Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology (KTH), Stockholm, Sweden.
Nucleic Acids Res. 2023 Mar 21;51(5):e27. doi: 10.1093/nar/gkac1255.
Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions. Evidence of how GGGGCC repeats form into G-quadruplexes in vivo is sparse. We devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. Thereby, folding states of cyanine-labeled RNA, and in particular G-quadruplexes, can be identified in a sensitive manner. The isomerization kinetics, monitored via fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA with GGGGCC repeats in aqueous solution using fluorescence correlation spectroscopy and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with laser excitation modulation characteristics, we could then detect folding states of fluorescently tagged RNA introduced into live cells.
鸟嘌呤（G）-rich 核酸容易组装成四链结构，即所谓的 G-四链体。异常的 GGGGCC 重复延伸，特别是它们的折叠状态，与肌萎缩侧索硬化症和额颞叶痴呆有关。然而，由于方法学的限制，大多数 G 四链体结构的研究仅限于体外条件。关于 GGGGCC 重复在体内如何形成 G-四链体的证据很少。我们设计了一种读出策略，利用花菁染料的反式-顺式异构化对局部粘度和空间位阻的敏感性。因此，可以以敏感的方式识别花菁标记 RNA 的折叠状态，特别是 G-四链体。通过荧光闪烁监测反式-顺式异构化的动力学，首次使用荧光相关光谱和瞬态（TRAST）监测在水溶液中对具有 GGGGCC 重复的 RNA 进行了表征。使用 TRAST，通过监测平均荧光强度随激光激发调制特性的变化来监测异构化动力学，我们可以检测引入活细胞的荧光标记 RNA 的折叠状态。
