date only acyl (carbonyl) groups and structurally similar sulfonyl groups are known to react with 2’-OH groups, giving limited structural diversity as adducts on the RNA. In the large majority of cases, the known reagents require a cosolvent like DMSO to keep them soluble along with the RNA, which can be toxic to cells, disrupt RNA folding, or disturb cell biology. The acyl reagents are often short-lived in water, limiting their usefulness as reactants. In somecases, the adducts on RNA after reaction can be unstable to hydrolysis, causing them to fall off RNA prematurely before their application is realized.

[0006] Compositions and methods for modification of RNA are provided herein.SUMMARY

[0007] Molecules that react with RNA are useful for modifying RNA’s properties and for probing RNA structure and function. Compositions and methods are provided relating to a class of electrophilic aromatic reagents that react in high yields with RNA, resulting in heteroaryl or aryl adducts at 2'-OH position of RNA. Multiple structural variants of reagents are described, along with applications in labeling, mapping, and profiling interactions of RNA. The reagents and reactive groups are easily synthesized, easily conjugated, function in pure water without organic solvents, are chemically stable for storage, and survive for extended times in water during reaction with RNA.

[0008] Reactants that selectively modify RNA at the 2'-OH groups are provided. In some embodiments the reactants have a structure (I)where LG is a leaving group, and may be halide, e.g. comprising F, Cl, Br, I, etc.; an ammonium salt, tertiary amines, e.g. triaklylamines, -N(CH3)3+, N-methyl morpholine (NMM);X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, with the proviso that at least one of X and Y is a nitrogen;R1 and R2 are independently selected and may be absent or present. If present R1 , R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, -NHR, and may comprise azide or alkyne reactant groups suitable for click chemistry, e.g. NHCH2CH2OCH2CH2N3.

[0009] In some embodiments the reactants have a structure (II), where LG, X, R1 and R2 are as defined above.

[0010] Examples of reactants include, without limitation, those set forth in FIG. 3, including without limitation:

[0011] In some embodiments an aromatic compound of interest for reaction in the methods of the disclosure is activated with a tertiary amine, for example by replacing a halogen leaving group on halogen-substituted heterocycles of Formula I or Formula II with N-methylmorpholine (NMM) or a trimethyl amine. In some embodiments a reactant comprises as one or both of Ri and R2an azide or alkyne functional group for click chemistry conjugation, e.g. using Cu- mediated or Cu-free click chemistry.

[0012] In some embodiments a composition is provided comprising a compound of structure (I). In some embodiments the compound is provided in a powdered form. In some embodiments the compound is provided in a solution, e.g. in an aqueous solution. In some embodiments the compound is provided in a kit. A kit may further comprise instructions for use, click chemistry reactants, e.g. a detectable label, therapeutic moiety, etc., RNA control samples, and the like.

[0013] The reactants of the disclosure selectively react with RNA 2'-OH groups, e.g. where the reactivity is at least 2X greater than reactivity with DNA, at least 5X greater than reactivity with DNA, and may be at least 10X or more greater than reactivity with DNA. The reaction can be performed at high efficiency in aqueous solutions, e.g. in the absence or organic solvents such as DMSO, where 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 about95%, or more. The reactants are selective for ssRNA relative to dsRNA. The aryl adducts on RNA may block enzymatic reactions, e.g. polymerase.

[0014] In some embodiments a method is provided for selective modification of RNA at 2’-OH groups, the method comprising contacting the RNA with a reactant of structure (I). In some embodiments the reaction is performed in an aqueous solution. In some embodiments the reaction is performed in vitro. In some embodiments the reaction is performed in vivo. In some embodiments the RNA is an mRNA and is selectively modified at the poly tail, e.g. to reduce enzymatic degradation and increase stability.

[0015] In some embodiments the reaction generates an RNA with a click chemistry adduct, e.g. azide or alkyne groups, including for example strained cyclooctyne. In some embodiments the RNA is further modified by conjugation at the click chemistry adduct, e.g. by conjugating to a detectable group, to a therapeutic moiety, to a drug for profiling, etc.

[0016] In some embodiments a method is provided for mapping RNA structure in vivo, e.g. in a living cell, or in vitro, where the RNA is reacted by the methods disclosed herein, such that the RNA is preferentially modified at unfolded (single stranded) regions. The regions may be mapped by labeling reacted sequences, by reaction with an enzyme that stalls at adduct sites, etc., and analysis by gel electrophoresis, sequencing, hybridization, etc., as known in the art.

[0017] In some embodiments methods are provided for reactivity-based RNA profiling (RBRP). To analyze interactions of drugs with RNAs throughout the cell, the RNA of interest is reacted with a reactant of structure (I) or (II), comprising as an Ri or R2 group a drug of interest that binds to the RNA. Exposure of the conjugate to cells comprising the RNA introduces an adduct at the site where the drug binds, which can be analyzed as described above. In some embodiments, the LG group comprises the drug of interest, leaving the aromatic adduct on the RNA as a tag after the drug binds and the aromatic group reacts.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0019] FIG. 1 : Initial set of reagents (all purchased from various commercial sources) screened for reaction with RNA 2'-OH in water. The numbers (in %) indicate conversion yield estimated by MALDI-TOF MS after ethanol precipitation: the percentage of all tRF3 RNA after reaction that has at least one 2'-OH covalently modified by electrophilic reagent, “n.d.” denotes no conversion observed, *: no RNA recovered after ethanol precipitation. All the moleculesdescribed in this figure displayed no reaction with a DNA oligonucleotide with the same sequence as tRF3.

