Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiolcontaining molecules as well as with cysteine-or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.
Abstract2‐Formyl‐2′‐deoxyadenosine triphosphate (dCHOATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHOATP was based on the preparation of protected 2‐dihydroxyethyl‐2′‐deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHOATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31‐mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single‐nucleotide extension followed by PEX was used for site‐specific incorporation of one aldehyde‐linked adenosine into a longer 49‐mer sequence. The reactive formyl group was used for cross‐linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647‐linked hydroxylamine.
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