FIBER (Frontier Institute for Biomolecular Engineering Research), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
FIRST (Graduate School of Frontiers of Innovative Research in Science and Technology), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Nucleic Acids Res. 2020 Dec 2;48(21):12042-12054. doi: 10.1093/nar/gkaa572.
The stability of Watson-Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na+ or K+ in the presence or absence of Mg2+). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°37 and Tm values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg2+, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR-Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.
Watson-Crick 配对的 RNA/DNA 杂交体的稳定性对于设计用于 ASO（反义寡核苷酸）和 CRISPR（成簇规律间隔短回文重复）-Cas9 技术的最佳寡核苷酸至关重要。然而，以前用于预测 1 M NaCl 溶液中杂交体稳定性的最近邻（NN）参数可能不适用于预测更接近生理条件的盐浓度下的稳定性（例如，存在或不存在 Mg2+时，约 100 mM 的 Na+或 K+）。在此，我们报告了在 100 mM NaCl 下 38 个 RNA/DNA 杂交体的测量热力学参数，并得出了新的 NN 参数来预测双链体稳定性。基于建立的 NN 参数预测的 ΔG°37 和 Tm 值与测量值吻合良好，偏差分别为 2.9%和 1.1°C。新结果还可用于对分别模拟细胞内和细胞外盐条件的 100 mM KCl 或 100 mM NaCl 中形成的双链体进行精确预测。使用 ASO 和 CRISPR-Cas9 比较预测的热力学参数与已发表的数据，可以设计出用于这些技术的更短的寡核苷酸，从而降低非特异性结合的可能性，并提高靶基因调控的效率。
