A Small Polymerase Ribozyme That Can Synthesize Itself And Its Complementary Strand

The emergence of a chemical system capable of self-replication and evolution is a critical event in the origin of life.

RNA polymerase ribozymes can replicate RNA, but their large size and structural complexity impede self-replication and preclude their spontaneous emergence. Here we describe QT45: a 45-nucleotide polymerase ribozyme, discovered from random sequence pools, that catalyzes general RNA-templated RNA synthesis using trinucleotide triphosphate (triplet) substrates in mildly alkaline eutectic ice.

QT45 can synthesize both its complementary strand using a random triplet pool at 94.1% per-nucleotide fidelity, and a copy of itself using defined substrates, both with yields of ~0.2% in 72 days. The discovery of polymerase activity in a small RNA motif suggests that polymerase ribozymes are more abundant in RNA sequence space than previously thought.

A small RNA motif with a functionally dense core encodes the triplet polymerase activity of the QT ribozyme. (A) Synthesis of 42 nt CGU repeat sequence by QT51 and its truncation variants. Reaction conditions: 0.25 μM primer F10, 0.25 μM template tP10CGU14, 0.25 μM ribozyme, 5 μM pppCGU triplet, 0.05% Tween 20, 50 mM MgCl2, 50 mM CHES-KOH, pH 9, 5 days at -7 °C frozen. (B) Synthesis of a mixed sequence template by the same ribozymes as in (A). Reaction conditions: 0.25 μM primer F10, 0.25 μM template t6FP10mix, 1.25 μM ribozyme, 5 μM each triplet, 0.05% Tween 20, 50 mM MgCl2, 50 mM CHES-KOH, pH 9, 5 days at -7 °C frozen. In both (A) and (B), ribozymes are not hybridized to template. (C) Predicted secondary structure diagrams of the ribozymes used in (A) and (B) (D) Heatmap of the primer extension activity for all measured single and double mutants of the QT45 ribozyme, with the first constituent point mutation indicated on the x-axis and the second one on the y-axis. Missing datapoints are shown in gray. (E) Predicted secondary structure of QT45 ribozyme with nucleotide color corresponding to its measured average activity on three different templates for each of the three possible mutations, in the same order as the 3 representative mutations provided, indicated in the same color scheme as (D). — biorxiv.org

Astrobiology, Genomics, evolution,