Thermodynamics and kinetics of Lesion Induced DNA Amplification (LIDA)

“…These simulations are consistent with previous work that showed a strong dependence of replication on the magnitude of k cat of ligation. [54] Taken together this work reveals two important take-home messages for developers of nonenzymatic self-replicating systems: they should select rapid ligation strategies and identify ways to stabilize the intermediate complex (as well as destabilize the product duplex). Surfaces [16,55] or viscous solvents [28,29] in possible prebiotic replication scenarios might facilitate the ligation reaction as well as interact more strongly with the intermediate complexes enabling turnover.…”

Section: Chemistry-a European Journalmentioning

confidence: 94%

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

Park

Parshotam

Hales

et al. 2023

Chemistry A European J

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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.

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“…These simulations are consistent with previous work that showed a strong dependence of replication on the magnitude of kcat of ligation. 51 These simulations reveal two important take-home messages for developers of nonenzymatic self-replicating systems: they should focus on ways to stabilize the intermediate complex as well as ways to destabilize the product duplex and select rapid ligation strategies. Regarding the latter, very little product was observed within the first 7200 seconds (2 hours) of reaction when kcat was reduced to 0.1kcat (simulation not shown).…”

Section: Table 2 Kinetic Parameters Of Dna Complexesmentioning

confidence: 99%

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

Park

Parshotam

Hales

et al. 2022

Preprint

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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.

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Thermodynamics and kinetics of Lesion Induced DNA Amplification (LIDA)

Cited by 4 publications

References 4 publications

Reaction kinetics exploration of a protocellular metabolism

Reaction kinetics exploration of a protocellular metabolism

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

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

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