# Overview of Methods for Large-Scale RNA Synthesis
## 2 Methods of RNA Synthesis
### In Vitro RNA Synthesis Using T7 RNA Polymerase  
In vitro transcription is a common method used for synthesizing RNA molecules. It utilizes a DNA template with a promoter sequence for T7 RNA polymerase followed by a sequence encoding the target molecule [75]. The enzyme binds the template at the promoter region and starts the RNA synthesis. The elongation of the RNA chain terminates when the enzyme drops off the 3'-end of the DNA template [75]. In vitro synthesis can be efficient, resulting in milligrams of RNA sample, but a high yield depends on several factors. The most important is the type of nucleotides located immediately downstream of the T7 promoter. The transcript should start from at least one guanosine residue, but having two or three consecutive Gs is better [75; 76; 77]. Usually, the RNA sequence can be adjusted and mutations can be easily introduced into the DNA template. If the RNA sequence cannot be changed, catalytic RNAs can be employed (see below).  
In structural studies, a serious drawback of in vitro RNA synthesis is the presence of short, abortive transcripts due to polymerase slippage and transcripts with additional nucleotides added to the 3'-end that are not encoded by the DNA template [78]. Separating short products from full-length oligomers is usually easy, while it is more difficult and time-consuming with transcripts extended by an additional one or two nucleotides [58]. The amounts of N+1 and N+2 products range from 10 to 50% of the whole synthesis, reducing the homogeneity, yield, and crystallization potential of the RNA sample [59].  
In vitro transcription is conducted with a mixture of nucleoside triphosphates (NTPs), resulting in RNA having tri-phosphate at the 5'-end. In some instances, it can negatively  
Figure 3: Examples of crystals that were grown from chemically synthesized RNAs.  
Figure 2: HPLC chromatograms of chemically synthesized RNAs: (**A,B**) 11 nt-long RNA oligomer synthesized in DMT-OFF and DMT-ON mode, respectively; (**C**) 45 nt-long RNA synthesized in DMT-ON mode.  
influence the crystallization potential of the RNA by preventing the packing of RNA molecules into a crystal lattice or introducing disorder [79]. Thus, the conversion of the 5' triphosphate into a 5' hydroxyl group may be required to improve the crystallization and quality of the crystals. This can be easily performed by in vitro transcription in the presence of guanosine residues or with the use of alkaline phosphatase [59]. If a 5' monophosphate group is required for further RNA modification, the transcription reaction can be supplemented with guanosine monophosphate (GMP).  
In vitro transcription can be employed for the production of capped RNAs. Cap is a modified nucleotide (7-methylguanosine, m7G) linked by the 5',5'-triphosphate bond to the first transcribed nucleotide [80, 81]. Cap structure increases the stability of cellular and exogenous mRNAs (such as vaccine mRNA) [82, 83]. It is also required for mRNA splicing, export and initiation of translation [82, 84, 85]. Therefore, capped RNAs are widely used in studies concerning vital cellular processes [86, 87, 88]. The increasing demand on simple and efficient methods for synthesis of capped mRNAs resulted in the development of several protocols [86, 89, 90, 91]. For long RNAs, cap structure is commonly introduced during in vitro transcription in the presence of chemically synthesized cap analogs such as m7G, anti-reverse cap analog (ARCA) and its derivatives [86, 90, 91, 92, 93, 94]. However, this method suffers from low efficiency which rarely exceeds 80% under optimized conditions. Moreover, it is limited to Cap 0 and the first transcribed nucleotide must be purine [95]. Recently, other capping reagents have been developed, extending the availability of different cap structures and increasing the yield of cap incorporation [95, 96]. Short capped RNAs are synthesized using solid-phase synthesis but it requires non-standard phosphoramidites or modified solid supports [97, 98, 99].