[0020] FIG 2: (A) Scheme for RNA 2'-OH modification using electrophilic aromatic reagents. (B) MALDI-TOF M / S analysis of tRF3 RNA reaction with 200 mM of reagent 11. Number of aryl adducts on RNA represented by labels in red. (C) MALDI-TOF M / S analysis of tRF3DNA (DNA oligonucleotide with same sequence as tRF3 RNA) reaction with 200 mM of 11 . No DNA modification is detected. Note: Reactions performed with 18-mer tRF RNA (or 1RF3DNA), reaction volume 10 pL, 20% DMSO, reaction time 24 h, temperature 37°C, [MOPS] = [NaCI] = 100 mM, [MgCh] = 6.06 mM, pH 7.5, MALDI-TOF M / S analysis after ethanol precipitation.

[0021] FIG 3: Structure and reactivity of cationic aryl reagents investigated. Note: Reactions performed with 18-mer tRF RNA (or tRF3DNA), reaction volume 10 pL, reaction time 24 h, temperature 37°C, [MOPS] = [NaCI] = 100 mM, [MgCI2] = 6.06 mM, pH 7.5, MALDI-TOF M / S analysis after ethanol precipitation.

[0022] FIG 4: Hydrolysis half-life measurement of DMTMM. This reagent is estimated to have a half-life of >10 days, by fitting the natural logarithm of the mole fraction of DMTMIVI to a straight line. NMR sample preparation: 15 mg of DMTMM is dissolved in 750 pL D2O containing 5 pL acetonitrile as an internal standard.

[0023] FIG 5: MALDI-TOF M / S analysis of tRF3 and 1RF3DNA after reaction with 200 or 100 mM of DMTMM under 0% or 20% DMSO conditions.

[0024] FIG. 6. Mass peaks.DETAILED DESCRIPTION

[0025] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0027] Unless defined otherwise, 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0028] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0029] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0030] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak 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 U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), 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. (Richmond VA), Novabiochem and Argonaut Technology.

[0031] As used herein, "methods known to one of ordinary skill in the art" may be identified through various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al.,"Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. 0. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C. may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0032] The term “Alkyl” refers to a C1-C20 alkyl that may be linear, branched, or cyclic. “Lower alkyl”, as in “lower alkyl”, or “substituted lower alkyl", means a C1-C10 alkyl. The term “alkyl”, “lower alkyl” or “cycloalkyl” includes methyl, ethyl, isopropyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, Ce to C12 spirocycles, cyclopropylethyl, cyclobutylethyl, decalinyl, Bicyclo-[1 .1 .1]-pentyl, norboranyl, bicylo-[2.2.2]-octyl, cubyl, adamantanyl and related cage hydrocarbon moieties. In certain embodiments, the alkyl is a C1-C20 alkyl. In certain embodiments the alkyl group is poly deuterated.

[0033] A “substituted alkyl” is an alkyl which is typically mono-, di-, or tri-substituted with 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 (wherein a counterion may be present), -CONR2, -NRCOR, -NHC(O)OR, -NHC(O)NR2, -NHC(NH)NR2, SO3, -SO2OR, -OSO2R, -SO2NR2, or -NRSO2R, where 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 alkyls which are substituted with one to three of the substituents selected from the group consisting of alkynyl, cyano, halo, alkyloxy, thio, nitro, amino, or hydroxy are particularly of interest.

[0034] The term “Aryl” refers to an aromatic ring having (4n+2) pi electrons that may contain 6 to 20 ring carbon atoms, and be composed of 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, which are linked by a single bond (e.g., biphenylyl). Incertain cases, the aryl is Ce-C or Ce to C . In certain embodiments the alkyl group has one or more hydrogen atoms replaced with deuterium.

[0035] Heteroaryl means an aromatic ring system containing (4n+2)pi electrons and comprised of 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from 0, N, S, Se, having a single ring (e.g., thiophene, pyridine, pyrazine, imidazole, oxazole, tetrazole, etc.), or two or more condensed rings, for example 2 to 3 condensed rings (e.g., indole, benzimidazole, quinolone, quinoxaline, phenothiazine, etc.), or two or more aromatic rings, such as 2 to 3 aromatic rings, which are linked by a single bond (e.g., bipyridyl). In some cases, the heteroaryl is C1-C16, and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and 0.

[0036] The term “heterocycloalkyl”, “heterocycle”, “heterocyclic group” or “heterocyclyl” refers to a saturated or unsaturated nonaromatic ring system containing 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from 0, N, S, Se, having a single ring (e.g., tetrahydrofuran, aziridine, azetidine, pyrrolidine, piperidine, tetrathiopyran, hexamethylene oxide, oxazepane, etc.), or two or more condensed rings, such as 2 to 3 condensed rings (e.g., indoline, tetrahydrobenzodiazapines, etc., including fused, bridged and spiro ring systems, having 3-15 ring atoms, included 1 to 4 heteroatoms. In certain cases, the heterocycloalky is C1-C16, and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and 0. In fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, - S(O)-, or -SO2- moieties.

[0037] Examples of heterocycles and heteroaryls include, but are not limited to, 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, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, benzoisothiazole, phenazine, isoxazole, benzoisooxazole, 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, thiomorpholinyl (also referred to as thiamorpholinyl), 1 ,1 -dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, benzotetrahydrofuranyl, and the like.

[0038] Substituted heterocycloalkyl, aryl, heteroaryl are optionally substituted with, hydrogen, 1 to 3 alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, substituted aryl, aryl(alkyl), -SO2NR5R5, -PO3H2, -NR5SO2R6or -NR5C(=O)R6, wherein R5and R6are independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, optionallysubstituted heterocycloalkyl, aryloxy, heteroaryl, heteroaryl(alkyl), or R5and R6together are -(CH2)3-6- or -(CH2)o3X(CH2)o 3- where X= NR, 0, S, S02, substituted aryl(alkyl), halo(alkyl), SF5, NR , azido, cyano (also referred to herein as nitrile), -OR5, -SR5, -NR5R6, halogen, nitro, SCH3, OCF3, SO2CH3, SCF3, SO2CF3, CF3, -SO2OR5, -OSO2R5, CCI3, -C(=O)R5, -C(=O)OR5; -C(=O)NR5R6, -OC(=O)R5.

