Genetically encoded chemical crosslinking of RNA in vivo

Sun, Wei; Wang, Nanxi; Liu, Hongjiang; Yu, Bingchen; Jin, Lihua; Greenleaf, W; Shen, Yin; Wang, Lei

doi:10.1038/s41557-022-01038-4

Cited by 39 publications

(47 citation statements)

References 67 publications

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“…Due to the limitations of C2 substituted imidazoles, we explored a wider variety of leaving group structures. Inspired by many literature examples of the wide applicability of SuFEx reagents as covalent tags in aqueous environments, particularly in protein profiling by reaction with nucleophilic side chains of amino acids 22,23,26 and a recent report of engineered proteindirected RNA modification, 21 we tested the simple pyridine-3sulfonyl fluoride 9. In addition, we tested an NHS sulfonyl ester (10).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…In particular, activated sulfonyl groups have proven highly useful recently in protein profiling, showing reactivity at tyrosine and other side chains in water. − Encouragingly, a recent study has also reported that RNA-binding proteins that are genetically encoded to possess fluorosulfate-containing modified tyrosine residues can direct the formation of sulfate ester linkages at RNA 2′-OH . Following these precedents, we undertook a study of the potential for small-molecule sulfonylation in RNA.…”

Section: Introductionmentioning

confidence: 99%

“…Our chief motivations were (1) finding reactive motifs beyond modified sulfur groups on proteins, which may have different reactivities than small alkyl/aryl sulfonyl compounds due to the macromolecular context; (2) discovering strategies to tune RNA reactivity of small-molecule sulfonylating reagents and establishing the scope of effective structures; and (3) investigating the potential for further application in RNA chemical biology research ( e.g ., RNA conjugation and structure analysis). Importantly, to establish a general approach with wide applicability, we tested small-molecule sulfonyl reagents whose reactivity would be independent of any RNA-macromolecular interactions, to reduce the possibility of reactions occurring by proximity effects and to remove the requirement for genetic encoding . Given the literature examples and our motivation, the oxophilic nature of sulfur and the hydrophilicity of sulfonyl groups, we wondered whether there might exist a small-molecule sulfonylating reagent structure that might enable aqueous reactivity with 2′-OH groups in RNA.…”

Section: Introductionmentioning

confidence: 99%

“…18−20 Encouragingly, a recent study has also reported that RNA-binding proteins that are genetically encoded to possess fluorosulfate-containing modified tyrosine residues can direct the formation of sulfate ester linkages at RNA 2′-OH. 21 Following these precedents, we undertook a study of the potential for small-molecule sulfonylation in RNA. Our chief motivations were (1) finding reactive motifs beyond modified sulfur groups on proteins, which may have different reactivities than small alkyl/aryl sulfonyl compounds due to the macromolecular context; (2) discovering strategies to tune RNA reactivity of small-molecule sulfonylating reagents and establishing the scope of effective structures; and (3) investigating the potential for further application in RNA chemical biology research (e.g., RNA conjugation and structure analysis).…”

Section: ■ Introductionmentioning

confidence: 99%

“…Importantly, to establish a general approach with wide applicability, we tested smallmolecule sulfonyl reagents whose reactivity would be independent of any RNA-macromolecular interactions, to reduce the possibility of reactions occurring by proximity effects and to remove the requirement for genetic encoding. 21 Given the literature examples and our motivation, the oxophilic nature of sulfur and the hydrophilicity of sulfonyl groups, we wondered whether there might exist a small-molecule sulfonylating reagent structure that might enable aqueous reactivity with 2′-OH groups in RNA. Desirable features for RNA 2′-OH-reactive reagents include sufficient reactivity to functionalize this relatively bulky secondary hydroxyl in high yields, selectivity for 2′-OH groups in unpaired structure over paired contexts, adequate lifetime and solubility in water to remain available for RNA, cell permeability for in vivo application, ease of synthesis and purification, versatility of structural modification, and tunability of electrophilicity.…”

Section: ■ Introductionmentioning

confidence: 99%

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Sulfonylation of RNA 2′-OH groups

2023

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The nucleophilic reactivity of RNA 2′-OH groups in water has proven broadly useful in probing, labeling, and conjugating RNA. To date, reactions selective to ribose 2′-OH have been limited to bond formation with short-lived carbonyl electrophiles. Here we report that many activated small-molecule sulfonyl species can exhibit extended lifetimes in water and retain 2′-OH reactivity. The data establish favorable aqueous solubility for selected reagents and successful RNA-selective reactions at stoichiometric and superstoichiometric yields, particularly for aryl sulfonyltriazole species. We report that the latter are considerably more stable than most prior carbon electrophiles in aqueous environments and tolerate silica chromatography. Furthermore, an azide-substituted sulfonyltriazole reagent is developed to introduce labels into RNA via click chemistry. In addition to high-yield reactions, we find that RNA sulfonylation can also be performed under conditions that give trace yields necessary for structure mapping. Like acylation, the reaction occurs with selectivity for unpaired nucleotides over those in the duplex structure, and a sulfonate adduct causes reverse transcriptase stops, suggesting potential use in RNA structure analysis. Probing of rRNA is demonstrated in human cells, indicating possible cell permeability. The sulfonyl reagent class enables a new level of control, selectivity, versatility, and ease of preparation for RNA applications.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Sulfonylation of RNA 2′-OH groups

2023

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Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development

Tang,

Wang,

Zhu

et al. 2024

Angewandte Chemie

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Advances in targeted covalent inhibitors (TCIs) have been made by using lysine‐reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell‐active TCIs are however available. We report herein lysine‐reactive activity‐based probes (ABPs; 2‐14) based on the chemistry of aryl fluorosulfates (ArOSO2F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase‐binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X‐ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX‐680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine‐reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine‐reactive ArOSO2F‐based TCIs.

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Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins

Cao,

Yu,

Klauser

et al. 2024

Angewandte Chemie

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Sulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

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Genetically encoded chemical crosslinking of RNA in vivo

Cited by 39 publications

References 67 publications

Sulfonylation of RNA 2′-OH groups

Sulfonylation of RNA 2′-OH groups

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development

Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins

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