Synthesis and engineering of circular RNAs

Circular RNAs (circRNAs) have been discovered in all kingdoms of life. They are produced from introns as well as from exons. However, strongest interest is in circRNAs that are transcribed and spliced from exons of protein and noncoding genes in eukaryotic cells including humans. Therefore, synthesis and engineering of circRNAs as models for structure and function studies are strongly required. In vitro, methods for RNA synthesis and circularization are available. Chemical synthesis allows for preparation of RNAs incorporating nonnatural nucleotides in small RNA segments, whereas enzymatic synthesis is advantageous for production of long RNAs, however, without the possibility for site-specific modification. Strategies for chemical and enzymatic RNA synthesis may be combined to obtain long modified linear RNA strands for subsequent circularization. Here, we describe two alternative protocols for synthesis and circularization in dependence on downstream applications and template structure.