Journal article

Pyranosyl-RNA. Further observations on replication. Part 5

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 2 mols. of pr(GCCC)-2',3'-cp was investigated in detail. In aq. 1.5 M LiCl soln. of pH 8.5 at room temp. (0.45 mM ligand, 0.15 mM template), the reaction proceeds in up to 60% yield within a week. It is limited by concomitant hydrolysis of cyclophosphate groups of both reactant and ligation product as the only efficient side reaction, the latter occurring .apprx.3 times more slowly than ligation. No ligation at all is obsd. 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 contg. a single enantio-ribopyranosyl unit, no ligation is obsd., except to a minor extent in the case of the diastereoisomer that has that unit at the 4'-end. Observations made in expts. involving 6 different octamer templates contg. 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 qual. 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 det. most of the characteristic properties of p-RNA. As regards the etiol. context of this work on nucleic-acid alternatives, it is essential that the chem. properties found for p-RNA be compared with the corresponding properties of RNA. In the RNA series, the 2 ligations of the replicative cycle r(GGGCGGGC) .tautm. r(GCCCGCCC) using the corresponding ribofuranosyl-tetramer 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. [on SciFinder (R)]


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