Orotidine-Containing RNA: Implications for the Hierarchical Selection (Systems Chemistry Emergence) of RNA

Abstract

The prebiotic synthesis of canonical nucleobases from HCN is a cornerstone for the RNA world hypothesis. However, their role in the primordial pathways to RNA is still debated. The very same process starting from HCN also gives rise to orotic acid, which (via orotidine) plays a crucial role in extant biology in the de novo synthesis of uridine and cytidine, the informational base-pairs in RNA. However, orotidine itself is absent in RNA. Given the prebiotic and biological relevance of orotic acid vis-à-vis uracil, we investigated orotidine-containing RNA oligonucleotides and show that they have severely compromised base-pairing properties. While not unexpected, these results suggest that the emergence of extant RNA cannot just be a consequence of the plausible prebiotic formation of its chemical constituents/building blocks. In combination with other investigations on alternative prebiotic nucleobases, sugars, and linkers, these findings imply that the selection of the components of extant RNA occurred at a higher hierarchical level of an oligomer/polymer based on its functional properties-pointing to a systems chemistry emergence of RNA from a library of precursors.

Keywords: RNA world; base pairing; orotic acid; orotidine; prebiotic systems chemistry.

Figure 1.

De novo biosynthetic pathway for the synthesis of pyrimidine nucleotides through the direct nucleosidation of orotic acid with 5-phosphoribosyl-diphosphate.

Figure 2.

Previous studies dealing with base-pairing properties of orotic acid derivatives attached to oligoamide and oligopeptide backbones.^[20,21]

Figure 3.

Thermal melts documenting the destabilizing effect of single orotidine (O) insert into a self-complementary-RNA 5′-AUAUAUAUAUAUAUAU-3′ duplex, equaling that of the purine–purine 5′-AUAUAUAAAUAUAUAU-3′ mismatch (top) and destabilizing effect of single orotidine (O) insert into a non-self-complementary-RNA 5′-AAAAUUUAUAUUAUUA-3′ + 3′-UUUUAAAUAUAAUAAU-5′ duplex, equaling that of the purine–purine 5′-AAAAAUUAUAUUAUUA-3′ + 3′-UUUUAAAUAUAAUAAU-5′ mismatch (bottom). For conditions see footnote of Table 1.

Figure 4.

Thermal melts documenting the destabilizing effect of single orotidine (O) insert into a self-complementary-RNA 5′-CGAAUUCG-3′ duplex, equaling that of the purine–purine 5′-CGAAAUCG-3′ mismatch. For conditions see footnote of Table 1.

Figure 5.

The syn-conformation of orotic acid (over the anti-conformation) within a duplex would destabilize not only by absence of base-pairing, but also by disturbing the base-stacks above and below. Circles represent 3′,5′-phosphodiester links.

Figure 6.

Extant nature uses a small subset of canonical/biological nucleo-bases (within the circle) out of a library of prebiotically plausible nucleobases.

Scheme 1.

Synthesis of orotidine methyl ester phosphoramidite 3; a) di-tert-butylsilyl ditriflate, DMF, 0°C, 1 h; b) tert-butyldimethylsilyl chloride, imidazole, 60°C, overnight; c) HF-pyridine, DCM, 0°C, 3 h; d) DMTrCl, pyridine, 4°C, overnight; e) 2-cyanoethyldiisopropylchlorophosphoramidite, DIPEA, DCM, 0°C to RT, overnight.