An ELISA-based platform for rapid identification of structure-dependent nucleic acid-protein interactions detects novel DNA triplex interactors.
An ELISA-based platform for rapid identification of structure-dependent nucleic acid-protein interactions detects novel DNA triplex interactors.
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DOI：
10.1016/j.jbc.2022.102398
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发表时间：
2022-10
影响因子：
4.8
通讯作者：
Glazer, Peter M.
中科院分区：
文献类型：
--
作者：
Economos, Nicholas G.;Thapar, Upasna;Balasubramanian, Nanda;Karras, Georgios I.;Glazer, Peter M.
不寻常的核酸结构在许多细胞过程中作为中间体发挥着重要作用，并且在肽核酸（PNA）介导的三链体的情况下，被用作治疗性基因编辑的工具。然而，由于它们的瞬时性质，对与动态核酸结构相互作用和处理动态核酸结构的因子的理解仍然有限。在这里，我们开发了snapELISA（结构特异性核酸结合蛋白ELISA），这是一种快速高通量平台，可以在体外询问和比较多达2688个平行的核酸结构-蛋白质相互作用。我们将该系统应用于形成三链体的阿托西肽诱导的DNA三链体和DNA结合的PNA异三链体，以描述这两种结构的先前已知的和新的相互作用的识别。对于PNA异源三链体识别分析，snapELISA鉴定了涉及核苷酸切除修复（XPA，XPC）、单链退火修复（RAD 52）和重组中间结构结合（TOP 3A，BLM，MUS 81）的因子。我们继续验证选择的因子定位于人类细胞中临床相关基因座内的基因组靶向PNA结构。令人惊讶的是，这些结果表明XRCC 5定位于基因组中的PNA三链体形成位点，表明存在双链断裂中间体。这些结果描述了一个强大的比较方法，用于识别结构特异性核酸相互作用，并扩大了我们对三链体结构识别和修复机制的理解。
Unusual nucleic acid structures play vital roles as intermediates in many cellular processes and, in the case of peptide nucleic acid (PNA)–mediated triplexes, are leveraged as tools for therapeutic gene editing. However, due to their transient nature, an understanding of the factors that interact with and process dynamic nucleic acid structures remains limited. Here, we developed snapELISA (structure-specific nucleic acid-binding protein ELISA), a rapid high-throughput platform to interrogate and compare up to 2688 parallel nucleic acid structure–protein interactions in vitro. We applied this system to both triplex-forming oligonucleotide–induced DNA triplexes and DNA-bound PNA heterotriplexes to describe the identification of previously known and novel interactors for both structures. For PNA heterotriplex recognition analyses, snapELISA identified factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We went on to validate selected factor localization to genome-targeted PNA structures within clinically relevant loci in human cells. Surprisingly, these results demonstrated XRCC5 localization to PNA triplex-forming sites in the genome, suggesting the presence of a double-strand break intermediate. These results describe a powerful comparative approach for identifying structure-specific nucleic acid interactions and expand our understanding of the mechanisms of triplex structure recognition and repair.
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DOI：
10.1073/pnas.262556899
发表时间：
2002-12-24
影响因子：
11.1
作者：
Rogers, FA;Vasquez, KM;Glazer, PM
通讯作者：
Glazer, PM
影响因子：
16.6
作者：
Graindorge, Antoine;Pinheiro, Ines;Shkumatava, Alena
通讯作者：
Shkumatava, Alena
影响因子：
14.9
作者：
Hansen ME;Bentin T;Nielsen PE
通讯作者：
Nielsen PE
影响因子：
14.9
作者：
Bentin T;Hansen GI;Nielsen PE
通讯作者：
Nielsen PE
影响因子：
16.6
作者：
Bahal, Raman;McNeer, Nicole Ali;Quijano, Elias;Liu, Yanfeng;Sulkowski, Parker;Turchick, Audrey;Lu, Yi-Chien;Bhunia, Dinesh C.;Manna, Arunava;Greiner, Dale L.;Brehm, Michael A.;Cheng, Christopher J.;Lopez-Giraldez, Francesc;Ricciardi, Adele;Beloor, Jagadish;Krause, Diane S.;Kumar, Priti;Gallagher, Patrick G.;Braddock, Demetrios T.;Saltzman, W. Mark;Ly, Danith H.;Glazer, Peter M.
通讯作者：
Glazer, Peter M.