Thermal, Autonomous Replicator Made from Transfer RNA

Evolving systems rely on the storage and replication of genetic information. Here we present an autonomous, purely thermally driven replication mechanism. A pool of hairpin molecules, derived from transfer RNA replicates the succession of a two-letter code. Energy is first stored thermally in metastable hairpins. Thereafter, energy is released by a highly specific and exponential replication with a duplication time of 30 s, which is much faster than the tendency to produce false positives in the absence of template. Our experiments propose a physical rather than a chemical scenario for the autonomous replication of protein encoding information in a disequilibrium setting.

Figure 1

Thermal replication using a cross-catalytic replication reaction. A purely thermally driven exponential replication of sequence succession $ab$ is expected from metastable hairpins and microscale convection.

Figure 2

Replication results. (a) As fuel, a typical tRNA is cut next to the anticodon, forming a hairpin with a toehold sequence $A$. (b) Quenching of 2-aminopurine rises as duplexes are formed in the full reaction with 500 nM of four hairpins $a,b,A,B$, and 35 nM template duplex $ab$ (red). The non-cross-catalytic mixture of two hairpins $A,B$ with template $ab$ shows no signal (gray). An explicit numerical model fits the fluorescence quenching accurately in both cases (black). (c) Gel electrophoresis of the resulting products. Slow annealing of single, two, or four hairpins leads to equilibrium expectation of hairpins, duplexes, and quadruples. Under thermal oscillation, only the full reaction transforms four hairpins into duplexes and quadruples. (d) Melting curves of quadruple ($37\pm 3°\mathrm{C}$, black), hairpin ($62\pm 2°\mathrm{C}$, blue), and duplex ($>75°\mathrm{C}$, purple).

Figure 3

Specificity. (a) No replication was found for a mutated toehold sequence (brown). (b) With isothermal driving, the complete reaction replicated slowly and showed no dependence on the template concentration ($\mathrm{\text{template duplex}}=\{5,25,\mathrm{\text{and}}45\mathrm{n}M\}$, $\mathrm{\text{hairpin}}=500\mathrm{nM}$). Isothermal reactions using a nonmatching toehold sequence (yellow) or without hairpins (blue) showed no signature of replication.

Figure 4

Exponential replication. (a) Measured (color) and simulated (dashed lines) quenching of complete reactions with different initial template concentration are in agreement. (b) The increase of the replicated product raised with increasing template concentration. (c) Time derivative of the modeled template concentration $dC/dt$ within the first three temperature cycles plotted versus the initial template concentration. A fit by Eq. (1) revealed a doubling time $\tau =28\pm 5\mathrm{s}$, matching the thermal cycling time of 30 s.