Oliynyk, R. T.; Church, G. M.

We demonstrate DNA circularization efficiencies nearly an order of magnitude greater than those forecasted by classical Jacobson-Stockmayer theory - a cornerstone physical model unchallenged for over seven decades - even at high DNA concentrations suitable for routine practical use. This breakthrough performance is enabled by simultaneous restriction cutting and ligation of double-stranded DNA fragments using the Type IIS enzyme BsaI-HFv2 in combination with T4 DNA ligase under finely optimized buffer conditions. These results unveil a biologically derived exception that decisively overcomes the longstanding physical barriers to DNA cyclization imposed by Jacobson-Stockmayer theory, while highlighting the potential for systematic exploration of enzyme combinations to identify additional systems achieving comparable or superior intramolecular ligation efficiency.