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 such as polymerases or ligases represents an important and poorly understood step in the origin of life. In fact, the self-replication of nucleic acids remains strongly hindered by product inhibition, even when enzymes are present. Studying one of the few successful examples of enzymatic DNA self-replication based on a simple ligation chain reaction can shed light on how this fundamental process may have originally evolved. Previously, our group reported lesion-induced DNA amplification (LIDA), which is an isothermal ligase chain reaction. It was proposed that the lesion, a destabilizing abasic group, played an important role in reducing product inhibition. However, using abasic groups in non-enzymatic ligation cycles did not yield appreciable amplification. In order to identify the unknown factors that overcome product inhibition in LIDA, we have performed a detailed kinetic and thermodynamic analysis of the process, employing isothermal titration calorimetry (ITC) and global fitting of PAGE fluorescence data to characterize the individual steps of the amplification process. We found that in the absence of an enzyme, the ligated product binds four to five orders of magnitude more tightly to the template than to the shorter unligated strands of the intermediate complex. In the presence of T4 DNA ligase, this stability gap was reduced by two orders of magnitude, such that the intermediate and product complexes were roughly equal in stability. Thus, the ligase helps overcome product inhibition by reducing the affinity difference between the DNA product duplex and the intermediate complex. Furthermore, kinetic simulations showed that the stability of the intermediate complex, as well as the rate constant of ligation, significantly impacts the rate of self-replication, suggesting that catalysts that stabilize the intermediate complex might be a route to efficient nonenzymatic replication.

Self-Replicating DNA-Based Nanoassemblies

“…Moreover, the self-replication of Triangle72 was faster than for the analogous 18 mer DNA duplex by LIDA (Figure S2), which could be attributed to a higher effective concentration of the ligating strands as the binding of one end of Linear72 to the Destabilizing Template likely facilitated the hybridization of the other terminus to form the Nicked Duplex (Scheme 1). Consistent with our work on DNA duplex self-replication, 18 [23][24] we found that the rate of self-replication of these nanoassemblies by LIDA was highly temperature dependent (Figure S3). At 22 °C, the kinetics of the reaction was very slow yielding ~40% conversion of Linear72 to Triangle72 after 240 minutes.…”

“…Our previous work on LIDA of DNA duplexes has consistently observed a background-triggered self-replication that initiates much slower than the templated reaction. 18,24 Here we observed the sigmoidal formation of Triangle72 when no initial Triangle72 was present albeit the onset was delayed significantly. Based on our previous work, 24 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 Linear72 (Figure 2A).…”

“…18,24 Here we observed the sigmoidal formation of Triangle72 when no initial Triangle72 was present albeit the onset was delayed significantly. Based on our previous work, 24 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 Linear72 (Figure 2A). This leads to the slow formation of the Destabilizing Template and Triangle72 in small quantities, which is enough to initiate the rapid cross-catalytic amplification process.…”

“…22 For a 66nt strand, a higher selectivity toward cyclization over polymerization was observed at ≤5 µM concentration, consistent with our results performed at 1.4 µM DNA. Significantly this concentration has been shown in LIDA to lead to rapid self-replication 18, [23][24] suggesting that this concentration should facilitate self-replication of nanoassemblies (Scheme 1) under these conditions at 26 °C with 1% of Triangle72 led to the sigmoidal growth of Triangle72 revealing that our strategy achieved rapid self-replication of triangle wireframe structures (Figure 2A, 14 nM Triangle72). Moreover, the self-replication of Triangle72 was faster than for the analogous 18 mer DNA duplex by LIDA (Figure S2), which could be attributed to a higher effective concentration of the ligating strands as the binding of one end of Linear72 to the Destabilizing Template likely facilitated the hybridization of the other terminus to form the Nicked Duplex (Scheme 1).…”