[0039] By "substituted" as in "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1 -C24 alkyl (including C1 -C18 alkyl, further including C1 -C12 alkyl, and further including C1 -C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and / or heteroatom-containing moieties, in addition to unsubstituted groups.

[0040] The term "water-soluble group" refers to a functional group that is well solvated in aqueous environments and that 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 saccharides, 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 glycols (PEG) and modified PEGs, and polyethers. In some instances, water-soluble groups are primary, secondary, tertiary, and quaternary amines, carboxylates, phosphonates, phosphates, sulfonates, sulfates, -N(H)0-I(CH2CH2OH)I-2, NHCH2CH2N(CH3)23, -NHCH2CH2SO3H, -NHCH2CH2PO3H2and -NHCH2CH2CO2H, - (CH2CH2O)yyCH2CH2XR -(CH2CH2O)yyCH2CH2X-, -X(CH2CH2O)yyCH2CH2-, glycol, oligoethylene glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NRZZ, and Rzzand RYYare independently selected from H and C1 -3 alkyl.

[0041] The term “carboxy isostere” refers to standard medicinal bioisosteric replacement groups for carboxylic acids, amides and ester. These include, but are not limited to: acyl cyanamide, tetrazoles, hydroxychromes, 3-hydroxy-1 ,2,4-triazoles, 1 -hydroxy pyrazoles, 2,4- dihydroxy imidazoles, 1 -hydroxy imidazole, 1 -hydroxy 1 ,2,3-triazole, alkylsulfonylcarboxamides, hydroxy isoxazoles, 5-hydroxy 1 ,2,4-oxadiazoles, thiazoles, 1 ,2,4- oxadiazoles, 1 ,2,4-oxadiazolones, oxazoles, triazoles, thiazoles, others hydroxamic acids, sulfonimide, acylsulfonamide, sulfonylureas, oxadiazoIone, thiazolidinediones, oxadiazole, thiadiazole, isothiazoles, difluorophenols, tetramic acids, tetronic acids, squaric acids, hydroxyquinoline-ones, hydroxyquinoline-2-ones, boronic acids and phosphoric acids.

[0042] As used herein the term “PEG” refers to a polyethylene glycol or a modified polyethylene glycol. Modified polyethylene glycol polymers include a methoxypolyethylene glycol, and polymers that are unsubstituted or substituted at one end with an alkyl, a substituted alkyl or a substituent (e.g., as described herein).

[0043] By the term “functional groups” is meant chemical groups such as 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), carbamide (-NH-(CO)-NH2), cyano (-C=N), isocyano (-N+=C-), cyanato (-O-C=N), isocyanato (-O-N+=C-), isothiocyanato (-S-C=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 termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), 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, mono- and di-(C5-C20 aryl)-substituted phosphine. In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above.

[0044] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" is to be interpreted as "substituted alkyl and substituted aryl."

[0045] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[0046] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =0, =NR70, =N-OR70, =N2 or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =0, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SON, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O M+, -SO2OR70, -OSO2R70, -OSO2O M+, -OSO2OR70, -P(O)(O" )2(M+)2, -P(O)(OR70)O M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -0(0)0" M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -00(0) 0 M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2"M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of 0, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]o.5, [Mg2+]0.5, or [Ba2+]os (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80is meant to include -NH2, -NH-alkyl, M-pyrrolidinyl, M-piperazinyl, 4N- methyl-piperazin-1 -yl, A / -morpholinyl, -N(H)o-i(CH2CH2OH)i-2, -NHCH2CH2N(CH3)2-3, - NHCH2CH2SO3H, -NHCH2CH2PO3H2and -NHCH2CH2CO2H.

[0047] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O M+, -OR70, -SR70, -S M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3" M+, -SO3R70, -OSO2R70, -OSO3"M+, -OSO3R70, -PO3'2(M+)2, -P(O)(OR70)O"M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2"M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -0C02M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2-M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -0 M+, -OR70, -SR70, or -S M+.

[0048] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O M+, -OR70, -SR70, -S M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O M+, -S(O)2OR70, -OS(O)2R70, -OS(O) 2O M+, -OS(O)2OR70, -P(O)(O )2(M+)2, -P(O)(OR70)O M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S) R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S )R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR 70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined.

[0049] Salts include but are not limited to: Na, K, Ca, Mg, ammonium, tetraalkyl ammonium, aryl and alkyl sulfonates, phosphates, carboxylates, sulfates, Cl, Br, and guanidinium.

[0050] Unless otherwise specified, reference to an atom is meant to include isotopes of that atom. For example, reference to H is meant to include1H,2H (i.e., D) and3H (i.e., T), and reference to C is meant to include12C and all isotopes of carbon (such as13C).

[0051] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1 , 2, 3, or 4 substituents, 1 , 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0052] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “heterocycloalkyl(alkyl)” refers to the group (heterocycloalkyl)-(alkyl)-.

[0053] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0054] In certain embodiments, a substituent may contribute to optical isomerism and / or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. Polymorphic, pseudo-polymorphic, amorphous and co-crystal forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus, the compounds described herein include salts, solvates, hydrates, prodrug and isomer formsthereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative.

[0055] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are commercially available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available. Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0056] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium 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 immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Formulations tobe used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0057] The term “sample” with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s diseased cell, e.g., a sample comprising polynucleotides and / or polypeptides that is obtained from a patient’s diseased cell (e.g., a cell lysate or other cell extract comprising polynucleotides and / or polypeptides); and a sample comprising diseased cells from a patient. A biological sample comprising a diseased cell from a patient can also include non-diseased cells.

