Nucleic acid-binding photoprobes and uses thereof

A method of determining the three-dimensional structure, binding site of a ligand of interest, or accessibility of a nucleotide in a target nucleic acid, comprising:

• contacting the target nucleic acid with a of Formula II or a pharmaceutically acceptable salt thereof;
• irradiating the compound;
• determining whether covalent modification of a nucleotide of the nucleic acid has occurred;
• and optionally deriving the pattern of nucleotide modification, the three-dimensional structure, ligand binding site, or other structural information about the nucleic acid;
• wherein the of Formula II is of the following structure:
• or a pharmaceutically acceptable salt thereof;
• wherein:
• Ligand is a small molecule RNA binder selected from the group consisting of a heteroaryldihydropyrimidine (HAP), a macrolide, an alkaloid, an aminoglycoside, a tetracycline, a SMN2 ligand, a pleuromutilin, theophylline, ribocil, a substituted anthracene, a substituted triptycene, an oxazolidinone, and CPNQ;
• wherein Ligand may be optionally substituted with one or more substituents;
• T1 is a bivalent tethering group selected from a C1-20 bivalent straight or branched hydrocarbon chain wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 methylene units of the chain are independently and optionally replaced with a natural or non-natural amino acid, —O—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)—, —C(O)N(R)—, —(R)NC(O)—, —OC(O)N(R)—, —(R)NC(O)O—, —N(R)C(O)N(R)—, —S—, —SO—, —SO2—, —SO2N(R)—, —(R)NSO2—, —C(S)—, —C(S)O—, —OC(S)—, —C(S)N(R)—, —(R)NC(S)—, —(R)NC(S)N(R)—, or -Cy-;
• and 1-20 of the methylene units of the chain are independently and optionally replaced with —OCH2CH2—;
• wherein each -Cy- is independently a bivalent optionally substituted 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, optionally substituted phenylene, an optionally substituted 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-3 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, an optionally substituted 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, an optionally substituted 8-10 membered bicyclic or bridged bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, or an optionally substituted 8-10 membered bicyclic or bridged bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur;
• each R is independently hydrogen or an optionally substituted group selected from the group consisting of C1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, and, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur;
• T2 is a covalent bond or a bivalent tethering group selected from a C1-20 bivalent straight or branched hydrocarbon chain wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 methylene units of the chain are independently and optionally replaced with a natural or non-natural amino acid, —O—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)—, —C(O)N(R)—, —(R)NC(O)—, —OC(O)N(R)—, —(R)NC(O)O—, —N(R)C(O)N(R)—, —S—, —SO—, —SO2N(R)—, —(R)NSO2—, —C(S)—, —C(S)O—, —OC(S)—, —C(S)N(R)—, —(R)NC(S)—, —(R)NC(S)N(R)—, or -Cy-;
• and 1-20 of the methylene units of the chain are independently and optionally replaced with —OCH2CH2—;
• RCG is a click-ready group selected from the group consisting of an azide, an alkyne, 4-dibenzocyclooctynol (DIBO) gem-difluorinated cyclooctynes (DIFO or DFO), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), a strained cyclooctyne, an oxime, and oxanorbornadiene;
• or a pull-down group selected from the group consisting of a hapten, a 14C, a 32P, and a 3H radiolabel;
• and
• Rmod is a photoactivatable group selected from the group consisting of
• wherein Y− is a pharmaceutically acceptable anion.