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The origin of molecular evolution required the replication of short oligonucleotides to form longer polymers.

Prebiotically plausible oligonucleotide pools tend to contain more of some nucleobases than others. It has been

unclear whether this initial bias persists and how it affects replication. To investigate this, we examined the

evolution of 12 mer biased short DNA pools using an enzymatic model system. This allowed us to study the

long timescales involved in evolution, since it is not yet possible with currently investigated prebiotic replication

chemistries. Our analysis using next-generation sequencing from different time points revealed that the initial

nucleotide bias of the pool disappeared in the elongated pool after isothermal replication. In contrast, the

nucleotide composition at each position in the elongated sequences remained biased and varied with both

position and initial bias. Furthermore, we observed the emergence of highly periodic dimer and trimer motifs

in the rapidly elongated sequences. This shift in nucleotide composition and the emergence of structure through

templated replication could help explain how biased prebiotic pools could undergo molecular evolution and lead

to complex functional nucleic acids.