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

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 agent thus retaining the physicochemical properties of the ODN bait; iii) ODN including this photochemical agent are easily accessible. In combination with mass spectrometry, the probes incorporating this nucleobase are powerful tools for PAL strategies and can be added in the toolbox of the traditional photocrosslinkers for studying DNA-protein interactions.

Keywords: 4-thiothymidine; Human replication protein A; Photoaffinity labeling; Single-stranded DNA binding proteins.