Structure-Function Relationship of Substituted Bromomethylcoumarins in Nucleoside Specificity of RNA Alkylation

Abstract

Selective alkylation of RNA nucleotides is an important field of RNA biochemistry, e.g. in applications of fluorescent labeling or in structural probing experiments, yet detailed structure-function studies of labeling agents are rare. Here, bromomethylcoumarins as reactive compounds for fluorescent labeling of RNA are developed as an attractive scaffold on which electronic properties can be modulated by varying the substituents. Six different 4-bromomethyl-coumarins of various substitution patterns were tested for nucleotide specificity of RNA alkylation using tRNA from Escherichia coli as substrate. Using semi-quantitative LC-MS/MS analysis, reactions at mildly acidic and slightly alkaline pH were compared. For all tested compounds, coumarin conjugates with 4-thiouridine, pseudouridine, guanosine, and uridine were identified, with the latter largely dominating. This data set shows that selectivity of ribonucleotide alkylation depends on the substitution pattern of the reactive dye, and even more strongly on the modulation of the reaction conditions. The latter should be therefore carefully optimized when striving to achieve selectivity. Interestingly, the highest selectivity for labeling of a modified nucleoside, namely of 4-thiouridine, was achieved with a compound whose selectivity was somewhat less dependent on reaction conditions than the other compounds. In summary, bromomethylcoumarin derivatives are a highly interesting class of compounds, since their selectivity for 4-thiouridine can be efficiently tuned by variation of substitution pattern and reaction conditions.

NAIL‐MS in E. coli Determines the Source and Fate of Methylation in tRNA

“…Figure A shows the common alkylation sites of the canonical nucleosides as defined in the literature. The alkylation of nucleosides has been exploited in vitro for the introduction of functional groups such as coumarin derivatives with and without clickable moieties for further functionalization (Figure A). Here, mainly uridine and thymidine (at position N3) were found to be alkylated under the alkaline reaction conditions.…”

General Principles for the Detection of Modified Nucleotides in RNA by Specific Reagents

“…For milder electrophiles like bromomethylcoumarins, strong influence of pH and solvent on the selectivity of alkylation have been demonstrated. [17] DMS, a popular reagent in RNA structural probing, [11b,18] and methyl methanesulfonate are known to alkylate several nitrogens to different degrees. This also includes, e.g., N 6 -A, a reaction which rarely appears in literature because detection for the purpose of structural probing is not straightforward.…”

“…While we are certain that several brands of vintage organic chemistry still await their application to deep sequencing, one must be aware that some reagents might simply not be selective enough for application to full-fledged epitranscriptomes, despite being able to discriminate short modified versus unmodified RNAs. [17,22] Rather, it might be helpful to the community to determine the size of a "limited transcriptome," [99] such as, e.g., the collective of tRNAs and rRNAs of ≈10 4 nucleotides, that can be meaningfully investigated with a given combination of reagent and reaction conditions. For example, at false positive rate of 1 in 1000, we expect 10 false positives in transcriptome of that size, which is a manageable number for validation by orthogonal methods.…”

A new strategy for site-specific alkylation of DNA using oligonucleotides containing an abasic site and alkylating probes

“…The 2-AVP derivatives are expected to form hydrogen bonds and to react selectively with the target base opposite the AP site. 75 The reactive base portion (6) was synthesized as shown in Scheme 1. The N9 position of chloropurine was alkylated with t- butyl bromoacetate to yield the t-butyl ester (5) in 75% yield.…”

“…The 2-AVP probe conjugated to penta-arginine was prepared by (Fmoc)based solid-phase synthesis. The protected penta-arginine on the resin was coupled with the 2-AVP derivative (6) at the Nterminal position. Subsequently, the stable precursor of the 2-35 AVP probe conjugated with the penta-arginine (7) was cleaved from the resin by treatment with TFA and triisopropylsilane.…”