Experimental demonstration of kinetic proofreading inherited in ligation-based information replication

TL;DR

This work demonstrates that templated ligation can inherit kinetic proofreading to achieve high-fidelity information replication in a cascade. Using a simple two-step ligation system on a 74-nt template with 20-nt monomers, the authors observe nonmonotonic intermediate dynamics and a pronounced, length-dependent reduction in error that outperforms chain-growth polymerization under nonequilibrium conditions. A mass-action kinetic model, fitted to experimental data, reveals a discrimination factor $f = (k_L/k_L')( \tilde{k}'_{off} / \tilde{k}_{off} ) > 1$, with large values for the two ligation steps, supporting robust error suppression across cascade stages. The findings suggest a plausible route to high-fidelity replication in prebiotic chemistry and offer strategies for improving fidelity in DNA-based technologies such as storage and genotyping, leveraging nonequilibrium kinetics and hierarchical cascade architectures.

Abstract

We experimentally demonstrate that information replication by templated ligation of DNA strands inherits a kinetic proofreading mechanism and achieves significant error suppression through cascade replication. A simple simulation model derived from the experimental results shows that templated ligation has a significant advantage over replication by polymerization for error suppression of long strands. Specifically, longer chains show lower error rates, significantly distinct from the chain-growth polymerization where errors typically accumulate with chain length. This mechanism provides a plausible route for high-fidelity replication in prebiotic chemistry and illustrates how physical principles such as nonequilibrium kinetics and network architecture can drive reliable molecular information replication. The approach also offers new strategies for error suppression in biotechnology.

Figures (11)

• Figure 1: Cascade replication by templated ligation. $\mathrm{A}$, $\mathrm{B}$, $\mathrm{B'}$, and $\mathrm{C}$ are 20-nt DNA oligomers. Two strands adjacently hybridize (bind) to a template strand and are covalently connected by a ligase enzyme. Inset: Wrong substrate $\mathrm{B'}$ has two nucleotide mutations indicated by underlines, which reduce the hybridization stability and also the ligation rate.
• Figure 2: Replication dynamics at $T_\mathrm{anneal}=66.0\degreeCelsius$. (a) Dynamics of product concentrations: ABC+AB'C (red), AB+AB' (green), and BC+B'C (blue). Solid curves are simulations with six fitting parameters, the hybridization rate $k_\mathrm{on}$, the free energy change for the wrong monomer $\Delta G_{\mathrm{mono},\mathrm{W}}^\circ$, and four types of ligation rates $k_\mathrm{L}$ corresponding to the connections AB, BC, $\mathrm{AB'}$, and $\mathrm{B'C}$ (see Sec. \ref{['SSec:FittingProcedure']}). The inset is a magnified view of low concentrations. $N=3$ or 4 independent experiments are averaged. (b) Single-site error ratios after 150 cycles (symbols) and the simulation (solid curves). The error bars correspond to the standard errors.
• Figure 3: Single-site error ratios (left) and product concentrations (right) after 150cycles for the middle (a) and terminal (b) errors. $\langle \mathrm{B} \rangle$ and $\langle \mathrm{B'} \rangle$ denote the amount of the products containing B and $\mathrm{B'}$ units, respectively (e.g. $\langle\mathrm{B}\rangle=[\mathrm{AB}]$ for ${\cal M}_1$ and $\langle\mathrm{B}\rangle=[\mathrm{ABC}]$ for ${\cal M}_2$ and ${\cal M}_\mathrm{cas}$). Error bars indicate standard errors. Curves in (a) are simulation results. The parameters are obtained by fitting the dynamics (see Fig. \ref{['Fig:2']}a). For (b), $k_\mathrm{L}$ for the $\mathrm{A'B}$ connection was additionally estimated. We assumed that $k_\mathrm{L}$ and $k_\mathrm{on}$ do not depend on $T_\mathrm{anneal}$.
• Figure 4: Kinetic proofreading in ligation. The diagram is drawn with a focus on a specific template strand, and only the main reaction pathway involving a wrong substrate is shown. The loop structures that reset the state are characteristic of the kinetic proofreading murugan2012. The "Reuse" process indicates the hybridization of intermediate products.
• Figure 5: Simulations of long-template replications with single-site errors (a--c) and multi-site errors (d, e). Cascade replication with stable monomer hybridization (blue closed circle) and less stable one (red closed triangle) with $\Delta \Delta G = 1\, k_\mathrm{B} T$, and corresponding conditions of chain-growth polymerization (open circle and triangle) are compared in (b), (c), and (e) [Sec. \ref{['SSec:SingleSite']}]. (a) Schematic of single-site error replication, where an error may occur at the central position in a template of length $l$. The example of $l=5$ is illustrated. (b) Error ratio. The error ratio for chain-growth polymerization is given by the product of $\exp(-\Delta\Delta G / k_\mathrm{B} T)$ and $k_{\mathrm{L}}' / k_{\mathrm{L}}$ and is 0.37, where $\Delta\Delta G=1\,k_\mathrm{B} T$ and $k'_{\mathrm{L}} / k_{\mathrm{L}}=1$ are assumed. (c) Discrimination factor $f$ defined by Eq. \ref{['Eq.condition']} for each length of substrates. (d) Schematic of multi-site error replication. Errors can occur at any site. (e) Success probability of replication in the multi-site error case.