Preparation of topoisomers of short circular dsDNA with defined linking number by accurate topological control

Short DNA catenanes [circular double-stranded DNA (dsDNA)] have attracted considerable interest for constructing nanostructures and nanomachines, as well as understanding DNA topology. The study of topoisomers of a circular dsDNA with a definite linking number (Lk) is essential but very difficult for simplifying the complex problems about DNA topology. The topoisomers are difficult to prepare, especially in the case that two strands are completely complementary. In this study, using a model system, we prepared all eight topoisomers (Lk0-Lk7) of a 79-bp-long circular dsDNA (8-14 nm in size) by utilizing aid-DNA to prevent undesired hybridization. By rapid ligation before strand displacement, high selectivity (>75%) for most topoisomers (31% for Lk1) was achieved under the strict topological control. All eight topoisomers with high purity were obtained after purification. Using a gel shift assay with Z-DNA-specific binding proteins, as well as by circular dichroism chromatography and enzymatic digestion, it was found that Z-DNA forms for topoisomers Lk0-Lk6, and Lk0-Lk5 can be converted to Lk6 by topoisomerase I. The approach developed in this study can significantly contribute to DNA or RNA topology, particularly the effect of topological constraints on DNA structures and functions.