Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80309, USA.
Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80309, USA.
J Colloid Interface Sci. 2020 Apr 15;566:369-374. doi: 10.1016/j.jcis.2020.01.070. Epub 2020 Jan 20.
Single-molecule Förster Resonance Energy Transfer was used to observe the adsorption of fluorescently-labeled "target" DNA oligonucleotides and their association and hybridization with complementary DNA "probes" tethered to the surface as a function of surface grafting density. Ionic strength was varied systematically to disentangle the potentially competing effects of probe accessibility and electrostatic repulsion. At high ionic strength, when the Debye length was ~1 nm, the adsorption of target DNA was not significantly inhibited by the presence of tethered probe DNA, even at high grafting density, and the fraction of adsorbed target strands undergoing hybridization increased systematically with grafting density, leading to a dramatic increase in the net hybridization rate at high grafting density. However, at lower ionic strength, when the Debye length was ≥3 nm, the adsorption rate of target DNA decreased and the fraction of adsorbed target strands undergoing hybridization saturated at high probe grafting density (≥7,000 strands/µm), presumably due to electrostatic repulsion. As a result, the net rate of hybridization exhibited a maximum as a function of grafting density. This has important consequences for the design of systems that optimize surface-mediated DNA hybridization under low-salt high-stringency conditions.
利用单分子Förster 共振能量转移技术，观察荧光标记的“靶”DNA 寡核苷酸的吸附及其与表面固定的互补 DNA“探针”的缔合和杂交，作为表面接枝密度的函数。系统地改变离子强度以解耦探针可及性和静电排斥的潜在竞争效应。在高离子强度下，当德拜长度约为 1nm 时，即使在高接枝密度下，固定的探针 DNA 的存在也不会显著抑制靶 DNA 的吸附，并且经历杂交的吸附靶链的分数系统地随接枝密度增加，导致在高接枝密度下净杂交速率急剧增加。然而，在较低的离子强度下，当德拜长度≥3nm 时，靶 DNA 的吸附速率降低，并且在高探针接枝密度（≥7,000 链/µm）下，吸附的靶链经历杂交的分数饱和，可能是由于静电排斥。因此，净杂交速率作为接枝密度的函数表现出最大值。这对于在低盐高严格条件下优化表面介导的 DNA 杂交的系统设计具有重要意义。
