School of Chemistry, University of Southampton, SO17 1BJ, UK.
Nucleic Acids Res. 2012 Aug;40(14):e108. doi: 10.1093/nar/gks303. Epub 2012 Apr 11.
Cy3B is an extremely bright and stable fluorescent dye, which is only available for coupling to nucleic acids post-synthetically. This severely limits its use in the fields of genomics, biology and nanotechnology. We have optimized the synthesis of Cy3B, and for the first time produced a diverse range of Cy3B monomers for use in solid-phase oligonucleotide synthesis. This molecular toolkit includes phosphoramidite monomers with Cy3B linked to deoxyribose, to the 5-position of thymine, and to a hexynyl linker, in addition to an oligonucleotide synthesis resin in which Cy3B is linked to deoxyribose. These monomers have been used to incorporate single and multiple Cy3B units into oligonucleotides internally and at both termini. Cy3B Taqman probes, Scorpions and HyBeacons have been synthesized and used successfully in mutation detection, and a dual Cy3B Molecular Beacon was synthesized and found to be superior to the corresponding Cy3B/DABCYL Beacon. Attachment of Cy3, Cy3B and Cy5 to the 5-position of thymidine by an ethynyl linker enabled the synthesis of an oligonucleotide FRET system. The rigid linker between the dye and nucleobase minimizes dye-dye and dye-DNA interactions and reduces fluorescence quenching. These reagents open up new future applications of Cy3B, including more sensitive single-molecule and cell-imaging studies.
Cy3B 是一种极其明亮和稳定的荧光染料，只能在合成后与核酸偶联。这严重限制了它在基因组学、生物学和纳米技术领域的应用。我们已经优化了 Cy3B 的合成，并首次生产了多种 Cy3B 单体，用于固相寡核苷酸合成。这个分子工具包包括与脱氧核糖、胸腺嘧啶 5 位以及己炔基连接的 Cy3B 连接的亚磷酰胺单体，以及将 Cy3B 连接到脱氧核糖的寡核苷酸合成树脂。这些单体已被用于将单个和多个 Cy3B 单元内部和两端掺入寡核苷酸中。已经成功合成了 Cy3B Taqman 探针、Scorpions 和 HyBeacons，并用于突变检测，并且还合成了双 Cy3B 分子信标，发现其优于相应的 Cy3B/DABCYL 信标。通过乙炔基连接体将 Cy3、Cy3B 和 Cy5 连接到胸腺嘧啶的 5 位，从而能够合成寡核苷酸 FRET 系统。染料和碱基之间的刚性连接体最小化了染料-染料和染料-DNA 的相互作用，减少了荧光猝灭。这些试剂为 Cy3B 的新应用开辟了道路，包括更敏感的单分子和细胞成像研究。
