Abiotic synthesis of RNase-resistant phosphodiester and pyrophosphate-linked polymers via thermodynamically controlled wet-dry cycling

We report the abiotic synthesis of structurally modified, RNase-resistant oligonucleotides with phosphodiester backbones from non-activated mononucleotides of adenosine monophosphate (AMP) and uridine monophosphate (UMP), and pyrophosphate linked D-ribose-5’-phosphate polymers under simulated wet-dry cycling conditions. These polymers were generated in a dedicated simulation chamber, under well controlled and reproducible thermodynamic conditions. The polymers lead to diffuse bands in electrophoresis gels using fluorescence detection with SYBR Gold at an excitation wavelength of 365 nm, a key spectral signature that allows us to distinguish them from standard nucleic acids. We show that these polymers are stable against enzymatic degradation by RNase 1, DNase 1, and DBR1, however partially degrade in alkaline hydrolysis. Evidence for polymerization was obtained in UV-vis and IR spectra. SAXS suggests aggregates in solution of approximately 120 nucleotide and ribose phosphate units. ³¹P NMR provided evidence for the formation of a phosphodiester backbone in cycled AMP/UMP mixtures, and the formation of a pyrophosphate backbone when cycling ribose phosphate. This suggests that these abiotically formed polymers are not perfect copies of canonical RNA; they were likely structurally modified (e.g., with missing bases), yet they are stable enough to persist and form distinct bands on a gel. These findings demonstrate that thermodynamic control within an environmental simulation chamber can drive the formation of complex, enzyme-resistant biopolymers without the use of activating agents, offering a robust model for exploring localized prebiotic synthesis pathways.