Post-synthetically Ligated Ribozymes:  An Alternative Approach to Iterative Solid-Phase Synthesis (original) (raw)

To improve the overall yield of ribozyme synthesis, a convergent approach, based on the post-synthetic formation of an amino linker between two half-ribozymes was investigated. Borane‚pyridine-mediated reductive amination of 3′-phosphoglycaldehyde-5′-half-ribozymes with 5′-aminohexyl-3′-half-ribozymes generated the corresponding amino-linked ribozymes in yields >77% on different scales. The investigation of a variety of reducing agents is discussed together with a kinetic analysis of the selected coupling reaction. These post-synthetically ligated ribozymes exhibited slightly reduced in vitro catalytic activity and cell efficacy. tance and enhanced catalytic activity . Because of the iterative nature of solid-phase oligoribonucleotide synthesis, only modest chemical yields of a 37-mer ribozyme can be achieved. Moreover, the necessary reversed-phase and anionexchange purification steps further diminish this yield due to the inherent difficulty in separating the full-length ribozyme from the failures. As part of an ongoing effort to overcome these limitations, our group has designed an alternative approach in which two half-ribozymes are synthesized using known solid-phase methodologies. These halves contain complementary chemical functionalities that allow post-synthetic chemical ligation through a covalent linkage. Due to their lengths and the iterative synthesis process, the halfribozymes are obtained in greater yield as compared to a full-length ribozyme. Therefore, this half-ribozyme approach has a theoretical advantage over the purely solid-phase procedure, with respect to yields, provided that the post-synthetic chemical ligation proceeds efficiently.