The Chemical Synthesis of DNA and RNA Oligonucleotides for Drug Development and Synthetic Biology Applications

Abstract

Synthetic DNA and RNA sequences have been and are still heavily used in the development of a plethora of therapeutic, diagnostic, and genomic applications. This report describes the details of the phosphoramidite approach to the solid-phase synthesis of DNA sequences including optimization of the critical steps that have been made over the years to improve the coupling kinetics and efficiency of deoxyribonucleoside phosphoramidite monomers. The use of deoxyribonucleoside H -phosphonate monomers, as an alternative to phosphoramidite monomers, is presented in sufficient detail to highlight the versatility of the H -phosphonate approach to the synthesis of native and modified oligonucleotides despite its inherent limitations. The solid-phase synthesis of RNA sequences is also reviewed with a particular emphasis on the selection of 2′-hydroxyl protecting groups for ribonucleosides and their phosphoramidite derivatives to underscore this critical issue in the synthesis of RNA sequences. The in situ synthesis of DNA sequences on microarrays using photolithographic, inkjet printing, electrochemical, and microfluidic processes is reported in the context of diagnostic, drug discovery, and gene assembly applications. The usefulness of DNA microarrays in the fabrication of RNA microarrays is demonstrated while the difficulties of in situ RNA synthesis on glass surfaces remain to be overcome.