Figure 7. Triplet-initiated template sequence copying.

(A) Extension by t5⁺¹ of fluorescein-labelled primers bound to either the 3’ (A10) or 5’ (^pppba) ends of a template (T8GAA, 10 μM ^pppGAA, 0.5 μM/RNA, −7˚C ice 69 hr), demonstrating extension in either 5’−3’ (A10) or 3’−5’ (^pppba) directions. (B) Synthesis of β⁺ on Tβ template via t5⁺¹-catalysed polymerisation of the substrates indicated on the right (2 μM t5⁺¹, 5 μM each triplet, 0.5 μM template (lane 2: 0.5 μM hexanucleotide), −7˚C ice 9 days). Extension products in lanes 2–5 were eluted from template, PAGE-separated and SYBR-Gold stained alongside in vitro transcribed full-length segment control (lane 1). Lane 3 shows full-length synthesis of β⁺ segment from triplets alone. (C) Triplet-based replication of the γ segment. Top left, synthesis of γ⁺ from the indicated substrates (1 μM t5⁺¹, 5 μM each triplet, 2 μM TγHP template (lane 2: 2 μM Bioγ7 primer), −7˚C ice 7 days). Biotinylated extension products in lanes 2–4 were isolated from template, PAGE-separated and SYBR-Gold stained alongside in vitro transcribed staining marker (Mγ⁺m1, lane 1). This indicated 10% yield (per template) of full-length synthesis of γ⁺ segment from triplets alone (the final band in lane 4), which was purified for use as a template in γ ^- segment synthesis (bottom right, 1 μM t5⁺¹, 5 μM each triplet, 0.05 μM template, with 0.05% Tween-20, –7˚C ice 27 days). Extension products in lanes 5 and 6 were eluted from template, PAGE-separated and SYBR-Gold stained alongside in vitro transcribed full-length segment control (Mγ ^-m1, lane 7). This indicated 6% yield (per template) of full-length synthesis of γ ^- segment from triplets alone (the final band in lane 6).