Institute of Organic Chemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Nucleic Acids Res. 2011 Aug;39(15):6825-34. doi: 10.1093/nar/gkr281. Epub 2011 May 6.
Guanosines with substituents at the 8-position can provide useful fluorescent probes that effectively mimic guanine residues even in highly demanding model systems such as polymorphic G-quadruplexes and duplex DNA. Here, we report the synthesis and photophysical properties of a small family of 8-substituted-2'-deoxyguanosines that have been incorporated into the human telomeric repeat sequence using phosphoramidite chemistry. These include 8-(2-pyridyl)-2'-deoxyguanosine (2PyG), 8-(2-phenylethenyl)-2'-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2'-deoxyguanosine (4PVG). On DNA folding and stability, 8-substituted guanosines can exhibit context-dependent effects but were better tolerated by G-quadruplex and duplex structures than pyrimidine mismatches. In contrast to previously reported fluorescent guanine analogs, 8-substituted guanosines exhibit similar or even higher quantum yields upon their incorporation into nucleic acids (Φ = 0.02-0.45). We have used these highly emissive probes to quantify energy transfer efficiencies from unmodified DNA nucleobases to 8-substituted guanosines. The resulting DNA-to-probe energy transfer efficiencies (η(t)) are highly structure selective, with η(t)(duplex) < η(t)(single-strand) < η(t)(G-quadruplex). These trends were independent of the exact structural features and thermal stabilities of the G-quadruplexes or duplexes containing them. The combination of efficient energy transfer, high probe quantum yield, and high molar extinction coefficient of the DNA provides a highly sensitive and reliable readout of G-quadruplex formation even in highly diluted sample solutions of 0.25 nM.
具有 8 位取代基的鸟嘌呤核苷可以提供有用的荧光探针，即使在高度要求的模型系统（如多态 G-四链体和双链 DNA）中，也能有效地模拟鸟嘌呤残基。在这里，我们报告了一小组 8-取代-2'-脱氧鸟苷的合成和光物理性质，这些鸟苷已通过亚磷酰胺化学方法被整合到人类端粒重复序列中。这些包括 8-(2-吡啶基)-2'-脱氧鸟苷（2PyG）、8-(2-苯乙烯基)-2'-脱氧鸟苷（StG）和 8-[2-(吡啶-4-基)-乙烯基]-2'-脱氧鸟苷（4PVG）。在 DNA 折叠和稳定性方面，8-取代的鸟嘌呤可以表现出上下文相关的效应，但比嘧啶错配更能耐受 G-四链体和双链结构。与之前报道的荧光鸟嘌呤类似物不同，8-取代的鸟嘌呤在整合到核酸中时具有相似甚至更高的量子产率（Φ=0.02-0.45）。我们已经使用这些高发光探针来量化未修饰的 DNA 碱基与 8-取代的鸟嘌呤之间的能量转移效率。所得的 DNA 到探针的能量转移效率（η(t)）具有高度的结构选择性，η(t)(双链)<η(t)(单链)<η(t)(G-四链体)。这些趋势与包含它们的 G-四链体或双链的精确结构特征和热稳定性无关。高效能量转移、高探针量子产率和 DNA 的高摩尔消光系数的组合，即使在 0.25 nM 的高度稀释的样品溶液中，也能提供对 G-四链体形成的高度敏感和可靠的读出。
