Nonenzymatic, Template-Directed Ligation of Oligoribonucleotides Is Highly Regioselective for the Formation of 3‘−5‘ Phosphodiester Bonds

We have found that nonenzymatic, template-directed ligation reactions of oligoribonucleotides display high selectivity for the formation of 3'−5' rather than 2'−5' phosphodiester bonds. Formation of the 3'−5'-linked product is favored regardless of the metal ion catalyst or the leaving group, and for several different ligation junction sequences. The degree of selectivity depends on the leaving group: the ratio of 3'−5'- to 2'−5'-linked products was 10−15:1 when the 5'-phosphate was activated as the imidazolide, and 60−80:1 when the 5'-phosphate was activated by formation of a 5'-triphosphate. Comparison of oligonucleotide ligation reactions with previously characterized single nucleotide primer extension reactions suggests that the strong preference for 3'−5'-linkages in oligonucleotide ligation is primarily due to the occurrence of ligation within the context of an extended Watson−Crick duplex. The ability of RNA to correctly self-assemble by template-directed ligation is an intrinsic consequence of its chemical structure and need not be imposed by an external catalyst (i.e., an enzyme polymerase); RNA therefore provides a reasonable structural basis for a self-replicating system in a prebiological world.