Single-Molecule Characterization of Cy3.5 -Cy5.5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes.
Single-Molecule Characterization of Cy3.5 -Cy5.5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes.
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DOI：
10.1007/s10895-022-03093-z
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发表时间：
2023-03
影响因子：
2.7
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单分子FRET(Forster Resonance Energy Transfer)是研究生物分子结合动力学和构象转变的一种非常有效的方法。只有少数供体-受体染料对被表征用于单分子FRET(SmFRET)研究。因此，为了扩大单分子FRET在生物分子研究中的应用范围，引入和表征额外的FRET染料对是很重要的。在这里，我们使用裸双链DNA(DsDNA)和核小体在单分子水平上表征了Cy3.5和Cy5.5染料对在FRET下的性质。我们证明了这对染料在连续光照下的光稳定性为~ 5分钟。我们还报告了在双链DNA背景下，在几种生化缓冲剂和光保护剂存在的情况下，Cy3.5-Cy5.5 FRET的邻近依赖性和稳定性。最后，在核小体的体外研究中，我们证明了Cy3.5-Cy5.5对smFRET的兼容性。在线版本包含补充材料，可在10.1007/s10895-022-03093-z上查阅。
Single molecule FRET (Forster resonance energy transfer) is very powerful method for studying biomolecular binding dynamics and conformational transitions. Only a few donor - acceptor dye pairs have been characterized for use in single-molecule FRET (smFRET) studies. Hence, introducing and characterizing additional FRET dye pairs is important in order to widen the scope of applications of single-molecule FRET in biomolecular studies. Here we characterize the properties of the Cy3.5 and Cy5.5 dye pair under FRET at the single-molecule level using naked double-stranded DNA (dsDNA) and the nucleosome. We show that this pair of dyes is photostable for ~ 5 min under continuous illumination. We also report Cy3.5-Cy5.5 FRET proximity dependence and stability in the presence of several biochemical buffers and photoprotective reagents in the context of double-stranded DNA. Finally, we demonstrate compatibility of the Cy3.5-Cy5.5 pair for smFRET in vitro studies of nucleosomes. The online version contains supplementary material available at 10.1007/s10895-022-03093-z.