A Highly Reactive Imidazolium-Bridged Dinucleotide Intermediate in Nonenzymatic RNA Primer Extension

摘要

Because of its importance for the origin of life, the nonenzymatic copying of RNA templates has been the subject of intense study for several-decades. Previous characterizations of template-directed primer extension using 5'-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3'-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concentration at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3'-hydroxyI of the primer displaces an activated nudeotide as the leaving group and results in extension of the primer by one nudeotide. Analysis of monomer solutions by NMR indicates formation of the proposed imidazolium-bridged dinudeotide in the expected pH-dependent manner. We have used synthetic methods to prepare material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinudeotide intermediate provides a mechanistic interpretation of previously observed catalysis by an activated nudeotide located downstream from the site of primer extension.