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

For more than 75 years, the Jacobson-Stockmayer J-factor has been regarded as the fundamental physical limit on DNA cyclization efficiency. Here, we show that this limit can be substantially exceeded by simultaneous restriction cutting and ligation with the Type IIS enzyme BsaI-HFv2 and T4 DNA ligase. We achieved 75% circularization efficiency at a practically relevant concentration of 120 ng/μL for 452 bp minicircles. Using DNA with pre-cut and purified overhangs, we calibrated the J-factor to establish the theoretical values expected under classical theory, and showed that cut-ligation with BsaI-HFv2 exceeds these expectations by 3.4-fold. Experimental results with additional enzymes (Esp3I and BbsI) provided insight into possible mechanisms underlying this outstanding performance. These findings demonstrate the existence of biological mechanisms that can dramatically increase effective local concentration beyond free-diffusion expectations, thereby exceeding long-standing theoretical limitations by over threefold and opening the way for systematic discovery of additional high-efficiency enzyme systems for DNA circularization.