Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution
Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution
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DOI：
10.1093/nar/gkaa133
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发表时间：
2020-05-21
影响因子：
14.9
通讯作者：
Saxena, Sunil
中科院分区：
文献类型：
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作者：
Ghosh, Shreya;Lawless, Matthew J.;Saxena, Sunil
电子顺磁共振（EPR）已成为探测核酸构象变化的重要工具。核酸的一系列EPR标签是可用的，但它们通常以长链为代价，依赖于特定核苷酸的存在，或者只能放置在末端。位点定向结合Cu2+-螯合到配体，2,2'二聚胺（DPA）是一个潜在的有吸引力的策略，位点特异性，核苷酸独立的Cu2+标记在DNA中。为了充分了解这个标签的潜力，我们使用EPR和分子动力学（MD）模拟对Cu2+-DPA基序进行了系统和详细的分析。我们利用连续波EPR实验来表征Cu2+与DPA的结合，并优化Cu2+的加载条件。我们在两个频率下进行了双电子-电子共振（DEER）实验，以阐明取向选择性效应。此外，DEER和MD模拟的距离分布的比较表明，在最可能的距离上有显著的一致性。结果表明Cu2+-DPA在报道足够长的碱基对分离的DNA主链构象方面是有效的。这种标记策略可以作为探测DNA与其他大分子相互作用时构象变化的重要工具。
Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2' dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.