Koh Kyung Duk, Chiu Hsiang-Chih, Riedo Elisa, Storici Francesca
School of Biology, Georgia Institute of Technology, Atlanta, GA, 30332, USA.
Methods Mol Biol. 2015;1297:43-57. doi: 10.1007/978-1-4939-2562-9_3.
Ribonucleotides, ribonucleoside monophosphates (rNMPs), have been revealed as possibly the most noncanonical nucleotides in genomic DNA. rNMPs, either not removed from Okazaki fragments during DNA replication or incorporated and scattered throughout the genome, pose a perturbation to the structure and a threat to the stability of DNA. The instability of DNA is mainly due to the extra 2'-hydroxyl (OH) group of rNMPs which give rise to local structural effects, which may disturb various molecular interactions in cells. As a result of these structural perturbations by rNMPs, the elastic properties of DNA are also affected. Here, we show the approach to test whether the presence of rNMPs in DNA duplexes could alter the elasticity of DNA by implementing atomic force microscopy (AFM)-based single molecule force-measurements of short rNMP(s)-containing oligonucleotides (oligos).
核糖核苷酸，即核糖核苷单磷酸（rNMPs），已被揭示可能是基因组DNA中最不典型的核苷酸。rNMPs要么在DNA复制过程中未从冈崎片段中去除，要么被掺入并散布于整个基因组中，这对DNA的结构造成干扰，并对其稳定性构成威胁。DNA的不稳定性主要归因于rNMPs额外的2'-羟基（OH）基团，该基团会产生局部结构效应，可能会扰乱细胞内的各种分子相互作用。由于rNMPs造成的这些结构扰动，DNA的弹性特性也会受到影响。在此，我们展示了一种方法，即通过对含短rNMP的寡核苷酸（oligos）进行基于原子力显微镜（AFM）的单分子力测量，来测试DNA双链体中rNMPs的存在是否会改变DNA的弹性。
