# Overview of Methods for Large-Scale RNA Synthesis
## 3 Discussion
An attractive alternative to the ribozyme approach is in vitro transcription of the C2\({}^{\prime}\)-methoxy-modified DNA. This does not have specific requirements for the proper folding of catalytic RNAs, and a DNA template can be obtained from external companies or synthesized in-house. Using this method, we observed significant improvement in terms of the RNA's 3\({}^{\prime}\)-end homogeneity (Figure 8). Although N+1 activity of RNA polymerase was still present, RNAs were easily separated using HPLC or gel electrophoresis (data not shown). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Method** & \multicolumn{2}{c}{**Important Factors**} \\ \hline Chemical synthesis & \(\bullet\) & Highly efficient for short oligomers (up to 20 nt) \\  & \(\bullet\) & High-grade phosphoramidites (preferably TOM protection) \\  & \(\bullet\) & Acetonitryl with lowest possible content of water (up to 20 ppm) \\  & \(\bullet\) & Preferable synthesis in DMT-ON mode \\ In vitro transcription & \(\bullet\) & Concentration of DNA template (50–100 \(\upmu\)g/mL) and reaction \\  & \(\bullet\) & Hammerhead ribozyme showed highest cleavage efficiency \\  & \(\bullet\) & DNA template with C2\({}^{\prime}\)-methoxy modifications reduces \\  & 3\({}^{\prime}\)-end heterogeneity \\ \hline \hline \end{tabular}
\end{table} 
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