NASA Exobiology Grant Proposal 2014 Title: Roles for Peptides in the Origin of Life My laboratory is focused on understanding how prebiotically available chemicals self-assembled into simple protocells capable of growth, division and Darwinian evolution. Over the past 5 years we have published a series of papers describing robust processes leading to growth and division of model protocell membranes. Much of our current work is aimed at solving problems related to non-enzymatic RNA replication, and the integration of RNA replication with vesicle replication. We recently described the first system allowing for non-enzymatic RNA template copying inside fatty acid vesicles (Science, 2013). In the course of our recent work, it has become apparent that the development of a replicating protocell may require several previously unanticipated roles for simple peptides. Here we consider possible functions for three broad classes of short peptides with restricted amino acid compositions. The prebiotic synthesis of such compositionally restricted peptides seems plausible, based on recent geochemical scenarios proposed by Sutherland (Angew. Chemie, 2013). 1. Basic peptides. One of the fundamental problems with non-enzymatic RNA replication is that following template copying and thermal strand separation, the separated strands will rapidly re-anneal, preventing subsequent rounds of RNA replication. We have recently found that short basic peptides can dramatically slow down re-annealing kinetics, without significantly interfering with template copying chemistry or vesicle integrity. Further exploration of this phenomenon will be a major portion of the proposal, including mechanistic studies, studies of variant peptide sequences and lengths, and efforts to demonstrate multiple cycles of non-enzymatic RNA replication. 2. Acidic peptides. Non-enzymatic RNA replication requires high concentrations of divalent metal ions such as Mg2+, but these are incompatible with fatty acid based protocell membranes. We recently showed that citrate could chelate Mg2+, thereby protecting membranes while allowing RNA replication to proceed. We plan to search for more prebiotically plausible solutions to this problem, and suggest that short acidic peptides might play such a role. We will screen acidic peptides for metal ion binding, compatibility with or catalysis of RNA chemistry, and ability to protect membranes from disruption. 3. Hydrophobic peptides. We have recently shown that hydrophobic dipeptides can localize to the membrane and drive competitive growth by increasing local membrane order, which decreases the off rate of fatty acids from the membrane (Nat. Chem, 2013). We plan to define the peptide sequence and length requirements for this effect. We will also attempt to evolve ribozymes that can catalyze the synthesis of hydrophobic dipeptides from suitable substrates such as NCAs. We also plan to explore the potential of membrane localized peptides as transporters of ions and ionic nutrients such as nucleotides. The proposed studies will contribute to our understanding of how primitive cells emerged from the chemical environments of the early earth.