Fig. 3: Open-ended exponential amplification of RNA.
a, Design of replication substrates and scheme for iterative replication of an N17 RNA random-sequence library. To start, library template LibN17 was mixed at 8 nM in replication buffer (including 0.9 mM KCl and 20 nM TPR) together with 20 nM each of the indicated primers (FITCrep, Cy5rep, pppGUAGC, pppGGACC) and 12 nM each of all 64 triplets (pppNNN). b, FITCrep extension products from this reaction analysed at each five-cycle interval before threefold serial dilution. c, Quantification of overall amplification of ‘(3)n + 5’-register products in b, calculated as the fold increase in band intensity versus five cycles, multiplied by reaction dilution versus five cycles. Exponential fits yielded the per-cycle amplification efficiencies described in the text. d, Reactions set-up as in a, but seeded with 0.8 nM of both strands of one emergent RNA duplex sequence from a–c (Rep(1–4)+ with Rep(1–4)−, detected in the sequencing of each product population of the final reactions in b) as a template, using their constituent triplets as substrates (middle lane: triplets from all four sequences but no template). e, Cycling as in a of 0.8 nM of both strands of one clone (shown here beneath its substrates) without dilution. Average strand copy numbers (filled orange circles, FITC strand; open blue circles, Cy5 strand; comprising full-length and starting template) are shown for three independent reactions per cycle (transparent circles). Dotted lines are exponential curves fitted up to four cycles (x): FITC strand = e0.38x, R2 = 0.997; Cy5 strand = e0.20x, R2 = 0.984.
