Cryo-EM structure of a 95-base-pair double-stranded DNA minicircle at 5.3 Å resolution

Double-stranded DNA minicircles have been observed in a variety of biological settings and are also widely employed in biotechnology, therapeutic applications, and basic research. Here, we report a cryo-EM structure of a 95-base-pair minicircle (dsMC95) at a 5.3 Å resolution. dsMC95 forms a closed ring as designed and no severe local duplex disruption is observed. The two DNA strands are fully resolved, with the major and minor grooves clearly distinguishable. Analysis reveals a nine-fold periodicity in the helical twist, which corresponds to approximately 10.56 base pairs per turn. Together with groove width analysis, the data indicate that dsMC95 maintains a B-DNA configuration. The dsMC95 ring exhibits modest in-plane ellipticity and small out-of-plane displacement, with outward-facing grooves widened and inward-facing ones compressed. The dsMC95 structure, which is the only free DNA cryo-EM structure with a resolution better than 6 Å to date, allows comparison to other structures to better understand DNA physical features such as bending. The findings advance our understanding of DNA structure under topological constraints and will inform studies of naturally occurring small circular DNA as well as the manipulation of DNA in nanotechnology applications.