A strategy for the construction of double-stranded DNA microcircles with circumferences less than 50 bp.

A procedure for the construction of double-stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. The assembly strategy needs only three components, i.e. two synthetic oligonucleotides containing any sequence of interest and T4 DNA ligase. All components can be provided in large quantities and only two enzymatic reaction steps are required. Such microcircles can serve as a model for topologically fixed and highly bent DNA molecules and the amounts needed for X-ray crystallography, n.m.r. spectroscopy or drug binding studies can readily be produced. The construction of a 42 base pair double-stranded DNA microcircle is presented, by far the smallest double-stranded DNA circle yet described either in vivo or in vitro.