Overview of Methods for Large-Scale RNA Synthesis

Abstract

In recent years, it has become clear that RNA molecules are involved in almost all vital cellular processes and pathogenesis of human disorders. The functional diversity of RNA comes from its structural richness. Although composed of only four nucleotides, RNA molecules present a plethora of secondary and tertiary structures critical for intra and intermolecular contacts with other RNAs and ligands (proteins, small metabolites, etc.). In order to fully understand RNA function it is necessary to define its spatial structure. Crystallography, nuclear magnetic resonance and cryogenic electron microscopy have demonstrated considerable success in determining the structures of biologically important RNA molecules. However, these powerful methods require large amounts of sample. Despite their limitations, chemical synthesis and in vitro transcription are usually employed to obtain milligram quantities of RNA for structural studies, delivering simple and effective methods for large-scale production of homogenous samples. The aim of this paper is to provide an overview of methods for large-scale RNA synthesis with emphasis on chemical synthesis and in vitro transcription. We also present our own results of testing the efficiency of these approaches in order to adapt the material acquisition strategy depending on the desired RNA construct.

Ushering in the era of tRNA medicines

“…To be cost-effective, the efficiency of each synthetic coupling reaction must be as high as possible (reviewed in Ref. ( 71 )). To produce a 25-nt oligonucleotide, 99% coupling efficiency results in 79% overall yield, whereas a relatively small drop to 97% coupling efficiency reduces the overall yield to 48%.…”

Utilization of Bacteriophage phi6 for the Production of High-Quality Double-Stranded RNA Molecules

“…In chemical synthesis, phosphoramidites are added to each other in a cyclic process occurring in an automated synthesizer on a solid support, such as controlled pore glass [ 25 ]. Chemical synthesis guarantees good yields and purity for RNA molecules under 50 nucleotides (nt), whereas, for longer RNAs, its application is impractical and can be justified only for specific reasons, such as, for instance, when modified RNA must be obtained [ 26 ]. If a long RNA molecule needs to be chemically synthesized, a range of short RNAs are produced, followed by ligation with T4 ligase [ 27 ].…”

“…The common method for the enzymatic production of RNA molecules is the in vitro transcription with T7 DdRp or with a similar enzyme from bacteriophage SP6 [ 26 , 28 ]. In this method, T7 DdRp binds to its promoter sequence at the 5′-end of a DNA template (linearized plasmid or DNA fragment produced by means of a polymerase chain reaction, PCR) and initiates RNA synthesis.…”

Enabling mRNA Therapeutics: Current Landscape and Challenges in Manufacturing

“…The method relies on phosphoramidite chemistry and involves the cyclical addition of nucleosides in a sequence-specific manner on a solid support [171,172]. However, this method for RNA production is only suitable for short oligomers, with some claiming its ability to form chains of up to 40 nucleotides and others using this method extending up to approximately 100-150 nucleotides [153,173,174]. This renders this method currently unsuitable for mRNA production, but rather, it is appropriate for the production of siRNA, miRNA and anti-sense oligonucleotide (ASO) molecules as a straightforward chemical synthesis.…”