Microsystem and Nanotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland.
Microsystem and Nanotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland.
Biophys J. 2022 Jun 7;121(11):1986-2001. doi: 10.1016/j.bpj.2022.05.008. Epub 2022 May 10.
Biomolecular thermodynamics, particularly for DNA, are frequently determined via van't Hoff analysis of optically measured melt curves. Accurate and precise values of thermodynamic parameters are essential for the modeling of complex systems involving cooperative effects, such as RNA tertiary structure and DNA origami, because the uncertainties associated with each motif in a folding energy landscape can compound, significantly reducing the power of predictive models. We follow the sources of uncertainty as they propagate through a typical van't Hoff analysis to derive best practices for melt experiments and subsequent data analysis, assuming perfect signal baseline correction. With appropriately designed experiments and analysis, a van't Hoff approach can provide surprisingly high precision, e.g., enthalpies may be determined with a precision as low as 10 kJ ⋅ mol for an 8-base DNA oligomer.
生物分子热力学，特别是 DNA 的热力学，通常通过对光测熔解曲线的范特霍夫分析来确定。对于涉及协同效应的复杂系统的建模，如 RNA 三级结构和 DNA 折纸，准确和精确的热力学参数是至关重要的，因为折叠能景观中每个基序的不确定性会累积，显著降低预测模型的能力。我们跟踪不确定性的来源，因为它们通过一个典型的范特霍夫分析传播，以得出熔解实验和随后的数据分析的最佳实践，假设信号基线的完美校正。通过适当设计的实验和分析，范特霍夫方法可以提供令人惊讶的高精度，例如，对于 8 个碱基的 DNA 寡聚物，焓可以以低至 10 kJ ⋅ mol 的精度确定。
