Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
Biochemistry. 2020 Oct 27;59(42):4072-4080. doi: 10.1021/acs.biochem.0c00657. Epub 2020 Oct 13.
Transient association of guanine-rich RNA and DNA in the form of hetero-G-quadruplexes (RDQs) has emerged as an important mechanism for regulating genome transcription and replication but relatively little is known about the structure and biophysical properties of RDQs compared with DNA and RNA homo-G-quadruplexes. Herein, we report the assembly and characterization of three RDQs based on sequence motifs found in human telomeres and mitochondrial nucleic acids. Stable RDQs were assembled using a duplex scaffold, which prevented segregation of the DNA and RNA strands into separate homo-GQs. Each of the RDQs exhibited UV melting temperatures above 50 °C in 100 mM KCl and predominantly parallel morphologies, evidently driven by the RNA component. The fluorogenic dye thioflavin T binds to each RDQ with low micromolar values, similar to its binding to RNA and DNA homo-GQs. These results establish a method for assembling RDQs that should be amenable to screening compounds and libraries to identify selective RDQ-binding small molecules, oligonucleotides, and proteins.
嘌呤丰富的 RNA 和 DNA 以异源 G-四联体 (RDQs) 的形式短暂结合，已成为调节基因组转录和复制的重要机制，但与 DNA 和 RNA 同源 G-四联体相比，人们对 RDQs 的结构和生物物理特性知之甚少。本文报道了基于人端粒和线粒体核酸中发现的序列基序构建和表征的三种 RDQs。使用双链支架组装稳定的 RDQs，防止 DNA 和 RNA 链分离成单独的同源 G-Qs。每个 RDQ 在 100 mM KCl 中表现出高于 50°C 的 UV 熔点，并且主要具有平行构象，显然是由 RNA 成分驱动的。荧光染料硫代黄素 T 与每个 RDQ 的结合值为低微摩尔，类似于其与 RNA 和 DNA 同源 G-Qs 的结合。这些结果建立了一种组装 RDQs 的方法，该方法应该适合筛选化合物和文库，以鉴定选择性 RDQ 结合的小分子、寡核苷酸和蛋白质。
