Novel synthetic pathways to cyclic oligoribonucleotides

In the frame of this project, novel sequence-unrestricted methods for the chemical syntheses of small- to medium-sized cyclic oligoribonucleotides (cRNA) will be developed. Their efficient synthesis has remained a major challenge and the current limited access to these compounds severely impedes progress in modern research areas of RNA biology. The envisaged synthetic pathways also aim at increasing the structural diversity of cRNAs; this will be achieved by modifications concerning the phosphate backbone through formation of alternative internucleotide linkages (e.g. 2`-5`-, 5`-5`(3`-3`)-phosphodiester, phosphorothioate, triazole, and amide linkages) and by insertion of distinct ribose modifications (e.g. 2`-OCH3, - F, -H, 2`-O4`-C methylene (LNA monomers), 2`-tethers bearing reactive groups such as alkyne, vinyl, or amino groups). Importantly, in collaborative efforts with the team of Virginijus Siksnys, these novel cRNA derivatives will be tested for their potential as activators/inhibitors of CRISPR-associated (Cas) Csm6 ribonucleases, and additionally, in crystallographic studies aiming at a high resolution structure of the corresponding protein complexes. This cRNAbased signaling pathway has been discovered very recently and coordinates components of CRISPR-Cas to prevent phage infection and propagation. The research proposed here therefore aims at solving a fundamental scientific problem that links advanced synthetic bioorganic chemistry to highly topical research on CRISPR-Cas systems. The expected results have potential to significantly impact on exploring new avenues to chemically programmed RNA-targeting interference technologies for applications in bioscience, biotechnology, and applied medicine.

The goal of the present project was to develop novel sequence-unrestricted methods for the chemical syntheses of small- to medium-sized cyclic oligoribonucleotides comprising natural phosphodiester linkages, and some unnatural analogs in the junction site. Their efficient synthesis has remained a major challenge and the currently limited access to these compounds severely impedes progress in modern research areas of RNA biology. The project was based on the original idea to combine standard solid-phase synthesis of a linear protected oligoribonucleotide and a few high-yielding liquid phase cyclization approaches. We developed novel approaches to the cyclization in solution by the reactions that 1) give high yields, 2) proceed under simple reaction conditions, which are compatible with oligoribonucleotide chemistry, and 3) generate insignificant byproducts. We believe that the strategies presented here, summarizing all the advantages of the solid phase and the liquid phase methods, significantly simplify the synthesis and result in increased yields of the desired cRNAs. The suggested here synthetic pathways also aimed at increasing the structural diversity of cRNAs through the formation of alternative internucleotide linkages and by insertion of distinct ribose modifications. Importantly, in collaborative efforts with the team of Virginijus Siksnys, these novel cRNA derivatives will be tested for their potential as activators/inhibitors of CRISPR-associated (Cas) Csm6 ribonucleases, and additionally, in crystallographic studies aiming at a high resolution structure of the corresponding protein complexes. This cRNA-based signaling pathway has been discovered recently and coordinates components of CRISPR-Cas to prevent phage infection and propagation. The research proposed here therefore aimed at solving a fundamental scientific problem that links advanced synthetic bioorganic chemistry to highly topical research on CRISPR-Cas systems. The obtained and further expected results have the potential to significantly impact exploring new avenues to chemically programmed RNA-targeting interference technologies for applications in bioscience, biotechnology, and applied medicine.