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The template-directed formation of phosphodiester bonds between two nucleic acid components is a pivotal process in biology. To induce such a reaction in the absence of enzymes is a challenge. This challenge has been met for the extension of a primer with mononucleotides, but the ligation of short oligonucleotides (dimers or trimers) has proven difficult. Here we report a method for ligating dimers and trimers of ribonucleotides using in situ activation in aqueous buffer. All 16 different dimers and two trimers were tested. Binding studies by NMR showed low millimolar dissociation constants for complexes between representative dimers and hairpins mimicking primer-template duplexes, confirming that a weak template effect is not the cause of the poor ligating properties of these short oligomers. Rather, cyclization was found to compete with ligation, with up to 90% of dimer being converted to the cyclic form during the course of an assay. This side reaction is strongly sequence dependent and more pronounced for dimers than for trimers. Under optimized reaction conditions, high yields were observed with strongly pairing purines at the 3'-terminus. These results show that short oligomers of ribonucleotides are competent reactants in enzyme-free copying.

Enzyme-free ligation of dimers and trimers to RNA primers