Ribosome-Free Translation up to Pentapeptides via Template Walk on RNA Sequences

The origin of translation is one of the most difficult problems of molecular evolution. Identifying molecular systems that translate an RNA sequence into a peptide sequence in the absence of ribosomes and enzymes is a challenge. Recently, single-nucleotide translation via coupling of 5' phosphoramidate-linked amino acids to 2'/3'-aminoacyl transfer-NMPs, as directed by the sequence of an RNA template, was demonstrated for three of the four canonical nucleotides. How single-nucleotide translation could be expanded to include all four bases and to produce longer peptides without translocation along the template strand remained unclear. Using transfer strands of increasing length containing any of the four bases that interrogate adjacent positions along the template, we now show that pentapeptides can be produced in coupling reactions and subsequent hydrolytic release in situ. With 2'/3'-aminoacylated mono-, di-, tri- and tetranucleotides we thus show how efficient translation can be without biomacromolecules.