Minimizing Product Inhibition in DNA Self‐Replication: Insights for Prebiotic Replication from the Role of the Enzyme in Lesion‐Induced DNA Amplification**

Abstract

Self‐replication of nucleic acids in the absence of enzymes represents an important and poorly understood step in the origin of life as such reported systems are strongly hindered by product inhibition. Studying one of the few successful examples of enzymatic DNA self‐replication based on a simple ligation chain reaction, lesion‐induced DNA amplification (LIDA), can shed light on how this fundamental process may have originally evolved. To identify the unknown factors that lead LIDA to overcome product inhibition we have employed isothermal titration calorimetry and global fitting of time‐dependent ligation data to characterize the individual steps of the amplification process. We find that incorporating the abasic lesion into one of the four primers substantially decreases the stability difference between the product and intermediate complexes compared with complexes without the abasic group. In the presence of T4 DNA ligase this stability gap is further reduced by two orders of magnitude revealing that the ligase also helps overcome product inhibition. Kinetic simulations reveal that the intermediate complex stability and the magnitude of the ligation rate constant significantly impact the rate of self‐replication, suggesting that catalysts that both facilitate ligation and stabilize the intermediate complex might be a route to efficient nonenzymatic replication.

Self-Replicating DNA-Based Nanoassemblies

“…Moreover, the self-replication of Triangle 72 was faster than for the analogous 18mer DNA duplex by LIDA (Figure S3), which could be attributed to a higher effective concentration from intramolecular cyclization. Consistent with our work on DNA duplex self-replication, ,, we found that the rate of self-replication of these nanoassemblies by LIDA was highly temperature dependent (Figure S4). At 22 °C, the kinetics of the reaction was very slow yielding ∼40% conversion of Linear 72 to Triangle 72 after 240 min.…”

“…Previous work indicated cyclization over polymerization was observed at ≤5 μM concentration, consistent with our results performed at 1.4 μM DNA. 24 Significantly this concentration has been shown in LIDA to lead to rapid self-replication 20,25,26 suggesting that this concentration should facilitate the selfreplication of nanoassemblies (Scheme 1).…”

“…Here we observed the sigmoidal formation of Triangle 72 when no initial Triangle 72 was present with a delay in the onset. Based on our previous work, we infer that the Fragments that constitute the Destabilizing Template can ligate via a pseudo blunt end ligation based on the single-base overhang between the abasic and adenosine phosphate upon hybridizing with Linear 72 (Figure A). This leads to the slow formation of the Destabilizing Template and Triangle 72 in small quantities, which is enough to initiate the rapid cross-catalytic amplification process.…”