Turaev Anton V, Isaakova Ekaterina A, Severov Vjacheslav V, Bogomazova Alexandra N, Zatsepin Timofei S, Sardushkin Makar V, Aralov Andrey V, Lagarkova Maria A, Pozmogova Galina E, Varizhuk Anna M
Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, 119435, Russia.
Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, 119435, Russia; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, 119435, Russia.
Biosens Bioelectron. 2021 Mar 1;175:112864. doi: 10.1016/j.bios.2020.112864. Epub 2020 Dec 2.
We report the design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration- or carcinogenesis-related genes. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the 'genome-inspired' design is a productive approach to the development of biocompatible molecular tools.
我们报告了基于在神经退行性变或致癌相关基因中发现的天然DNA i-基序（iM）设计的用于测量细胞内pH值的稳健传感器。这些iM似乎是基因组调控元件，可能会响应pH刺激调节转录。鉴于它们对生理范围内微小pH变化的内在敏感性，这种非经典DNA结构可作为传感器核心元件，除荧光标记或猝灭剂外无需其他模块。我们聚焦于几种表现出快速折叠/解折叠动力学的iM。使用停流技术和荧光共振能量转移（FRET）熔解/退火分析，我们证实了温度驱动的iM-单链DNA（ssDNA）转变速率与pH驱动的转变速率相关。因此，我们提出基于FRET的滞后分析作为选择具有所需动力学特性传感器的快速方法。对于领先的快速响应传感器，我们优化了标记方案并进行了细胞内校准。与常用的小分子pH指示剂不同，该传感器能有效转运至细胞核。考虑到其良好的动力学特性，该传感器可用于监测细胞核中的质子动力学。这些结果表明，“受基因组启发”的设计是开发生物相容性分子工具的有效方法。
