Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
Nucleic Acids Res. 2023 Nov 10;51(20):11298-11317. doi: 10.1093/nar/gkad807.
We conducted a thermodynamic analysis of RNA stability in Eco80 artificial cytoplasm, which mimics in vivo conditions, and compared it to transcriptome-wide probing of mRNA. Eco80 contains 80% of Escherichia coli metabolites, with biological concentrations of metal ions, including 2 mM free Mg2+ and 29 mM metabolite-chelated Mg2+. Fluorescence-detected binding isotherms (FDBI) were used to conduct a thermodynamic analysis of 24 RNA helices and found that these helices, which have an average stability of -12.3 kcal/mol, are less stable by ΔΔGo37 ∼1 kcal/mol. The FDBI data was used to determine a set of Watson-Crick free energy nearest neighbor parameters (NNPs), which revealed that Eco80 reduces the stability of three NNPs. This information was used to adjust the NN model using the RNAstructure package. The in vivo-like adjustments have minimal effects on the prediction of RNA secondary structures determined in vitro and in silico, but markedly improve prediction of fractional RNA base pairing in E. coli, as benchmarked with our in vivo DMS and EDC RNA chemical probing data. In summary, our thermodynamic and chemical probing analyses of RNA helices indicate that RNA secondary structures are less stable in cells than in artificially stable in vitro buffer conditions.
我们对 Eco80 人工细胞质中的 RNA 稳定性进行了热力学分析，该细胞质模拟了体内条件，并将其与 mRNA 的全转录组探测进行了比较。Eco80 含有 80%的大肠杆菌代谢物，具有生物浓度的金属离子，包括 2 mM 游离 Mg2+和 29 mM 代谢物螯合的 Mg2+。荧光检测结合等温线（FDBI）用于对 24 个 RNA 螺旋进行热力学分析，发现这些螺旋的平均稳定性为-12.3 kcal/mol，比 ΔΔGo37 低约 1 kcal/mol。FDBI 数据用于确定一组 Watson-Crick 自由能最近邻参数（NNP），结果表明 Eco80 降低了三个 NNP 的稳定性。利用 RNAstructure 包对 NN 模型进行了调整。体内样调整对体外和计算机预测的 RNA 二级结构影响最小，但显著提高了大肠杆菌中 RNA 碱基配对分数的预测，与我们的体内 DMS 和 EDC RNA 化学探测数据进行了基准测试。总之，我们对 RNA 螺旋的热力学和化学探测分析表明，与人工稳定的体外缓冲条件相比，细胞内的 RNA 二级结构不太稳定。
