Z-DNA Formation in the Hybrid between Two Circular ssDNAs Involving Hairpin Structures

Z-DNA, a left-handed DNA conformation, plays critical roles in transcriptional regulation, genetic recombination, genomic instability, immunity, and human diseases. In 2019, a stable LR-chimera containing Z-DNA (Lk = 0) under physiological ionic conditions was prepared by hybridizing two complementary circular ssDNAs. However, the difficulty in preparing circular ssDNA precursors and the excessively long Z-DNA segment in the chimera limit its applications. In this study, using a splint-free circularization method, we prepared two circular ssDNAs (each with a hairpin structure). Hybridization of these two circles whose loops are complementary (but not the two hairpins) yielded a Stem-LR chimera containing short Z-DNA and B-DNA and two hairpins that could not hybridize with each other. Stability analysis revealed that the 18-34 bp Z-DNA segment with only unmodified nucleotides in the Stem-LR chimera remained stable under physiological conditions (10 mM Mg²⁺, 37 °C). When hairpins were far apart (180°), multiple Stem-LR chimera isomers (varying in B-Z junction numbers and Z-DNA lengths) formed. Intriguingly, higher hybridization temperatures (60 °C) favored continuous B-DNA and Z-DNA segments (minimal B-Z junctions). When hairpins were adjacent (0° orientation), exclusively continuous B-DNA/Z-DNA was obtained, even for hybridization at 10 °C. As expected, Stem-LR chimeras exhibited enhanced resistance to topoisomerase I compared to chimeras without hairpins. This approach holds promise for delivery into cells or organisms to investigate the impact of Z-DNA and its biological functions under physiological conditions.