Department of Physics, PhD School in Nanotechnology, University of Trieste, 34127 Trieste, Italy.
Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy.
Molecules. 2022 Aug 17;27(16):5262. doi: 10.3390/molecules27165262.
Cleavage of DNA at noncanonical recognition sequences by restriction endonucleases (star activity) in bulk solution can be promoted by global experimental parameters, including enzyme or substrate concentration, temperature, pH, or buffer composition. To study the effect of nanoscale confinement on the noncanonical behaviour of BamHI, which cleaves a single unique sequence of 6 bp, we used AFM nanografting to generate laterally confined DNA monolayers (LCDM) at different densities, either in the form of small patches, several microns in width, or complete monolayers of thiol-modified DNA on a gold surface. We focused on two 44-bp DNAs, each containing a noncanonical BamHI site differing by 2 bp from the cognate recognition sequence. Topographic AFM imaging was used to monitor end-point reactions by measuring the decrease in the LCDM height with respect to the surrounding reference surface. At low DNA densities, BamHI efficiently cleaves only its cognate sequence while at intermediate DNA densities, noncanonical sequence cleavage occurs, and can be controlled in a stepwise (on/off) fashion by varying the DNA density and restriction site sequence. This study shows that endonuclease action on noncanonical sites in confined nanoarchitectures can be modulated by varying local physical parameters, independent of global chemical parameters.
在溶液中，限制内切酶（星活性）在非规范识别序列处切割 DNA，可以通过全局实验参数来促进，包括酶或底物浓度、温度、pH 值或缓冲液组成。为了研究纳米尺度限制对 BamHI 的非规范行为的影响，BamHI 可以切割 6 个碱基的单一独特序列，我们使用 AFM 纳米嫁接技术在不同密度下生成横向限制的 DNA 单层（LCDM），形式可以是几微米宽的小斑块，也可以是金表面上硫醇修饰 DNA 的完整单层。我们专注于两个 44 碱基对的 DNA，每个都包含一个非规范的 BamHI 位点，与同源识别序列相差 2 个碱基。形貌 AFM 成像用于通过测量 LCDM 相对于周围参考表面的高度降低来监测末端反应。在低 DNA 密度下，BamHI 有效地仅切割其同源序列，而在中等 DNA 密度下，非规范序列切割发生，并且可以通过改变 DNA 密度和限制位点序列以逐步（开/关）方式进行控制。这项研究表明，在纳米结构中，内切酶在非规范位点的作用可以通过改变局部物理参数来调节，而与全局化学参数无关。
