Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

The Salk Institute

Informational macromolecules in biology are composed of subunits of a single handedness, d-nucleotides in nucleic acids and l-amino acids in proteins. Although this chiral uniformity may be expedient, it is not a chemical necessity, as demonstrated by the recent example of an RNA enzyme that catalyzes the RNA-templated polymerization of RNA molecules of the opposite handedness. This reaction, when carried out iteratively, can provide the basis for exponential amplification of RNA molecules and the information they contain. By carrying out thermal cycling, analogous to the polymerase chain reaction, and supplying oligonucleotide building blocks that comprise both the functional strand of RNA and its complement, cross-chiral exponential amplification was achieved. This process was used to amplify the l-RNA form of the hammerhead ribozyme, catalyzed by the d-RNA form of the polymerase. The resulting l-hammerhead exhibits the expected activity in cleaving a corresponding l-RNA substrate. Exponential amplification was also carried out within individual droplets of a water-in-oil emulsion. The ability to amplify enantio-RNAs, both in bulk solution and within compartments, provides a means to evolve cross-chiral RNA polymerases based on the function of the RNAs they produce.

Grant A. L. Bare

The Salk Institute

Chemical Triphosphorylation of Oligonucleotides