[0058] RNA. The methods and reactants of the disclosure can be used to modify any form of single-stranded RNA, or single-stranded region of an RNA, including 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 synthetic or a natural product. In some embodiments the RNA is an mRNA of eukaryotic or prokaryotic origin. An mRNA may or may not have a cap and / or polyA tail. An RNA may or may not contain unnatural modified nucleobases. An RNA may be at least 12 nt in length, at least about 15, at least about 20, at least about 25, and may be greater than about 100 nt, 500 nt, 750 nt, 1 kb, 1 .5 kb, 2 kb, or larger. An RNA can be linear or cyclic.

[0059] Selective modification of a region of RNA, e.g. the poly-A tail, can be achieved by hybridizing the RNA to complementary DNA specific for sequences other than the region selected for modification, for example the 5'-UTR, open reading frame, and 3'-UTR of an mRNA. In some embodiments the 5'-UTR, open reading frame, 3'-UTR of mRNA are hybridized with complementary DNA oligos, with a length ranging from about 18 nt to about 120 nt. In some embodiments, substantially the entire mRNA sequence, apart from the poly- A tail is hybridized. In alternative embodiments a single strand of complementary DNA that hybridizes to the 5'-UTR, open reading frame, and 3'-UTR of the mRNA, e.g. synthesized byreverse transcriptase. Subsequent removal of the DNA strand with DNases produces mRNA with modifications at the targeted region.

[0060] Click chemistry is a concept in organic chemistry that describes a set of highly efficient and selective chemical reactions that can be employed to quickly and reliably generate molecular assemblies or conjugates. The most well-known example of a click reaction is the copper-catalyzed azide-alkyne cycloaddition (CuAAC), where an azide group reacts with an alkyne group to form a stable triazole linkage. Other click reactions include the strain-promoted azide-alkyne cycloaddition (SPAAC) that eliminates the need for copper catalysts, and the Diels-Alder reaction, among others.

[0061] The facile scalability, modularity and biocompatibility of click reactions allows application in the field of nucleic acids, enabling, for example, labeling of oligonucleotides (ODNs) with small-molecular probes; joining 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; etc.

[0062] For modifying nucleic acids, click chemistry reactions offer bio-orthogonal and highly specific options that are compatible with DNA and RNA. In some embodiments a reactant of Formula (I) or Formula (II) comprises as one or both of Ri and R2 a functional group for click chemistry conjugation, including, for example, azide, alkynes, e.g. for copper-catalyzed azide- alkyne cycloaddition (CuAAC); strained alkynes, e.g., DBCO for SPAAC, tetrazines and transcyclooctenes (TCO), thiol and alkene for UV-initiated thiol-ene reaction; oxime and hydrazone; aminooxy or hydrazine; isothiocyanates; etc., as known in the art. See, for example Fantoni et al. (2021 ) Chem Rev. 121 122-7154, herein specifically incorporated by reference.

[0063] In some embodiments click chemistry reactants provide for metal-free chemistry, e.g. SPAAC, conjugation between oxanorbornadiene derivatives and azides; reaction between strained alkenes and tetrazines; and alkene-tetrazole photoclick reaction. The SPAAC reaction involves the cycloaddition between a strained cyclooctyne and an organic azide, where RNA modified by the methods of the disclosure may comprise a strained cyclooctyne or an azide group, usually an azide.

[0064] Click chemistry reactions may be used to label RNA with a detectable group, a radioisotope, a therapeutic moiety, and the like. In some embodiments a detectable label is a fluorescent dye or fluorophore. Examples include, without limitation fluorescein, rhodamine, cyanine dyes such as Cy3 and Cy5, Alexa fluor series, e.g. Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647, green fluorescent protein (GFP) and variants thereof, Boron- dipyrromethene (BODIPY) dyes, Texas red, Dylight fluorophores; Atto dyes; Tetramethylrhodamine, etc.

[0065] Therapeutic moieties refer to pharmacologically active molecules or substances that can elicit a specific biological response in the body to treat or alleviate a particular medical condition. These moieties can be diverse, encompassing small organic molecules, peptides, nucleic acids, or even larger biologies such as proteins and antibodies.Compositions

[0066] Reactants that selectively modify RNA at the 2'-OH groups are provided. In some embodiments the reactants have a structure (I)where LG is a leaving group, and may be halide, e.g. comprising F, Cl, Br, I, etc.; an ammonium salt, tertiary amines, e.g. triaklylamines, -N(CH3)3+, N-methyl morpholine (NMM);X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, with the proviso that at least one of X and Y is a nitrogen;R1 and R2 are independently selected and may be absent or present. If present R1 , R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, -NHR, and may comprise groups suitable for click chemistry, e.g. NHCH2CH2OCH2CH2N3.

[0067] In some embodiments the reactants have a structure (II), where LG, X, R1 and R2 are as defined above.

[0068] Examples of reactants include, without limitation:In some embodiments an aromatic compound of interest for reaction is activated with a tertiary amine, for example by replacing the halogen on halogen-substituted heterocycles with NMM or trimethyl amine. In some embodiments a reactant comprises as one or both of Ri and R2 an azide or alkyne functional group for RNA conjugation using Cu-free click chemistry.

[0069] In some embodiments a composition is provided comprising a compound of structure (I) or (II). In some embodiments the compound is purified, e.g. at least about 75% of a composition is the compounds of interest, at least about 80%, at least about 90%, at least about 95% or more. In some embodiments the compound is provided in a powdered form. In some embodiments the compound is provided in a solution, e.g. in an aqueous solution. In some embodiments the compound is provided in a kit. A kit may further comprise instructions for use, click chemistry reactants, e.g a detectable label, RNA control samples, and the like.

