Enzyme‐Free Replication with Two or Four Bases

All known forms of life encode their genetic information in a sequence of bases of a genetic polymer and produce copies through replication. How this process started before polymerase enzymes had evolved is unclear. Enzyme‐free copying of short stretches of DNA or RNA has been demonstrated using activated nucleotides, but not replication. We have developed a method for enzyme‐free replication. It involves extension with reversible termination, enzyme‐free ligation, and strand capture. We monitored nucleotide incorporation for a full helical turn of DNA, during both a first and a second round of copying, by using mass spectrometry. With all four bases (A/C/G/T), an “error catastrophe” occurred, with the correct sequence being “overwhelmed” by incorrect ones. When only C and G were used, approximately half of the daughter strands had the mass of the correct sequence after 20 copying steps. We conclude that enzyme‐free replication is more likely to be successful with just the two strongly pairing bases than with all four bases of the genetic alphabet. Enzyme free replication with nucleotides was achieved by using chemically activated aminonucleotides and reversible termination. Mass spectrometric monitoring showed the mutation levels for each step. With all four bases (A/C/G/T), an “error catastrophe” occurred, whereas when only C and G were used, approximately half of the daughter strands had the correct mass after 20 copying steps.