Kretzer Balázs, Kiss Bálint, Tordai Hedvig, Csík Gabriella, Herényi Levente, Kellermayer Miklós
Department of Biophysics and Radiation Biology, Semmelweis University, 1094 Budapest, Hungary.
Micromachines (Basel). 2020 Feb 19;11(2):212. doi: 10.3390/mi11020212.
Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.
单分子实验为生物分子现象的机制提供了独特的见解。然而，由于改变溶质浓度通常需要更换分子周围的整个溶液，因此对同一分子进行配体浓度依赖性测量具有挑战性。在本工作中，我们利用了层流微流控装置中会出现依赖扩散的浓度梯度这一事实，该浓度梯度可用于控制机械操纵的单个分子所接触的配体浓度。我们通过用双阱光镊仪器固定一个λ噬菌体双链DNA分子，并使其暴露于扩散控制浓度的SYTOX Orange（SxO）和四（4-N-甲基）吡啶基卟啉（TMPYP）中来测试这种实验方法。我们证明，该实验设计允许对同一单个分子进行瞬态动力学、平衡和配体浓度依赖性的力学实验。