[0070] Also provided are modified RNAs comprising an aryl adduct, e.g. an adduct of structure (I) without the leaving group. As discussed above, any RNA comprising at least a region that is single stranded is suitable for this purpose.Reactions

[0071] In some embodiments a method is provided for selective modification of RNA at 2’-OH groups, the method comprising contacting the RNA with a reactant of structure (I) or (II). In some embodiments the reaction is performed in an aqueous solution. In some embodiments the reaction is performed in vitro. In some embodiments the reaction is performed in vivo. In some embodiments the RNA is an mRNA and is selectively modified at the poly A tail, e.g. to reduce enzymatic degradation and increase stability.

[0072] In some embodiments the reaction generates an RNA with a click chemistry adduct, e.g. azide or alkyne groups. In some embodiments the RNA is further modified by conjugation at the click chemistry adduct, e.g. by conjugating to a detectable group, to a therapeutic moiety, to a drug for profiling, etc.

[0073] The reactants of the disclosure selectively react with RNA 2'-OH groups, e.g. where the reactivity is at least 2X greater than reactivity with DNA, at least 5X greater than reactivity with DNA, and may be at least 10X or more greater than reactivity with DNA. The reaction can be performed at high efficiency in aqueous solutions, e.g. in the absence or organic solvents such as DMSO, where 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 reactants are selective for ssRNA relative to dsRNA. The aryl adducts on RNA may block enzymatic reactions, e.g. polymerase.Kits

[0074] Kits may be provided. Kits may include reactants suitable for modifying RNA. A kit may further comprise, for example, click chemistry reactants, e.g a detectable label, RNA control samples, and the like. Components may be separately packaged in two or more containers suitable for use in the methods disclosed herein. Kits may also include tubes, buffers, etc., and instructions for use.EXPERIMENTAL

[0075] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1

[0076] 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), were screened against an 18-mer RNA oligonucleotide tRF3. All the RNA reactions were performed in pH 7.5 SHAPE buffer ([MOPS] = [NaCI] = 100 mM, [MgCI2] = 6.06 mM) over 24 h at 37°C. The concentration of RNA in these 10 pL reactions was fixed at 10 pM, and the concentration of electrophilic reagents was 200 mM. All RNA screening reactions for this set contained 20% DMSO by volume, and parallel control reactions were performed with DNA (under identical conditions) to determine RNA selectivity of these reagents.

[0077] The screening of this set revealed that several reagents react selectively with RNA 2'- OH in water in low to medium yields. Reagents 11 -21 are halogen-substituted pyrimidines, with electron-withdrawing groups at various positions on the heterocycle. In contrast to similar pyrimidine motifs in reagents 1 -6, which do not possess electron withdrawing substituents on the aromatic ring and do not react with RNA under the screening conditions, reagents 11-21 (except 19) demonstrate significant RNA conversion. The presence of a strong electron withdrawing group at the para position (relative to halogen substituent) significantly improves the RNA reactivity of these reagents, which is likely due to reduced electron density (via resonance) at the electrophilic carbon. Molecules 7-10 demonstrate that pyridine and benzene motifs do not result in RNA modification, likely due to a combination of poor solubility and lower reactivity compared to pyrimidine reagents. Surprisingly, reagents 22-24 do not display any detectable RNA modification despite being pyrimidine reagents with a highly electrophilic carbon center and containing strong electron withdrawing substituents. This is possibly due to the steric hindrance caused by large electron-rich substituents ortho to the electrophilic center. Finally, triazine and tetrazine reagents 27-32 have poor aqueous solubility, and lead to little or no detectable RNA modification. Interestingly, CDMT, reported as a peptide coupling reagent, resulted in RNA 2'-OH selective modification (13% conversion). CDMT has not been previously reported to react with alcohol functional motifs and is considered to be selective for carboxylic acid modification.

[0078] We noted that N-methylmorpholine has been used previously to convert the chlorotriazine species CDMT to a more water-soluble peptide coupling reagent, DMTMM. The cationic reagent DMTMM (Figure 3) is commercially available for that purpose; it reacts with carboxylic acids to make an active ester intermediate that reacts well with primary amines. We tested DMTMM and tested whether it reacts with RNA. Surprisingly, the reagent provided >98% conversion of 1RF3 RNA, and <6% 1RF3DNA modification under identical reaction conditions (20% DMSO, total reaction volume = 10 pL, [RNA] = [DNA] = 10 pM, [DMTMM] = 200 mM, [MOPS] = [NaCI] = 100 mM, [MgCI2] = 6.06 mM, pH 7.5, 24 h reaction at 37°C). However, it is reported that DMTMM is much more soluble, and stable, in water compared to DMSO. Thus, we decided to eliminate DMSO from the reaction workflow, and prepared DMTMM stock solutions in RNAse-free water for use in RNA reactions. Upon optimization of reaction conditions, we obtained >99% 1RF3 RNA conversion with 100 mM DMTMM, and <5% 1RF3DNA modification under identical conditions (0% DMSO, total reaction volume = 10 pL, [RNA] = [DNA] = 10 pM, [MOPS] = [NaCI] = 100 mM, [MgCI2] = 6.06 mM, pH 7.5, 24 h reaction at 37°C).

[0079] Next, we tested whether other halogen-substituted heterocycles could also be reacted with NMM to form an RNA-reactive species. The pyrimidine reagent 17 was chosen as a test substrate, as it contains an ester group that could inspire design and facile synthesis ofanalogs with useful functional handles. Gratifyingly, NMM-activated reagent 2PMM (Figure 3) was synthesized by simply adding 2 molar equivalents of N-methylmorpholine into a solution of 17 in anhydrous THF (50-100 mg scale, initial

[0017] = 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 filtration of 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 utilized for DMTMM experiments, we observed >98% RNA conversion and <1% DNA conversion with 200 mM 2PMM. Thus, we demonstrate that activation of triazine and pyrimidine heterocycles with N-methylmorpholine is an effective strategy to design molecules that selectively react with RNA 2'-OH in aqueous environments. Importantly, these molecules can perform RNA modification in total absence of DMSO, which is a useful property for future biological applications.

