Summary of Large-Scale RNA Synthesis Methods

May 16, 2023

RNA molecules are involved in almost all important cellular processes and pathogenesis of human diseases. The functional diversity of RNA comes from its structural richness, and despite being composed of only four nucleotides, RNA molecules exhibit a plethora of secondary and tertiary structures, which are essential for interaction with other RNAs and ligands (proteins, small metabolic Intramolecular and intermolecular contacts are crucial. To fully understand the function of RNA, it is necessary to define its spatial structure. Crystallography, NMR, and cryo-electron microscopy have had considerable success in determining the structures of biologically important RNA molecules. However, these powerful methods require a large number of samples. Despite their limitations, chemical synthesis and in vitro transcription are routinely used to obtain milligram quantities of RNA for structural studies, providing simple and efficient methods for large-scale production of homogeneous samples.


The structural study of RNA molecules begins with the design, synthesis, and purification of macromolecules. During this step, the purity and homogeneity of the sample is critical for successful crystallization and diffraction experiments. Another hurdle is the amount of material required for crystallization, often requiring milligrams of homogeneous RNA. Although many proven protocols exist, obtaining sufficient quantities of pure RNA is challenging. While chemical synthesis can produce large amounts of RNA, it is only applicable to relatively short RNA oligomers and requires a laboratory equipped with specific infrastructure (synthesizers, fume hoods, vacuum evaporators, etc.).


RNA can be obtained in three ways: purified from biological sources, chemically synthesized using solid-phase methods, or enzymatically synthesized by in vitro transcription. Isolation from cells is suitable for complex or large and abundant RNAs (ribosomes or tRNAs), resulting in native samples with all important post-transcriptional modifications. Shorter or smaller RNAs can be synthesized chemically or enzymatically. These methods are more general and better suited for structural studies of RNA molecules.


Chemical synthesis is suitable for relatively short oligomers (up to 40 nt). While yields of 99% can be achieved in a single extension cycle, the overall efficiency drops systematically with each cycle. For a 25 nt long oligomer, the synthesis was performed in approximately 79% overall yield, assuming an average single coupling efficiency of 99%. When the coupling efficiency drops to 97%, the overall yield is only around 48%. For the longer oligomers (50 nt), the overall yield was only 37%, even though the average performance of the individual couplings was 98%.


Therefore, chemical synthesis of longer RNAs is impractical unless other reasons, such as the introduction of modified nucleotides, are considered. In contrast to in vitro transcription, chemical modifications can be easily incorporated into the RNA strand during automated synthesis. If the oligomer is too long to be efficiently synthesized, two shorter strands of the modified RNA can be obtained separately and ligated using split ligation or T4 RNA ligase. Another advantage of chemical synthesis is that it is less restrictive and heterogeneous to the RNA sequence compared to in vitro transcription.


Chemical synthesis can prepare sufficient quantities of RNA up to 20 nucleotides. Longer (>40 nt) oligomers can be synthesized, but at much lower efficiency. Chemically synthesized RNAcan also be purchased from outside companies. However, since crystallization experiments often require testing a large number of different structures, the use of in-house synthesizers can significantly reduce RNA production costs. In vitro transcription is suitable for longer RNAs, but its main disadvantage is the non-specific activity of RNA polymerase that generates 3'-end heterogeneity. Homogenized samples can be obtained by cleavage at the 3' site of the RNA construct using ribozymes. 


RNA is involved in the regulation of nearly all aspects of cellular physiology. The structural richness of RNA molecules is not only explored in nature, but also adopted by humans in the construction of tools in molecular biology, biomedicine, and nanotechnology. Therefore, studying the three-dimensional structure of RNA is very important and is the key to understanding the RNA-function relationship. In recent years, many new methods for large-scale RNA synthesis have emerged, such as circular RNAs, and position-selectable markers for RNA and polymerase chain transcription. Although still under development, they demonstrate new avenues of research and have great potential to open up the field of RNA structure research.