DNA Cut-Ligation Cyclization Surpasses Jacobson-Stockmayer J-Factor Expectations by Over Threefold

We demonstrate DNA circularization efficiencies more than threefold greater than those predicted by classical Jacobson-Stockmayer theory for the ligation of linear double-stranded DNA. To quantify deviations from classical expectations, we experimentally calibrated the J-factor by ligating 452 bp DNA fragments bearing pre-cut, purified 4-nt overhangs at the optimal shortest minicircle length and at the highest DNA concentration; this reference value then enabled accurate calculation of expected cyclization efficiencies across the full range of DNA lengths (452-952 bp) and the three concentrations examined. These elevated cyclization efficiencies are enabled by simultaneous restriction cutting and ligation using the Type IIS enzyme BsaI-HFv2 in combination with T4 DNA ligase under finely optimized buffer conditions, yielding a 3.4-fold improvement over the classical estimate and achieving 75% cyclization at a high, practically relevant DNA concentration of 120 ng/{micro}l. We also identified a second enzyme, Esp3I, that exceeds the classical expectations by 2.3-fold (though less efficiently than BsaI-HFv2), while BbsI cut-ligation systematically underperformed expectations, providing insight into possible mechanisms underlying the outstanding performance of the first two enzymes. These results reveal a biologically mediated exception that overcomes the long-standing mechanistic expectations of Jacobson- Stockmayer theory and highlight the value of systematically screening enzyme combinations to discover additional systems capable of highly efficient intramolecular ligation.