Continuing his work studying the prebiotic synthesis of RNA, Orgel explored mechanisms by which inorganic phosphate and nucleotide phosphoryl groups could be chemically activated for condensation into nucleic acid polymers. Starting in the 1960s, Orgel explored a variety of cyanide-based activating agents which could have plausibly been present on a young earth. A carbodiimide reagent was found to be effective at activating nucleotide phosphoryl groups and promoting the formation of short Adenosine dimers and trimers. In 2018, John D. Sutherland and co-workers proposed that methyl isocyanide and acetaldehyde could combine to form a pre-biotic phosphate activating agent which could plausibly have formed under early-earth conditions.
Orgel also theorised that one single strand of RNA could have been the template for the first life on Earth and that these imidazole-activated nucleotides could have used this RNA template strand to polymerise and replicate. Lohrmann and Orgel reported that the phosphorimidazolide derivative of adenosine monophosphate (in which a phosphoryl group oxygen is substituted by an imidazole ring) forms short adenosine oligomers in the presence of poly-uridine templates. They further discovered that the divalent metal cation used to catalyze the reaction influenced the regiochemistry of the inter-nucleotide linkage. Pb gave primarily 5’-2’ linked nucleotides while Zn gave primarily 5’-3’ linked nucleotides from guanosine phosphorimidazolides in the presence of a poly-cytidine template. Montmorillonite clay was also shown to promote the polymerization of adenosine phosphorimidazolide into oligonucleotides tens of bases in length starting from a poly-adenosine 10-mer primer. In the absence of montmorillonite, the primer was capped through the formation of a 5’ adenosine pyrophosphate.
The oligonucleotide products in early studies were typically characterized through a combination of C radiolabeling, gel electrophoresis, and paper electrophoresis. Enzymatic digestion was used to differentiate regioisomers. The advent of HPLC allowed the characterization of long oligomers of guanosine.