Fig. 1.

Nucleopeptide replication networks. (A and B) NCL reactions forming the R^AA and R^TT conjugates from their respective electrophile and nucleophile precursors and time-dependent formation of these conjugates in template-free (bg), autocatalytic (ac), and cross-catalytic (cc) reactions. Note that at pH 7.4, the Glu side chain carboxylic acids would be in their deprotonated anionic form. Reactions were carried out with 100 µM E^AA or E^TT and 100 µM N, either in the absence of a template (bg) or when seeded with the designated amount of template at initiation (ac/cc). Insets show the early stages of the background reactions, highlighting the lag phase typical of product formation through autocatalysis (Top insets) and the rate enhancement (percent) in cross-catalytic reactions seeded with 60 or 30 µM template or in autocatalytic reactions seeded with 60 µM template (Bottom insets). (C and D) A time-dependent analysis of the replicator-assisted product formation of the conjugates R^AA (green) and R^TT (red) in network reactions initiated with E^AA (50 µM), E^TT (50 µM), and N (100 µM) and seeded with 20 (dashed lines) or 60 μM (solid lines) R^TT (C) or R^AA (D). HPLC chromatograms (Top) indicate the increase in R^AA and R^TT product over time in representative reactions seeded with 60 μM R^TT (C) or 60 μM R^AA (D); note that R^TT and R^AA peaks have initial intensity due to seeding (in C and D, respectively), and the * symbols denote minor (≥15%) branched product peaks, removed for clarity (see also SI Appendix, Fig. S20). All reactions were carried out in duplicate, in Hepes buffer (pH 7.4), in the presence of TCEP as a reducing agent (5 mM) and with ABA (30 μM) as the internal standard. Data were acquired by HPLC analysis of aliquots collected at the designated times (SI Appendix, Figs. S17–S20).