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.

单分子 FRET（福斯特共振能量转移）是研究生物分子结合动力学和构象转变的非常有效的方法。只有少数供体-受体染料对已被表征用于单分子 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 染料对非常重要。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 FRET 下 Cy3.5 和 Cy5.5 染料对的特性。我们证明这对染料在连续照明下可耐光约 5 分钟。我们还报告了 Cy3.5-Cy5。5 在双链 DNA 的背景下，存在多种生化缓冲液和光保护试剂时的 FRET 邻近依赖性和稳定性。最后，我们证明了 Cy3.5-Cy5.5 对在核小体 smFRET 体外研究中的相容性。