Replication (single-turnover) of pyranosyl-RNA (= p-RNA) sequences can be accomplished reliably by template-directed ligation of 2′, 3′-cyclophosphates of short oligomers. The ligation process was studied using (mostly) octamers as templates and tetramers as ligands. The transcription of the sequence pr(GGGCGGGC) into the (antiparallel) complementary sequence pr(GCCCGCCC) by ligation of two molecules of pr(GCCC)-2′, 3′-cp was investigated in detail; In aqueous 1.5M LiCI solution of pH 8.5 at room temperature (0.45 mM ligand, 0.15 m,M template), the reaction proceeds in up to 60% yield within a week. It is limited by concomitant hydrolysis of cyclophosphate groups of both reactand and ligation product as the only efficient side reaction, the latter occurring ca. three times more slowly than ligation. No ligation at all is observed in the absence of template. The reaction is highly regioselective: the (4′ → 2′) phosphodiester junction is formed exclusively; no isomeric (4′ → 3′) junctions are found. For ligation to occur, template and ligand must be homochiral and must have the same sense of chirality; with chiro-diastereoisomeric tetramer-2′, 3′-cyclophosphates containing a single enantio-ribopyra-nosyl unit, no ligation is observed, except to a minor extent in the case of the diastereoisomer that has that unit at the 4′-end. Observations made in experiments involving six different octamer templates containing isomeric base sequences indicate that the ligation process does not tolerate a mismatch at ligation sites. However, ligation still takes place when a mismatch occurs at either end of the (octamer) template. Ligation efficiencies differ widely, depending on the nature, as well as the sequence, of participating bases. These differences can be understood qualitatively by considering the relative stability of ternary pre-ligation complexes, together with the differences in interstrand base stacking at ligation sites, Dominance of the latter over intrastrand base stacking is the feature of the p-RNA structure that appears to determine most of the characteristic properties of p-RNA.
As regards the etiological context of our work on nucleic-acid alternatives, it is essential that the chemical properties found for p-RNA be compared with the corresponding properties of RNA. In the RNA series, the two ligations of the replicative cycle r(GGGCGGGC) ⟺ r(GCCCGCCC) using the corresponding ribofuranosyl-te-tramer 2′, 3′-cyclophosphates as ligands are found to proceed also, though somewhat less efficiently than in the p-RNA series; however, the ligation step produces exclusively the unnatural (5′ → 2′) phosphodiester junctions instead of the natural (5′→ 3′) junctions. This is in sharp contrast to p-RNA, where template-controlled 2′, 3′-cyclophosphate ligations produce the ‘correct’ phosphodiester junctions.