Photoactivatable oligonucleotide probes to trap single-stranded DNA binding proteins: Updating the potential of 4-thiothymidine from a comparative study

Biochimie
Tom Gérard-HirneDominique Guianvarc'h

Abstract

Photoaffinity labeling (PAL) in combination with recent developments in mass spectrometry is a powerful tool for studying nucleic acid-protein interactions, enabling crosslinking of both partners through covalent bond formation. Such a strategy requires a preliminary study of the most judicious photoreactive group to crosslink efficiently with the target protein. In this study, we report a survey of three different photoreactive nucleobases (including a guanine functionalized with a benzophenone or a diazirine and the zero-length agent 4-thiothymine) incorporated in 30-mer oligonucleotides (ODN) containing a biotin moiety for selective trapping and enrichment of single-stranded DNA binding proteins (SSB). First, the conditions and efficiency of the photochemical reaction with a purified protein using human replication protein A as the relevant model was studied. Secondly, the ability of the probe as bait to photocrosslink and enrich SSB in cell lysate was addressed. Among the different ODN probes studied, we showed that 4-thiothymine was the most relevant: i) it allows efficient and specific trapping of SSB in whole cell extracts in a similar extent as the widely used diazirine, ii) it features the advantages of a zero-length a...Continue Reading

Citations

Feb 6, 2020·Photochemistry and Photobiology·Nadejda I Rechkunova, Olga I Lavrik
Mar 2, 2021·Chemical Society Reviews·Luke K McKenzieMarcel Hollenstein

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