A Universal Assay for Making DNA, RNA, and RNA–DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation

作者：Yajun Yang, Lun Song, Xiaocong Zhao, Chen Zhang, Wenqiang Wu, Huijuan You, Hang Fu, Er-Chi Zhou, Xinghua Zhang · 发表于：ACS Synthetic Biology · 年份：2019 · DOI：10.1021/acssynbio.9b00241 · 被引用次数：17 · 研究领域：Nanofabrication and Lithography Techniques、Force Microscopy Techniques and Applications、Molecular Junctions and Nanostructures

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.