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.

光亲和标记（PAL）与质谱技术的最新发展相结合，是研究核酸-蛋白质相互作用的强大工具，可通过共价键形成来实现两个伙伴的交联。这种策略需要对最明智的光反应基团进行初步研究，以有效地与目标蛋白交联。在这项研究中，我们报告了对三种不同的光反应性核碱基（包括用二苯甲酮或二嗪胺功能化的鸟嘌呤和零长度剂4-硫代胸腺嘧啶）进行的一项调查，该碱基掺入含有生物素部分的30-聚体寡核苷酸（ODN）中，以进行选择性捕集以及单链DNA结合蛋白（SSB）的富集。第一的，以人复制蛋白A为模型，研究了纯化蛋白与光化学反应的条件和效率。其次，探讨了探针作为诱饵在细胞裂解液中进行光交联和富集SSB的能力。在研究的各种ODN探针中，我们表明4-硫代胸腺嘧啶是最相关的：i）可以在全细胞提取物中有效和特异性地捕获SSB，其程度与广泛使用的重氮类似。ii ）具有零位剂的优点，从而保留了ODN诱饵的理化特性；iii）包括该光化学试剂在内的ODN易于获得。与质谱相结合，掺入该核碱基的探针是用于PAL策略的强大工具，可以添加到传统光交联剂的工具箱中以研究DNA与蛋白质的相互作用。