FIBER (Frontier Institute for Biomolecular Engineering Research) , Konan University , 7-1-20 Minatojima-Minamimachi , Chuo-ku, Kobe 650-0047 , Japan.
Biomedical Research Institute , National Institute of Advanced Industrial Science and Technology (AIST) , 1-1-1 Higashi , Tsukuba , Ibaraki , 305-8566 , Japan.
Anal Chem. 2019 Feb 19;91(4):2586-2590. doi: 10.1021/acs.analchem.8b04177. Epub 2019 Jan 10.
Molecular crowding creates a unique environment in cells and imposes physical constraints such as the excluded volume effect, water activity, and dielectric constant that can affect the structure and function of biomolecules. It is therefore important to develop a method for quantifying the effects of molecular crowding in cells. In this study, we developed a Förster resonance energy transfer (FRET) probe based on a guanine-quadruplex (G4) DNA motif that shows distinct FRET signals in response to crowding conditions in the presence of salt and poly(ethylene glycol). FRET efficiencies varied in different solutions, reflecting the dependence of G4 stability and topology on salt concentration and water activity. In living cells, FRET signals in the nucleus were higher than those in the cytosol; the signals in membraneless nuclear compartments (i.e., nucleolus) were especially high, suggesting that a decrease in water activity is important for the crowding effect in the nucleus. Thus, the use of DNA sensors with variable structures can elucidate the local effects of molecular crowding in cells.
分子拥挤在细胞中创造了独特的环境，并施加了物理约束，如排除体积效应、水活度和介电常数，这些都会影响生物分子的结构和功能。因此，开发一种量化细胞中分子拥挤效应的方法非常重要。在这项研究中，我们开发了一种基于鸟嘌呤四链体（G4）DNA 基序的荧光共振能量转移（FRET）探针，该探针在存在盐和聚乙二醇的情况下，对拥挤条件表现出明显的 FRET 信号。FRET 效率在不同溶液中有所变化，反映了 G4 稳定性和拓扑结构对盐浓度和水活度的依赖性。在活细胞中，核内的 FRET 信号高于细胞质内的信号；无膜核区（即核仁）内的信号特别高，这表明水活度的降低对核内拥挤效应很重要。因此，使用具有可变结构的 DNA 传感器可以阐明细胞中分子拥挤的局部效应。