[0080] Encouraged by these results, we designed and synthesized an NMM-activated triazine reagent containing an azide functional group that could be used for RNA conjugation using Cu-free click chemistry. We designed the reagent AzTMM (Figure 3), which was synthesized in 2 steps: (1 ) conjugation of the N3-(PEG)2-NH2linker to molecule 32 using reported procedures,11and (2) activation of azide-containing chloro-triazine intermediate with N- methylmorpholine to synthesize AzTMM using standard procedures for NMM activation of triazines. Although AzTMM was successfully synthesized and characterized by LCMS, this reagent has poor solubility in both water and DMSO. Regardless of DMSO or water as cosolvent, AzTMM reaction with RNA only results in <60% RNA conversion even when [AzTMM] = 200 mM under RNA reaction conditions previously described. Gratifyingly, the reagent AzTN3 (Figure 3) synthesized by activation of 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 utilized for DMTMM experiments, we observed >93% RNA conversion and <5% DNA conversion with 200 mM AzTN3.

[0081] Encouraged by the favorable solubility properties of trimethylamine-activated aryl species, the reagents DMTN3 and 2PN3 were also synthesized using similar procedures. Interestingly, these highly water-soluble reagents maintain RNA 2'-OH reactivity in total absence of DMSO, demonstrating >99% RNA conversion in water. Thus, we demonstrate that N-methylmorpholine is not the only activating motif for high RNA reactivity in these cationic molecules. In general, activation by any tertiary amine may lead to high RNA 2'-OH reactivity, if water solubility can be maintained.

[0082] Stability in water is an important desirable property for RNA-reactive molecules, to maintain high effective reagent concentration for significant levels of RNA modification. In general, useful 2'-OH acylating reagents are highly reactive, and possess aqueous half-livesin the range of a few seconds to minutes. Recently reported sulfonylating 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 is 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 measured the mole fraction of DMTMM at various timepoints by1H NMR. This analysis revealed that DMTMM is surprisingly stable in water, with an aqueous half life of >10 days (Figure 4). This reagent is thus >66,000 fold more stable in water compared to 1 M7, >450 fold more stable than NAI, and >170 fold more stable compared to P3S (1 M7, NAI: common acylating agents, P3S: sulfonylating agent).

[0083] In summary, we have shown that tertiary amine-activated aryl groups can react in very high yields with RNA at 2'-OH groups. The resulting aryl ether adducts are stable on RNA, and are the first reactive species that do not react as carbonyl or sulfonyl structures. We have shown that the reagents can be synthesized and purified very easily, and that an NMM- activated species is unusually stable in water. We further report that these cationic reagents are sufficiently water soluble that there is no need for organic solvents when reacting with RNA. Finally, we have shown a number of applications of these groups, including labeling RNA and mapping RNA folded structure.Example 2. Preparation of a NMM reagent

[0084] 50 mg of compound 17 (0.29 mmol, Commercial Source: AA Blocks) was dissolved in580 pL of anhydrous THF in a 20 mL glass scintillation vial equipped with a magnetic stirrer. 58.7 mg of N-methylmorpholine (0.58 mmol, Commercial Source: TCI America) 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 washed with >5 mL THF to remove excess N-methylmorpholine. The residue was dried under vacuum to afford 79 mg of 2PMM (quantitative yield).

[0085] 1H NMR (300 MHz, DMSO-d6): 09.55 (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

[0086] ESI-MS [M-CI]+: Calculated: 238.12; Observed: 238.14Example 2. Synthesis of a trimethylamine triazine conjugate

[0087] AzTC. To a solution of 32 (2 g, 11.1 mmol) in anhydrous THF (80 mL) was added N,N- diisopropylethylamine (4.3 g, 33.3 mmol) in a 500 mL round bottom flask equipped with a magnetic stirrer. After stirring for 10 min, 2-(2-azidoethoxy)ethan-1 -amine (1.44 g, 11.1 mmol). After being stirred for 48 h, the solvent was removed in vacuo, and the resulting crude product was dissolved in ethyl acetate, followed by washing with 100 mM HCI and water (1x each). The organic phase was dried over Na2SO4, and concentrated in vacuo. Silica gel column chromatography (Hexane:EtOAc 1 :1 ) yielded AzTC (2.43 g, 80%)

[0088] 1H NMR (400 MHz, CDC ): [a mixture of conformational isomers] 6 6.05 (s, N / - / of major isomer), 5.94 (s, N H of minor isomer), 3.99 (s, OC / - / 3of major isomer), 3.94 (s, OC / - / 3of minor isomer), 3.70-3.63 (m, 6H), 3.41 -3.37 (m, 2H) ppm

[0089] ESI-MS [M+H]+: Calculated: 274.08; Observed: 274.11

[0090] AzTN3. 50 mg of AzTC (0.18 mmol) was dissolved in 364 pL of anhydrous THF in a 20 mL glass scintillation vial equipped with a magnetic stirrer. 180 pL of 2 M trimethylamine in dry THF (0.36 mmol, Commercial Source: TCI America) 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 washed with >5 mL THF to remove excess trimethylamine. The residue was dried under vacuum to afford 60 mg of AzTN3 (quantitative yield).

