# Overview of Methods for Large-Scale RNA Synthesis
## 2 Methods of RNA Synthesis
### Ribozymes for RNA Production with Homogeneous Ends  
Ribozymes are self-cleaving RNAs found mainly in small-RNA pathogens of plants [100, 101, 102]. They are involved in the replication process, with a rolling-circle mechanism. As a result, long, linear concatamers of replicated genome of the pathogen are produced, which are cleaved by the catalytic domain of the ribozyme into unit-length progeny [103, 15, 101]. There are four types of small ribozyme motifs: hammerhead, hairpin, Varkud satellite (VS), and hepatitis delta virus (HDV) [104]. The glucosamine-6-phosphate riboswitch (GlnS) ribozyme is also classified as a small ribozyme, but in contrast to other catalytically active RNAs, it is activated by the binding of the ligand molecule [105]. All ribozymes catalyze the RNA-cleavage reaction, producing a 5' hydroxyl group and 2',3'-cyclic phosphate (Figure 4) [102]. For optimal activity, they require divalent metal ions such as Mg2+. Ribozymes usually act in _cis_ mode when a substrate and ribozyme are present in single RNA chains, and cleavage separates RNA into two species (the catalytic domain of the ribozyme and target RNA). Ribozymes can also act in trans, recognizing a substrate by hybridizing to the complementary sequence and cleaving the RNA. Although ribozymes are commonly applied in different research areas, each type has its own specific requirements, mostly concerning the sequence of the RNA substrate (see below).  
Figure 4: Ribozyme-mediated cleavage of RNA.  
Their relatively small size makes ribozymes attractive tools for use in structural studies for the production of RNA molecules with homogeneous ends or for enhancing the efficiency of in vitro transcription [61; 63]. In the first case, they are incorporated into a DNA template upstream, downstream, or at both positions of the target sequence. During in vitro transcription, the RNA chain is cleaved, resulting in a mixture of target RNA and the catalytic domain of the ribozyme [61]. They need to be separated and purified using PAGE, gel filtration chromatography, or affinity tags [106; 107].  
When the ribozyme is located downstream of the target sequence, the 3\({}^{\prime}\)-end of the transcript RNA is homogeneous, but it possesses a 2\({}^{\prime}\),3\({}^{\prime}\)-cyclic phosphate group (Figure 5A). The phosphate group can be removed with T4 polynucleotide kinase if the 3\({}^{\prime}\) OH group is required for downstream experiments [108]. The ribozyme is placed at the 5\({}^{\prime}\)-end of the target RNA to increase the yield of in vitro transcription or to produce RNA with homogeneous 5\({}^{\prime}\)-ends (Figure 5B). Higher efficiency in RNA synthesis is achieved by introducing the GGGAGA sequence at the beginning of the catalytic domain of the ribozyme, which ensures high processivity for the RNA polymerase [109]. This approach is also used when the sequence of the target RNA is critical and cannot be adjusted according to the T7 RNA polymerase's requirements. Since homogeneity and high yield in RNA synthesis are key factors for successful crystallization, one should consider RNA constructs with ribozymes at both ends (Figure 5C) [61]. Ribozymes can also be used in trans. This requires the co-transcription of the catalytic domain of the ribozyme and target RNA [110; 111].