Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites

Kueck, Nadine A; Ovcharenko, Anna; Hartstock, Katja; Cornelissen, Nicolas V.; Rentmeister, Andrea

doi:10.1016/bs.mie.2021.06.006

Cited by 2 publications

(1 citation statement)

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“…Chemical agents that can covalently modify RNA offer the potential for conjugation with labels. For example, RNA can be covalently tagged with fluorophores for detection, imaging, and FRET analysis, − or with biotin for pull-down and separation protocols. , However, most RNA covalent labeling methods involve enzymatic nucleotide incorporation protocols or engineered RNA sequences, − and only a few reagents based on short-lived activated carbonyl species have been tested for high-yield labeling. , Given the stability and ease of synthesis of sulfonyltriazoles, we asked whether such species could be used to introduce a reactive handle in RNA. For our first test, we designed a sulfonyl triazole-containing reagent functionalized with an azide functional group that could potentially react with RNA 2′-OH groups, potentially allowing for further RNA conjugation.…”

Section: Resultsmentioning

confidence: 99%

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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