Nonhomologous RNA Recombination in a Cell-Free System: Evidence for a Transesterification Mechanism Guided by Secondary Structure

Extensive nonhomologous recombinations occur between the 5′ and 3′ fragments of a replicable RNA in a cell-free system composed of pure Qβ phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3′ hydroxyl group of the 5′ fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3′ hydroxyl on phosphoester bonds within the 3′ fragment.