Enhanced Golden Gate Assembly: evaluating overhang strength for improved ligation efficiency

Molecular cloning, a routine yet essential technique, relies heavily on efficient ligation, which can be significantly improved using Golden Gate Assembly (GGA). A key component of GGA is the use of type IIS enzymes, which uniquely cleave downstream of their recognition sequences to generate various overhangs, including non-palindromic ones. Recent advancements in GGA include the development of newly engineered enzymes with enhanced activity. Additionally, high-throughput GGA assays, which allow for the simultaneous study of all possible overhangs, have identified optimal GGA substrates with high efficiencies and fidelities, greatly facilitating the design of complex assemblies. Interestingly, these assays reveal unexpected correlations between ligation efficiencies and overhang stabilities. One hypothesis for this observation is that newly hydrolyzed DNA fragments with strong overhangs can readily re-ligate, thereby slowing down the overall process. In this paper, we employ a combination of gel electrophoresis and numerical calculations to test this hypothesis, ultimately determining that it does not hold true under the conditions established by conventional GGA assays. Using an assembly of 10 fragments, we demonstrate that strong overhangs yield higher GGA efficiency, while weak overhangs result in lower efficiency. These findings enable us to propose optimal overhangs for efficient GGA assays, significantly increasing yield.