# Overview of Methods for Large-Scale RNA Synthesis
## 1 Introduction  
Ribonucleic acid (RNA) is one of the major players involved in cell homeostasis [1]. Specific chemical features make RNA an important biological molecule, an interesting research object, and a tool adapted for human use [2, 3]. In early nucleic acid research, there was no clear distinction between RNA and deoxyribonucleic acid (DNA); they were seen as \"nuclein\", a phosphor-rich substance different from proteins and present in large quantities in every living organism [4]. The pioneering work of many researchers, including [PERSON], [PERSON], and their co-workers, allowed to distinguish DNA and RNA molecules and to identify their basic components, such as ribose rings, nitrogen-containing bases, and phosphate groups [5, 6, 7]. Since then, RNA has been revealed as a multifaceted molecule that can form complex three-dimensional structures, presents catalytic properties, and serves as a regulatory element for governing gene expression [8, 9, 10, 11, 12, 13, 14, 15, 16]. On the other hand, RNA has also been identified as a toxic factor involved in the pathogenesis of numerous human disorders [17, 18, 19]. Finally, RNA became a tool applied in biotechnology and medicine [20, 21, 22, 23, 24, 25]. The best-known examples are the mRNA-based vaccines used to fight the COVID-19 pandemic [26].  
Knowledge of the three-dimensional structure of RNA is crucial for explaining its biological functions and conformational dynamics and for providing model platforms for drug development [15, 27, 28]. The pioneering studies on nucleic acid structure originated from research by [PERSON], [PERSON], and [PERSON], who proposed the double-DNA-helix model [29, 30]. The first atomic transfer RNA (tRNA) structure was determined by [PERSON]'s group using single-crystal x-ray crystallography, opening up the field of structural studies of RNA molecules [31, 32, 33]. Subsequent discoveries of RNA properties (e.g., catalytic activity) and the development of methods for chemical and enzymatic synthesis increasedinterest in RNA and RNA-protein research, resulting in the three-dimensional structures of ribozymes, riboswitches, ribosomes, small nucleolar RNAs (snoRNAs), and other types of RNA being discovered [34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56]. These fundamental findings helped to explain the principles of RNA functions, folding pathways, the roles of water and ion molecules, and to reveal ligand-binding sites [57].  
Structural studies of RNA molecules start with the design, synthesis, and purification of the macromolecule. In this step, the purity and homogeneity of the sample are critical for successful crystallization and diffraction experiments [58; 59]. Another obstacle is the amount of material required for crystallization; milligrams of homogeneous RNA are usually needed [60; 61; 62; 63]. Although a number of well-established protocols exist, obtaining a sufficient amount of pure RNA is challenging [60; 61; 62; 63]. While chemical synthesis allows the production of large quantities of RNA, it is suitable only for relatively short RNA oligomers and requires a laboratory equipped with specific infrastructure (synthesizer, fume hood, vacuum evaporator, etc.). Moreover, modified phosphoramidites can be expensive and often non-commercially available. Longer RNAs can be effectively synthesized using in vitro transcription but this method suffers from considerable 3\({}^{\prime}\)-end heterogeneity and requires specific sequence requirements at the 5\({}^{\prime}\)-end. This paper provides an overview of large-scale RNA-synthesis methods used in structural studies, supplemented by our own results with the practical implementation of available protocols. In the first part, we describe methods for RNA synthesis and discuss their advantages and limitations. Next, we present the implementation of these methods and guidelines for adapting RNA-synthesis strategies.