Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria.
Sci Rep. 2022 Aug 31;12(1):14803. doi: 10.1038/s41598-022-19069-9.
Fluorescence is an ideal tool to see and manipulate nucleic acids, and engage in their rich and complex biophysical properties. Labeling is the preferred approach to track and quantify fluorescence with nucleic acids and cyanine dyes are emblematic in this context. The fluorescent properties of cyanine dyes are known to be sequence-dependent, with purines in the immediate vicinity increasing the fluorescence intensity of Cy3 and Cy5 dyes, and the ability of nucleobases to modulate the photophysical properties of common fluorophores may influence fluorescence measurements in critical assays such as FISH, qPCR or high-throughput sequencing. In this paper, we comprehensively map the sequence-dependence of Cy3 and Cy5 dyes in 3'-fluorescently labeled single-stranded DNA by preparing the complete permutation library of the 5 consecutive nucleotides immediately adjacent to the dye, or 1024 sequences. G-rich motifs dominate the high fluorescence range, while C-rich motifs lead to significant quenching, an observation consistent with 5'-labeled systems. We also uncover GCGC patterns in the extreme top range of fluorescence, a feature specific to 3'-Cy3 and Cy5 oligonucleotides. This study represents the final piece in linking nucleotide identity to fluorescence changes for Cy3, Cy5 and fluorescein in all 3', 5', single-stranded and double-stranded DNA formats.
荧光是观察和操纵核酸并研究其丰富而复杂的生物物理特性的理想工具。标记是跟踪和定量核酸荧光的首选方法，在这种情况下，氰基染料是标志性的。氰基染料的荧光性质已知是序列依赖性的，紧邻的嘌呤增加 Cy3 和 Cy5 染料的荧光强度，并且碱基改变常见荧光团的光物理性质的能力可能会影响关键测定（如 FISH、qPCR 或高通量测序）中的荧光测量。在本文中，我们通过制备紧邻染料的 5 个连续核苷酸的完整排列文库，即 1024 个序列，全面绘制了 Cy3 和 Cy5 染料在 3'荧光标记单链 DNA 中的序列依赖性。富含 G 的基序占据高荧光范围，而富含 C 的基序导致显著猝灭，这一观察结果与 5'标记系统一致。我们还在荧光的极端顶端范围内发现了 GCGC 模式，这是 3'-Cy3 和 Cy5 寡核苷酸特有的特征。这项研究代表了将核苷酸身份与 Cy3、Cy5 和荧光素在所有 3'、5'、单链和双链 DNA 格式中的荧光变化联系起来的最后一块。
