School of Physics & Astronomy, University of Leeds, Woodhouse Lane, Leeds, West Yorkshire LS2 9JT, UK.
Methods. 2013 Apr 1;60(2):122-30. doi: 10.1016/j.ymeth.2013.03.002. Epub 2013 Mar 14.
A polymerase chain reaction (PCR) based method of adding a single-stranded DNA (ssDNA) hairpin loop to one end of linear double-stranded (ds) DNA templates was developed. The loop structure serves as a fiducial marker in single molecule imaging by atomic force microscopy (AFM) and can be applied to study DNA-protein interactions. The nucleic acid end-labels allow discrimination of the polarity of the DNA template in the AFM while limiting non-specific interactions which might occur from non-nucleic acid labels. Homo-polynucleotide ssDNA loops made up of 20 base-pairs (bp) for each of the four bases (A, T, G, C) were investigated to determine the effects of sequence on template labelling. The products were produced with high efficiency and high yield with the loop readily distinguished from the dsDNA template by height and diameter in the AFM. The application of the method to study DNA transcription was investigated by firing Escherichia Coli RNA polymerase (RNAP) from a λPR promoter in the direction of the loop-labelled end. The ssDNA loops captured elongating complexes of RNAP, arresting transcription and preventing dissociation. The dual role of the loop as a polarity marker and retainer of previously active RNAP will allow mechanisms of gene expression to be studied with single molecule sensitivity by AFM. This will enable insight into molecular interactions of RNAP on single DNA templates in convergent or tandem transcription configurations.
开发了一种聚合酶链反应（PCR）方法，可将单链 DNA（ssDNA）发夹环添加到线性双链（ds）DNA 模板的一端。该环结构可作为原子力显微镜（AFM）中单分子成像的基准标记，可用于研究 DNA-蛋白质相互作用。核酸末端标记允许在 AFM 中区分 DNA 模板的极性，同时限制可能由非核酸标记引起的非特异性相互作用。研究了由四个碱基（A、T、G、C）中的每一个组成的 20 个碱基对（bp）的同聚核苷酸 ssDNA 环，以确定序列对模板标记的影响。该方法可高效高产地产生产物，并且通过 AFM 中的高度和直径很容易将环与 dsDNA 模板区分开来。该方法在研究 DNA 转录中的应用是通过从 λPR 启动子沿环标记端的方向引发大肠杆菌 RNA 聚合酶（RNAP）来研究的。ssDNA 环捕获了正在延伸的 RNAP 复合物，阻止了转录并阻止了复合物的解离。环作为极性标记和先前活跃的 RNAP 的保持器的双重作用将允许通过 AFM 以单分子灵敏度研究基因表达的机制。这将使我们能够深入了解在收敛或串联转录配置中 RNAP 在单个 DNA 模板上的分子相互作用。
