# Overview of Methods for Large-Scale RNA Synthesis
## 2 Methods of RNA Synthesis
### Chemical Synthesis  
Solid-phase chemical synthesis using an automated synthesizer was established by [PERSON] and was further applied for the synthesis of deoxyribonucleic and ribonucleic acids [64; 65]. It is based on the cyclic elongation of the DNA/RNA chain on a solid support, e.g., controlled-pore glass or highly crosslinked polystyrene. It uses phosphoramidites decorated with different protection groups in order to block their reactivity during chemical synthesis [66]. There are several types of commercially available phosphoramidites. Most of them possess mild base-labile protection of exocyclic amino group of nucleobase and phosphate moiety blocked by 2-cyanoethyl \(N\),\(N\),\(N^{\prime}\),\(N^{\prime}\)-tetra-isopropyl [66]. In the case of 2\({}^{\prime}\)-hydroxyl, the most widely employed class of protection groups are TOM (triisopropylsilyl-oxy-methyl) and TBDMS (tert-butyldimethylsilyl). They are fluoride-labeled groups which are removed after synthesis using TBAF (tetra-n-butylammonium fluoride) or TEA-3 HF (triethylamine trihydrofluoride). The 5\({}^{\prime}\) OH group is usually blocked by DMT (dimethoxyttriyl) which is compatible with 2\({}^{\prime}\)OH silyl blockage [67]. The selection of the 2\({}^{\prime}\)-protection group is the most critical step affecting the yield and time of RNA synthesis. Recently, more potent activators such as ETT (5-ethylthio-1H-tetrazole) and BTT (5-benzylthio-1H-tetrazole) helped to increase the rate of the coupling step [68]. Other strategies of phosphoramidites protection have also been developed, resulting in shorter and higher efficiency of RNA synthesis. However, they are expensive, non-commercially available or require different protocols of solid-phase synthesis (for review see [66; 69]). The standard solid-phase synthesis occurs from the 3\({}^{\prime}\) to 5\({}^{\prime}\)-end, and one elongation cycle results in a chain extension by one nucleotide. Each cycle includes four steps: detritylation, coupling, capping, and oxidation (Figure 1) [66]. It starts with removing the dimethoxyttriyl (DMT) protection group from the 5\({}^{\prime}\) OH group of the nucleotide attached to the solid support. This is followed by a coupling reaction, resulting in the chain elongation of oligomers with a 5\({}^{\prime}\) DMT group originating from added phosphoramidite. Since the yield of coupling is less than 100%, not all RNA chains are elongated. Thus, in the next step, the non-extended oligomers are 5\({}^{\prime}\) capped, preventing their further extension in the next cycle. Finally, the phosphate group is oxidized and converted into a stable moiety [62,66,70]. Chemical synthesis is suitable for relatively short oligomers (up to 40 nt-long). Although the yield of a single elongation cycle can reach 99%, the overall efficiency drops systematically with each cycle. For 25 nt-long oligomers, the overall yield of synthesis is about 79%, assuming an average single coupling efficiency of 99%. When the coupling efficiency drops to 97%, the overall yield is only approximately 48%. For longer oligomers (50 nt), the overall yield is only 37%, even if the average performance of a single coupling is 98% [71]. Therefore, chemical synthesis for longer RNAs is impractical, unless other reasons are considered, such as the introduction of modified nucleotides. In contrast to in vitro transcription, chemical modifications can be easily incorporated into RNA chains during automated synthesis. If the oligomer is too long to be efficiently synthesized, two shorter modified RNA chains can be obtained separately and ligated using split-join ligation or T4 RNA ligase [72, 73, 74]. The other advantage of chemical synthesis is fewer constraints on the sequence of RNA and lower heterogeneity in comparison to in vitro transcription. In our laboratory, chemical synthesis is routinely used for the preparation of RNA oligomers.