Fig. 4

Potentiating DNA self-replication with 5′-end pre-bound TP. a IVTTR reaction scheme using the TP-oriLR-p2-p3 DNA template. Short amplification products are not represented. The detailed experimental workflow, including preparation of the TP-oriLR-p2-p3 DNA, is shown in Supplementary Fig. 12a. b The replication products of the TP-oriLR-p2-p3 DNA template (∼75 ng input, equiv. ∼1.9 nM) expressed in PUREfrex were visualized on agarose gel after RNase and Proteinase K treatments, followed by RNeasy clean-up column purification. When indicated the p6-p5 DNA (70 ng input, equiv. ∼5.7 nM) was co-expressed. The results from two independent IVTTR experiments are shown in Supplementary Fig. 12c, f. For direct comparison of the amplification yield with and without parental TP, similar amounts of input DNA were used, the end-point reaction solutions were loaded on the same gel and the band intensities were analysed (Supplementary Fig.13). Clearly, replication of the TP-oriLR-p2-p3 DNA template is more efficient. c Samples were further incubated with λ-exonuclease to remove TP-uncapped DNA. Note that the overall amount of DNA on the gel is reduced (to the extent that the band corresponding to the input TP-oriLR-p2-p3 DNA in the –dNTPs control sample is no longer visible) after nuclease treatment due to dilution during the cleaning/purification steps. d De novo synthesized DNA was subsequently used as a template for a third IVTT reaction. The translation products were visualized by PAGE with GreenLys labeling. The protein gel analysis from an independent IVTTR experiment is shown in Supplementary Fig. 12d. e Autocatalytic IVTTR cycles realized in this study. A first IVTTR reaction was performed using oriLR-p2-p3 as input DNA and producing larger amount of TP-oriLR-p2-p3 (Supplementary Fig. 12b, e). The purified TP-oriLR-p2-p3 DNA was subsequently used as template for a second IVTTR (b). Finally, the purified DNA products from IVTTR 2 was used for a third IVTT (d)