Figure 5: Biochemical assessment of the genomic RNA clones chosen from rounds 0, 32, 80 and 128.

(a) Time course data for TcRR of the plus-strand RNA clones. The TcRR reaction was performed with each plus-strand (0.1 nM) in the emulsion at 37 °C. At each time point, plus-strand concentrations in the droplets were measured. The error bars indicate the s.d. (n=2). (b) Competition experiment between genomic plus-strand RNA and a parasitic RNA. TcRR reactions were performed with each clone (50 nM) and a parasitic RNA (10 fM) without compartmentalization at 37 °C for 4 h. The replicated RNAs were detected by radioisotope labelling, followed by non-denatured polyacrylamide gel electrophoresis and autoradiography. The full image of the gel is shown in the Supplementary Fig. S9. (c) A kinetic model of TcRR. A Michaelis–Menten-type reaction was assumed for each RNA replication reaction (that is, the replication rate is represented as k_rep [RNA][Replicase]/(K_M+[RNA]), where k_rep and K_M are the maximum replication rate constant and the Michaelis constant, respectively). During replication, template RNA and newly synthesized RNA hybridize to form double-stranded RNA, a dead-end product of this system⁴¹. Here R_ss represents the fraction of synthesized RNA that is in the single-stranded form. For simplicity, the formation of double-stranded RNA of the parasitic RNA is neglected, and the same parameters are assumed for both complementary strands of the parasitic RNA. Double-stranded RNA formation by collision between the plus- and minus-strand RNA was omitted in this model because the rates are extremely small for all clones (Supplementary Fig. S8).