# Overview of Methods for Large-Scale RNA Synthesis
## 3 Discussion  
This paper provides an overview of methods for large-scale RNA synthesis. We also describe our experience during the preparation of homogeneous samples for crystallization experiments (summarized in Table 2). Many factors should be taken into account when selecting methods for the production of milligram quantities of RNA suitable for structural studies. They include the length of the oligomer, the presence of specific chemical modifications, the oligomer manufacturing time, cost, and equipment availability. Figure 8: In vitro transcription of RNA using C2\({}^{\prime}\)-methoxy-modified DNA. (**A**) Schematic representation of DNA template. Modified nucleotides are marked with red Xs. Chromatograms showing RNA synthesis with (**B**) unmodified template, (**C**) C2\({}^{\prime}\)-methoxy-modified template, (**D**) C2\({}^{\prime}\)-methoxy-modified template in the presence of 10 mM guanosine, and (**E**) C2\({}^{\prime}\)-methoxy-modified template in the presence of 15 mM GTP. From our experience, chemical synthesis allows the preparation of sufficient quantities of RNA up to 20 nucleotides. Longer (>40 nt) oligomers can be synthesized but with much lower efficiency. Chemically synthesized RNA can also be purchased from external companies. However, since crystallization experiments often require testing large numbers of different constructs, using in-house synthesizers significantly reduces the cost of RNA production. We observed that in order to obtain a high yield of chemically synthesized RNA, it is crucial to dissolve reagents in acetonitrile with the lowest possible water content (up to 20 ppm). We recommend TOM rather than TBDMS protected phosphoramidites. They are characterized by higher coupling efficiency due to lower steric hindrance and prevented relocation from 2\({}^{\prime}\) to 3\({}^{\prime}\) moiety of ribose ring. The 2\({}^{\prime}\) to 3\({}^{\prime}\) migration can take place when using the TBDMS group, leading to the formation of non-biologically active 2\({}^{\prime}\)-5\({}^{\prime}\) linkages. The costs of TOM and TBDMS phosphoramidites are comparable. When modified phosphoramidites are produced in-house they should have the highest possible purity, because some H-phosphonate or other impurities can still be present, reducing the coupling efficiency. RNA oligomers should be synthesized with the DMT-ON method, which significantly increases the homogeneity of the sample. DMT-ON purification on dedicated columns can remove abortive products, and the resulting RNA is suitable for crystallization or nuclear magnetic resonance measurements (Figure 2). With longer oligomers, additional purification steps are usually required to obtain homogeneous RNA. In vitro transcription is suitable for longer RNA, but its main drawback is the non-specific activity of the RNA polymerase generating 3\({}^{\prime}\)-end heterogeneity. Homogeneous samples can be achieved by using ribozymes cleaving at the 3\({}^{\prime}\) site of the RNA construct. In our hands, the hammerhead ribozyme was characterized by the highest (~90%) cleavage efficiency, while the HDV-like ribozyme cleaved RNA less efficiently (Figure 7). Despite the careful design of RNA constructs to fulfill all the requirements for the proper folding of the catalytic domains of ribozymes, the cleavage efficiency was low when ribozymes were present at both ends of the RNA. An extreme example is the HP-RNA-HH construct, where the replacement of the 5\({}^{\prime}\) hammerhead with the hairpin ribozyme led to the complete inhibition of cleavage (Figure 7). The presence of the 5\({}^{\prime}\) hairpin ribozyme could interfere with the folding of the 3\({}^{\prime}\) hammerhead ribozyme. As a result, both ribozymes became inactive. The ribozyme approach requires the purification of the RNA product from uncleaved RNA and the catalytic domain of the ribozyme. Therefore, the lengths of the ribozyme and the RNA of interest should differ in such a way that they can be separated with gel electrophoresis or HPLC.