Experimental and theoretical aspects of Golden Gate Assembly assays

Molecular cloning is a routine yet essential technique. Its efficiency relies on ligation and can be greatly improved when using a procedure known as Golden Gate Assembly (GGA). Essential to GGA are type IIS enzymes that have the unique property to cleave downstream their recognition sequence and thus generate any non-palindromic overhangs. Today, GGA benefits from new engineered enzymes with enhanced activity. Concomitantly, high throughput GGA assays (involving the simultaneous study of all overhangs at a time) have proposed optimal GGA substrates with high efficiencies and fidelities. Surprisingly, those assays show either no or unexpected correlation between ligation efficiencies and overhang stabilities. To explain those observations, we present here experiments involving one or two substrates (overhangs) only. When performing GGA at a stable temperature of 37°C, we found that GGA efficiency strongly correlates with overhang stability. Combining those experimental results with a kinetic model, we were able to determine how relevant parameters (time, temperature, molarity, stoichiometry, stacking energy) influence GGA. This work provides a comprehensive view of low-assembly GGA, defines the required optimal conditions and substrates (allowing the fabrication of constructs for single-molecule experiments with unprecedented yields) and gives new insights into DNA ligation that are crucial in biology. ### Competing Interest Statement The authors have declared no competing interest.