[0091] 1H NMR (400 MHz, D2O): [a mixture of conformational isomers] 5 4.07 (s, OCH3 of major isomer), 4.02 (s, OC / - / 3of minor isomer), 3.79-3.72 (m, 6H), 3.54 (s, N(CH3)3 of minor isomer), 3.50 (s, N(C / - / 3)3of major isomer), 3.50-3.45 (m, 2H) ppm

[0092] ESI-MS [M-CI]+: Calculated: 297.18; Observed: 297.26Example 4. Reaction of NMM activated aryl groups with RNA in pure water or DMSO

[0093] In a sterile 200 pL PCR tube, 3.3 pL of SHAPE 3.3X Buffer (333 mM MOPS pH 7.5, 333 mM NaCI, 20 mM MgCI2in water), was mixed with 4.7 pL 21.3 pM tRF3 RNA stock solution. Fresh stocks of DMTMM reagent were prepared in DMSO or water; and 2 pL of 1 Mor 500 mM stock, were added to 200 mM or 100 mM reactions respectively. These reactions were incubated for 24 h at 37°C and the RNA was subsequently purified by ethanol precipitation. The level of RNA modification was measured by MALDI-TOF M / S and analyzed using MestReNova™ software. Note: For every RNA reaction, a parallel reaction with 1RF3DNA was undertaken under identical conditions.

[0094] Ethanol precipitation of RNA reactions: For an RNA reaction with total volume X L, 9X L RNA precipitation solution (0.33 M NaOAc (pH 5.2) in water containing 0.2 mg / mL glycogen) was added and mixed well. 30X pL of ice-cold absolute ethanol was then added, and the mixture mixed by vortexing for at least 30 s. After storage at -80°C overnight, the mixture was centrifuged at 14.8k RPM for 60 mins at 4°C. The supernatant was discarded to obtain a pellet, which was washed with 70% ethanol. The obtained pellet was air dried for 15 min and subsequently either stored at -80°C for future use or dissolved in water / PBS for direct use in further experiments.

[0095] MALDI-TOF MS: All MALDI-TOF spectra were recorded at the Stanford University Mass Spectrometry facility, using the Bruker Daltonik Microflex MALDI-TOF spectrometer equipped with an N2laser. All spectra were recorded in linear negative mode and samples were plated on an MSP Anchorchip 96 target plate. 0.3 M trihydroxyacetophenone in EtOH (matrix) and 0.1 M aqueous ammonium citrate (co-matrix) were mixed in a 2:1 ratio by volume to be used as a matrix mix for MALDL This mix was always freshly prepared before analysis. After RNA precipitation, the RNA pellet was redissolved in RNAse-free water to prepare 10 pM sample solution. 1 pL of this solution was transferred to the target plate and dried under an Ar stream. 1 pL of the matrix mix was then added directly on top of the dried sample and completely dried under Ar stream. The spectral data was then recorded using Flex Control software (Bruker), and analyzed using MNova (Mestrenova).Example 5Structure mapping RNA in vitro and in vivo

[0096] RNA in a buffer that supports folding is shown to react preferentially with DMTMM and other ammonium triazine and pyrimidine reagents at unfolded regions of RNA compared with double-stranded regions. When human 5S RNA (either in vitro or in living cells) is exposed to one of these reagents at 50-200 mM, it is used to map folded structure. The time of reaction is adjusted to yield approximately one aryl group per RNA strand. A fluorophore-labeled DNA primer that binds specifically to 5S RNA is hybridized to the reacted RNA after this reaction, and a reverse transcriptase enzyme (such as Superscript 3) is added along with nucleoside triphosphates to support cDNA synthesis. The enzyme stalls at sites where arylation has occurred, producing a range of cDNAs of varied length. Analysis by gel electrophoresis showsa banding pattern consistent with the known folded structure of 5S RNA. This shows that the arylation agents can be used to map RNA folded structure.Example 6Labeling RNA with triazine conjugate

[0097] In a sterile 200 pL PCR tube, 10 M tRF3 RNA was treated with 2 pL of 1 M stock solution of AzTN3 in water (or just water for control reactions) following the RNA reaction protocol for 0% DMSO RNA reactions. 50 picomoles of treated tRF3 RNA was then dissolved in 40 pL 1X PBS (pH 7.4), and incubated at room temperature for 10 mins. 10 pL of 250 pM TAMRA-DBCO stock solution in DMSO was then added to the RNA solution, and mixed well. After incubation for 2 h at 37°C, the samples were subjected to ethanol precipitation to isolate the RNA and remove unreacted TAMRA reagent. The obtained pellet was then either dissolved in water for MALDI-TOF and PAGE analysis, or 1X PBS (pH 7.4) for fluorimeter experiments.

[0098] Mass peaks index:5662 Naked RNA6422adducts6838click adduct7601 RNA + 3 AzTN3 adducts + 1 (AzTN3+TAMRA) click adduct8013click adducts8772 RNA + 3 AzTN3 adducts + 2 (AzTN3+TAMRA) click adducts9190click adductsExample 7 Drug conjugate profiling

[0099] Reactivity-Based RNA Profiling (RBRP) can be used to analyze the interactions of drugs with RNAs throughout the cell. Previously, an acylimidazole reactive group was used to react with RNA; however, these reactive groups have short half-lives, making the reagents unstable and lowering yields in reactions with RNA. In new experiments, we choose a drug to be profiled that has an amine sidechain, and react it with the above reagent AzTC, producing an RNA-reactive drug conjugate. Exposure of this conjugate with human cells allows the drug to enter cells, bind RNAs in the cell, and react with RNAs where it binds. Isolation of the reacted RNAs and analysis by sequencing (following the published RBRP methodology) enables determination of where in the human transcriptome the drug binds. Control experiments with unmodified drug as a competitor in excess confirms drug-specific interactions.Example 8RNA modifications for enhanced biological properties

[0100] An arylation reagent such as DMTMM is reacted with messenger RNA such as eGFP mRNA at 100-200 mM in the presence of DNA that is hybridized to the whole mRNA except the polyA tail. This leaves the polyA region open to selective arylation. The DNA is then digested following the published TRAIL method. The resulting mRNA is transfected into cells using a cationic lipid formulation. Analysis of eGFP fluorescence in the cell is performed to determine if arylation enhances the total level of expression of this protein.References

[0101] Zubradt, M. etal. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat Methods 14, 75-82 (2017).

