Extended Data Fig. 3: Influence of substrate GC-content upon inhibition of reannealing.
One cycle of replication in replication buffer, carried out as in Fig. 2c, but on RNA single strands or duplexes of different sequence compositions, supplying their constitutive triplet substrates and primers from the start (Sequence A = A+/AD template (>50% GC), FITCA & Cy5A primers, constitutive triplets as in Fig. 1b; Sequence B = B+/BD template (50% GC), FITCB & Cy5B primers, constitutive triplets; Sequence C = C+/CD template (40% GC), FITCC & Cy5C primers, constitutive triplets). Although the TPR can synthesise full-length extension products of FITC-labelled primers on all three single-stranded templates, it could only do so on double-stranded duplexes of 50% GC composition or higher. The capacity of low concentrations of GC-rich triplets – but not high concentrations of AU-rich triplets – to support duplex copying likely reflects the extreme cooperativity of strand coating (via Watson-Crick pairing and base stacking) needed to maintain a kinetic barrier to reannealing.
