Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.

下一代测序技术已经在生物科学中带来了一个新的定量维度。特别是，将测序技术与生物物理工具相结合，可以进行序列相关的机制研究。利用测序过程中产生的数百万个 DNA 簇进行高通量结合亲和力和动力学测量，可以在序列空间中构建能量景观，揭示序列、结构和功能之间的关系。在这里，我们回顾了在下一代测序芯片上针对 DNA、RNA 和蛋白质序列的变异进行整体荧光实验的方法。作为下一步，我们预计这些荧光实验将被推向单分子水平，它可以以前所未有的高通量方式直接揭示动力学和分子异质性。序列空间中的分子生物物理学，无论是在整体还是单分子水平上，都带来了新的机制见解。生物学和医学中的广泛应用范围从对进化途径的基本理解到新疗法的开发。