Efficient Assembly of Functional RNA by <i>in Situ</i> Phosphate Activation and Loop-Closing Ligation

How ribozymes might have emerged prior to the evolution of an efficient RNA replicase remains an open question. A potential solution involves the nonenzymatic ligation of RNA oligomers that are short enough to have been replicated by nonenzymatic chemistry. However, the high concentrations of metal ions that facilitate ligation are incompatible with primitive protocells. In addition, the mode of oligoribonucleotide 5'-phosphate activation is unclear, and in an aqueous environment the competing hydrolysis of such activated intermediates limits ligation efficiency. In this study, we have investigated the template-independent assembly of structured RNAs via loop-closing ligation using a potentially prebiotic <i>in situ</i> phosphate activation approach. We demonstrate that the 5'-phosphate of oligonucleotides can be activated through an <i>N</i>-methylimidazole-intercepted phospho-Passerini reaction, enabling subsequent loop-closing ligation to efficiently form hairpin structures. Remarkably, this reaction proceeds in the absence of divalent metal ions and remains effective even at low millimolar concentrations of the activating reagent. We identify a novel pathway leading to a phosphorimidazolium species, mediated by an <i>N</i>-imidoyl-<i>N</i>'-methylimidazolium intermediate. Furthermore, we show that two successive <i>in situ</i> activation/loop-closing ligations enable the assembly of a functional RNA Flexizyme with a near-quantitative yield. Our findings provide insight into potential mechanisms for the prebiotic origin of functional RNAs and offer a viable strategy for the chemical synthesis of long RNA molecules.