Institute of Chemical Biology and Fundamental Medicine SB RAS , Novosibirsk 630090, Russia.
Novosibirsk State University , Novosibirsk 630090, Russia.
J Phys Chem B. 2018 Jan 11;122(1):137-143. doi: 10.1021/acs.jpcb.7b10689. Epub 2017 Dec 20.
Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date, existing approaches to site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotides, thus hindering a majority of demanded applications. Herein, we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then postsynthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of measuring interspin distances up to ∼5-6 nm at 298 K using double quantum coherence electron paramagnetic resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.
三芳基甲基（三苯甲基，TAM）自由基是一类很有前途的自旋标记物，可用于在生理温度下测量生物分子中的纳米级距离。然而，迄今为止，用于 DNA 定点 TAM 标记的现有方法仅限于寡核苷酸末端的标记附着，从而阻碍了大多数所需的应用。在此，我们报告了一种在核酸任意位置附着 TAM 的新的多功能策略。它利用一种手性非核苷亚磷酰胺单体用于寡核苷酸的自动化固相合成，然后用 TAM 对其进行后期功能化。我们展示了一组寡核苷酸复合物的合成，这些复合物在内部或末端位置进行了 TAM 标记，并且还可以使用双量子相干电子顺磁共振（EPR）在 298 K 下测量高达约 5-6nm 的自旋间距离。所开发方法的实施大大拓宽了可用于室温下纳米级结构 EPR 研究的核酸和核蛋白复合物的范围。
