RNA-catalysed RNA replication is widely considered a key step in the emergence of life’s first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5’ and 3’ ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

RNA from primer extension reactions catalysed by the triplet polymerase ribozyme were PAGE purified, adaptor ligated and prepared for illumina sequencing as described in the main text.

Illumina Sequencing data were acquired and processed as FASTQ files using Terminal (and available software packages such as FASTX-toolkit). Prior to analysis the whole output file form illumina sequencing runs (containing also unrelated sequences) was split based on barcodes identifying the individual samples and trimmed starting with the original (P9₁) primer sequence (GAAGAACTG). After the primer sequence, the triplets at positions 1, 2 ,3 etc. would be identifiable representing extension products made by the ribozyme. The presence for the 3’ adapter sequence (GTCGAATAT…) in the aligned sequences marked the end of the original RNA extension product.

File 1 include the split and trimmed sequence data for circular and linear one-pot analysis (Circular_onepot and Linear_onepot, respectively), File 2 include the split and trimmed sequence data for branched RCS analysis (B3)).

File_1_2_ReadMe.txt (uploaded to Zenodo) describes the simple code executed to analyze the deep-sequencing datasets by counting the number of times the sequences in the List-files was present.

File_1 includes deep-sequencing datasets from sequencing of the circular and linear onepot reactions (Circular_onepot.fastq and Linear_onepot.fastq), the list (List.txt) containing the sequences that was counted in the deep-sequencing datasets to allow analysis, and the results (results_circular and results_linear) containing the number of times (count) each sequence in the list appered in the deep-sequencing datasets (arranged in the same order as the corresponding sequence in List.)

File_2 includes deep-sequencing dataset from sequencing of the branched RCS reaction (B3.fastq), the list (List.txt) containing the sequences that was counted in the deep-sequencing datasets to allow analysis, and the results (result) containing the number of times (count) each sequence in the list appered in the deep-sequencing datasets (arranged in the same order as the corresponding sequence in List.)