Development of HPLC-free system for solid phase RNA synthesis

Suny At Albany, Albany NY

Investigators

Abstract

The Chemical Synthesis Program of the Chemistry Division supports this project by Professor Maksim Royzen. Professor Royzen is a faculty member in the Department of Chemistry at the State University of New York at Albany. Professor Royzen is developing improved processes for the synthesis of RNA. Synthetic RNA occupies a very special place in modern research. Custom synthesis of 20-30 nucleotide-long RNA strands has become routine, propelling many fields of biochemical and pharmaceutical research. Research in the twenty-first century, however, creates a strong need for a robust solid phase synthesis of longer RNA strands ranging from 50-150 nucleotides (-nt) in length. Despite highly impressive efforts to take the field to the next level, synthesis of long RNA strands is still very challenging, even by the commercial leaders in the field. The biggest obstacle lies in purification of the target RNA strand from the impurities created in every cycle of the solid-phase synthesis. This proposal develops an approach towards chromatography-free synthesis and purification of RNA that is applicable to any strand regardless of its size. To demonstrate the versatility of the method, syntheses of incrementally longer RNA strands are being pursued, including a 58-nt long strand and 112-nt long non-coding RNA. The project has been designed to provide educational and research activities for graduate and undergraduate students. Outreach activities include the participation of local high school teachers in the research project. The standard solid phase RNA synthesis consists of four steps. It begins with activation of the solid phase-immobilized first subunit, typically achieved by cleavage of a protecting group, followed by coupling of the second subunit. Failure strands, generated due to incomplete coupling, are capped to prevent further propagation. After oxidation of the phosphite triester, the dimer strand is carried over to the next cycle. The most challenging aspect of this approach is purification of the final product from the failure sequences that occur to some extent during every coupling step. In a typical synthesis of 20-nt long RNA, failure sequences may constitute as much as 50-60% of the total oligonucleotide content. The standard purification of RNA is achieved through reverse phase High Performance Liquid Chromatography (HPLC), which is time and labor intensive and increasingly problematic with longer strands. The strategy, involving bio-orthogonal inverse electron demand Diels-Alder chemistry, is a transformative improvement over the existing approach. The final coupling step is carried out using a unique subunit functionalized with a releasable trans-cyclooctene (TCO) group. Because all failure sequences from previous cycles are capped, only the desired strand incorporates the releasable TCO. Upon completion of the final cycle, the synthetic oligonucleotide is cleaved from the original solid support and purified using bio-orthogonal "click-to-release" chemistry involving immobilized tetrazine. The bio-orthogonal purification procedure is fully compatible with larger RNA strands. The educational plan includes outreach to underrepresented minorities, who are introduced to solid phase RNA synthesis, as well as organic synthesis.