RNA/DNA 杂交双链体的细胞内稳定性预测，用于设计治疗寡核苷酸,Journal of the American Chemical Society

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RNA/DNA 杂交双链体的细胞内稳定性预测，用于设计治疗寡核苷酸
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2023-10-24 , DOI: 10.1021/jacs.3c06706
Dipanwita Banerjee ₁ , Hisae Tateishi-Karimata ₁ , Maria Toplishek ₂ , Tatsuya Ohyama ₁ , Saptarshi Ghosh ₁ , Shuntaro Takahashi ₁ , Marko Trajkovski ₂ , Janez Plavec _{2,

3,

4} , Naoki Sugimoto _{1,

5}

Affiliation

在细胞中，RNA/DNA 杂合双链体的形成调节基因表达和修饰。细胞器内的环境异质地挤满了高浓度的生物分子，这些生物分子影响 RNA/DNA 杂合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，通过使用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机制细节，揭示了拥挤条件下双链体的结构依赖性不稳定。由于嘌呤-嘧啶不对称性引导的 DNA 链构象变化，从 B 型杂合双链体转变为 A 型杂合双链体，显着增加了水合数，从而导致添加共溶质导致更大的不稳定。通过量化环境因素和双链体整体结构的个体贡献，我们开发了一组参数来预测在不同拥挤条件下具有构象差异的杂合双链体的稳定性。使用福斯特共振能量转移技术比较活细胞和体外拥挤溶液中的环境条件对混合双链体形成的影响，确定了我们的参数对活细胞的适用性。此外，我们得出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基因表达的细胞机制的可视化和针对不同细胞的基于核酸的疗法的开发的有用性。

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In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics

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 in vitro 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.

更新日期：2023-10-24

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