[0102] 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).

[0103] Spitale, R. C. ef al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature 2015519:7544519, 486-490 (2015).

[0104] Chatterjee, S., Shioi, R. & Kool, E. T. Sulfonylation of RNA 2'-OH groups. ACS Cent. Sci. 9, 531-539 (2023).

[0105] 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).

[0106] 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).

[0107] Fang, L. et al. Pervasive transcriptome interactions of protein-targeted drugs. Nat. Chem. 15, 1374-1383 (2023).

[0108] Kaminski, Z. J. 2-Chloro-4,6-dimethoxy-1 ,3,5-triazine. A New Coupling Reagent for Peptide Synthesis. Synthesis 1987, 917-920 (1987).

[0109] 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).

[0110] 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.

[0111] Kunishima, M. et al. Development of chlorotriazine polymer dehydrocondensing reagents (Poly-Trzs). Tetrahedron 63, 2604-2612 (2007).

[0112] Spitale, R. C. etal. RNA SHAPE analysis in living cells. Nature Chemical Biology 2012 9:1 9, 18-20 (2012).

[0113] Jahn, K. et al. Site-Specific Chemical Labeling of Long RNA Molecules. Bioconjugate Chem. 22, 95-100 (2011).

[0114] 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).

[0115] 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).

[0116] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

THAT WHICH is CLAIMED IS:1 . A method of selectively modifying RNA at a 2’OH group, the method comprising: reacting an RNA of interest with a reactant having a structure (I):wherein is a leaving group (LG) selected from a F, Cl, Br, I; an ammonium salt; or a tertiary amine;X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, with the proviso that at least one of X and Y is a nitrogen;R1 and R2are independently selected and may be absent or present, wherein if present R1, R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, or -NHR; to generate an aryl modified RNA.

2. The method of claim 1 , wherein the reactant has a structure (II):

3. The method of claim 1 or claim 2, wherein the tertiary amine is a trialkylamine.

4. The method of claim 3, wherein the trialkylamine is -N(CH3)3+.

5. The method of claim 1 or claim 2, wherein the tertiary amine is N-methyl morpholine (NMM).

6. The method of any of the preceding claims, wherein one or both of R1 and R2 comprise a click chemistry reactant.

7. The method of claim 6, wherein the click chemistry reactant comprises an azide or an alkyne.

8. The method of claim 1 or claim 2, wherein the reactant is selected from DMTMM, 2PMM, DMTN3, 2PN2, AzTMM, AzTN3.

9. The method of claim 6 or claim 7, further comprising the step of reacting the aryl modified RNA with a click reactant.

10. The method of claim 9, wherein the aryl modified RNA comprises an azide group and the click reactant comprises a constrained alkyne.

11. The method of claim 9 or claim 10, wherein the click reactant conjugates a detectable label to the aryl modified RNA.

12. The method of claim 9 or claim 10, wherein the click reactant conjugates a drug to the aryl modified RNA.

13. The method of claim 9 or claim 10, wherein the click reactant conjugates a therapeutic moiety to the aryl modified RNA.

14. The method of any of the previous claims wherein the reacting step is performed in vitro.

15. The method of any of the previous claims wherein the reacting step is performed in a living cell.

16. The method of any of the previous claims wherein the reacting step is performed in an aqueous solution.

17. A compound, comprising a structure (I):wherein is a leaving group (LG) selected from a F, Cl, Br, I; an ammonium salt; or a tertiary amine;X and Y are independently selected from N, C, CH, C-CO2R, C-CO2-, C-CN, C-NO2, C-SO3R, with the proviso that at least one of X and Y is a nitrogen;R1 and R2 are independently selected and may be absent or present, wherein if present R1, R2 are alkyl, heteroalkyl, halide, -OCH3, -NR2, or -NHR.

18. The compound of claim 17, having a structure (II):

19. The compound of claim 17 or 18, wherein the tertiary amine is a trialkylamine.

20. The compound of claim 19, wherein the trialkylamine is -N(CH3)3+.21 . The compound of claim 17 or 18, wherein the tertiary amine is N-methyl morpholine (NMM).

22. The compound of any of claims 17-21 , wherein one or both of R1 and R2 comprise a click chemistry reactant.

23. The compound of claim 17, wherein the click chemistry reactant comprises an azide or an alkyne.

24. The compound of claim 17 or 18, wherein the reactant is selected from DMTMM, 2PMM, DMTN3, 2PN2, AzTMM, AzTN3.

25. The compound of any of claims 17-24, conjugated to a drug at any of R1 , R2, or LG.

26. A kit comprising a compound of any of claims 17-25, and instructions for use in modifying RNA.

27. The kit of claim 26, further comprising a click chemistry reactant.

28. An RNA modified by the method of any of claims 1 -16.

29. The RNA of claim 28, wherein the RNA is selectively modified in a region of interest.

30. The RNA of claim 29, wherein the region of interest is a poly-A tail of an mRNA.

31. The RNA of any of claims 28-30, wherein the RNA has increased stability, expression, and / or decreased immunogenicity relative to the unmodified RNA.

32. The RNA of any of claims 28-30, wherein the aryl modification comprises a drug conjugate.

33. A method is for mapping RNA structure, comprising: modifying an RNA by the method of any of claims 1 -16, wherein the RNA is selectively aryl-modified at single stranded regions; binding primers to the RNA for initiation of polymerization wherein the polymerase is stalled at aryl adducts, to generate a population of polymerization products; analyzing the polymerization products for size and / or sequence.

Images