Thermodynamics and kinetics of DNA and RNA dinucleotide hybridization to gaps and overhangs
Thermodynamics and kinetics of DNA and RNA dinucleotide hybridization to gaps and overhangs
复制标题
DOI：
10.1016/j.bpj.2023.07.009
复制
发表时间：
2023-08-22
影响因子：
3.4
通讯作者：
Tokmakoff，Andrei
中科院分区：
文献类型：
--
作者：
Ashwood，Brennan;Jones，Michael S.;Tokmakoff，Andrei
短链核酸片段(~lt；4nt)与单链模板的杂交是非酶核酸复制和脚趾介导链置换等过程中的关键中间产物。这些模板通常包含相邻的双链片段，这些片段通过单链间隙或悬垂稳定碱基配对，但由于探测弱结合和快速结构动力学的实验挑战，在这种情况下杂交的热力学和动力学尚不清楚。在这里，我们开发了一种使用稳态和温度跳跃红外光谱直接测量DNA和RNA二核苷酸去杂交的热力学和动力学的方法。我们的结果表明，二核苷酸结合是通过与相邻双链片段的同轴堆积作用以及分子动力学模拟揭示的潜在的非正则碱基配对构型和间隙模板和悬垂模板的结构动力学来稳定的。根据模板和温度的不同，我们测量的解离时间从0.2-40μS不等。二核苷酸杂交和去杂交涉及一个显著的自由能屏障，其特征类似于规范的寡核苷酸。总之，我们的工作为预测短核酸片段与各种模板之间杂交的稳定性和动力学提供了初步的步骤。
Hybridization of short nucleic acid segments (<4 nt) to single-strand templates occurs as a critical intermediate in processes such as nonenzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood because of the experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential noncanonical base-pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2–40 μs depending on the template and temperature. Dinucleotide hybridization and dehybridization involve a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates.