# Overview of Methods for Large-Scale RNA Synthesis
## 2 Methods of RNA Synthesis
### Ribozymes for RNA Production with Homogeneous Ends
#### 2.3.5 GlmS Ribozyme  
The GlmS ribozyme is a widespread, 145 nt-long, conserved element in Gram-positive bacteria located upstream of the glmS gene (encoding glutamine/tructose-6-phosphate aminotransferase) [105; 135]. It was discovered as a riboswitch, but further characterization revealed that, instead of RNA protection, it induces RNA decay in the presence of metabolites. Thus, GlmS has been classified as a ribozyme activated by the binding of the ligand (glucosamine-6-phosphate, GlcNc6P). The secondary structure of the GlmS ribozyme is composed of four double-stranded regions connected by four junctions, while its tertiary fold forms a compact structure with three near-parallel stacks comprising a total of three pseudoknots [105; 136]. The cleavage site is located between conserved guanosine and adenosine residues, which are also involved in co-factor binding and the positioning of a scissile phosphate group [105]. GlmS has been successfully used for crystallographic studies of Xrn1-resistant RNAs (xrRNAs) from Flaviviridae (PDB: 4 PQV) [137]. In order to test the efficiency of in vitro transcription using ribozymes, we designed and synthesized several constructs differing by location, type of ribozyme, and length and sequence of the target RNA (Table 1 and Supplementary Materials). The RNA-HH 1 to 6 constructs possessed the hammerhead (HH) ribozyme at the 3\({}^{\prime}\)-end and HH-RNA-HH hammerhead ribozyme at both ends. The HH-RNA-HP construct had the hammerhead ribozyme at the 5\({}^{\prime}\) and hairpin (HP) ribozyme at the 3\({}^{\prime}\)-end, and RNA-HDV was equipped with the HDV-like ribozyme at the 3\({}^{\prime}\)-end. The constructs were prepared as chemically synthesized DNA or obtained by polymerase chain reaction (PCR). \begin{table}
\begin{tabular}{c c c} \hline
**Construct** & **Ribozyme** & **Length of Target RNA** \\ \hline RNA-HH 1 & 3\({}^{\prime}\) hammerhead & 47 nt \\ RNA-HH 2 & 3\({}^{\prime}\) hammerhead & 30 nt \\ RNA-HH 3 & 3\({}^{\prime}\) hammerhead & 83 nt \\ RNA-HH 4 & 3\({}^{\prime}\) hammerhead & 86 nt \\ RNA-HH 5 & 3\({}^{\prime}\) hammerhead & 113 nt \\ RNA-HH 6 & 3\({}^{\prime}\) hammerhead & 43 nt \\ HH-RNA-HH & 5\({}^{\prime}\) hammerhead/3\({}^{\prime}\) hammerhead & 43 nt \\ HH-RNA-HP & 5\({}^{\prime}\) hammerhead/3\({}^{\prime}\) hairpin & 43 nt \\ RNA-HDV 1 & 3\({}^{\prime}\) HDV & 47 nt \\ RNA-HDV 2 & 3\({}^{\prime}\) HDV & 51 nt \\ \hline \end{tabular}
\end{table} 
Table 1: RNA constructs used in this study. They were cloned into plasmid DNA, which was linearized prior to in vitro transcription. All the RNA-HH constructs showed a high cleavage efficiency independent of the sequence of the target RNA (Figure 7A-C). We estimated that more than 95% of the transcripts were cleaved. We added a 5\({}^{\prime}\)-end hammerhead ribozyme to the RNA-HH6 (a new construct, named HH-RNA-HH) to increase the yield from in vitro transcription. Surprisingly, the efficiency of the cleavage was decreased to about 50% (Figure 7C, line 2). We observed a band corresponding to an uncleaved intermediate RNA product attached to the 3\({}^{\prime}\) hammerhead ribozyme. This suggests that the presence of the 5\({}^{\prime}\) ribozyme affects the proper folding of 3\({}^{\prime}\) hammerhead, reducing its cleavage activity. We replaced the 3\({}^{\prime}\) hammerhead with the hairpin ribozyme in order to test whether other types of ribozymes at the 3\({}^{\prime}\)-end could increase the yield of the RNA product. However, this modification resulted in no cleavage of the HH-RNA-HP construct by either 5\({}^{\prime}\)- or 3\({}^{\prime}\)-end-located ribozymes (Figure 7D, lane 1). Optimizing the reaction conditions by increasing the concentration of Mg\({}^{2+}\) ions or performing cycles of denaturation-renaturation did not improve the cleavage efficiency (Figure 7D, lanes 2-6). Finally, we tested the RNA-HDV 1 and 2 constructs with the HDV-like ribozyme at the 3\({}^{\prime}\)-end. Although we detected RNA products, the cleavage efficiency was very low (Figure 7E, lane 1, and Figure 7F).