Banerjee Dipanwita, Tateishi-Karimata Hisae, Toplishek Maria, Ohyama Tatsuya, Ghosh Saptarshi, Takahashi Shuntaro, Trajkovski Marko, Plavec Janez, Sugimoto Naoki
Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.
J Am Chem Soc. 2023 Nov 1;145(43):23503-23518. doi: 10.1021/jacs.3c06706. Epub 2023 Oct 24.
In cells, the formation of RNA/DNA hybrid duplexes regulates gene expression and modification. The environment inside cellular organelles is heterogeneously crowded with high concentrations of biomolecules that affect the structure and stability of RNA/DNA hybrid duplexes. However, the detailed environmental effects remain unclear. Therefore, the mechanistic details of the effect of such molecular crowding were investigated at the molecular level by using thermodynamic and nuclear magnetic resonance analyses, revealing structure-dependent destabilization of the duplexes under crowded conditions. The transition from B- to A-like hybrid duplexes due to a change in conformation of the DNA strand guided by purine-pyrimidine asymmetry significantly increased the hydration number, which resulted in greater destabilization by the addition of cosolutes. By quantifying the individual contributions of environmental factors and the bulk structure of the duplex, we developed a set of parameters that predict the stability of hybrid duplexes with conformational dissimilarities under diverse crowding conditions. A comparison of the effects of environmental conditions in living cells and crowded solutions on hybrid duplex formation using the Förster resonance energy transfer technique established the applicability of our parameters to living cells. Moreover, our derived parameters can be used to estimate the efficiency of transcriptional inhibition, genome editing, and silencing techniques in cells. This supports the usefulness of our parameters for the visualization of cellular mechanisms of gene expression and the development of nucleic acid-based therapeutics targeting different cells.
在细胞中，RNA/DNA杂交双链体的形成调节基因表达和修饰。细胞器内部的环境中生物分子浓度很高，呈异质性拥挤状态，这会影响RNA/DNA杂交双链体的结构和稳定性。然而，具体的环境影响仍不清楚。因此，通过热力学和核磁共振分析在分子水平上研究了这种分子拥挤效应的作用机制细节，揭示了在拥挤条件下双链体结构依赖性的去稳定化。由于嘌呤-嘧啶不对称性引导的DNA链构象变化，从B型到A型杂交双链体的转变显著增加了水合数，这导致添加共溶质时去稳定化程度更高。通过量化环境因素和双链体整体结构的各自贡献，我们开发了一组参数，用于预测在不同拥挤条件下具有构象差异的杂交双链体的稳定性。使用Förster共振能量转移技术比较活细胞和拥挤溶液中环境条件对杂交双链体形成的影响，证实了我们的参数对活细胞的适用性。此外，我们推导的参数可用于估计细胞中转录抑制、基因组编辑和沉默技术的效率。这支持了我们的参数对于可视化基因表达的细胞机制以及开发针对不同细胞的基于核酸的疗法的有用性。
