Table 2.

Summary of RNA circularization methods.

Method	Advantages	Disadvantages
Chemical ligation	Effective for very small circRNAs (2–100 nucleotides)	Competition from intermolecular ligations and formation of 2′–5′ phosphodiester bonds
Enzymatic ligation by DNA ligase 1	Effective for larger circRNAs; ligation is sequence-specific; agnostic to terminal nucleotide identity; can produce exact sequence mimics	Competition from intermolecular ligations; requires optimization of the splint with each new substrate; higher amounts of the enzyme are needed due to low turnover
Enzymatic ligation by T4 RNA ligase 1	Effective for larger circRNAs; higher efficiency; splint not needed; easier to purify due to minimal reaction components; can produce exact sequence mimic	The efficiency of circularization is highly dependent on the structure of linear precursor; competition from intermolecular ligations; lower sequence specificity; strong nucleotide preference at 3′ and 5′ ends
Enzymatic ligation by T4 RNA ligase 2	Effective for larger circRNAs; greater substrate flexibility; splint not needed; agnostic to terminal nucleotide identity; can produce exact sequence mimic	The efficiency of circularization is highly dependent on the structure of linear precursor; competition from intermolecular ligations
PIE system with group I introns	Effective for various RNA sizes (100 nucleotides–5 kb); can be applied both in vivo and in vitro	CircRNAs retain portions of the exons from the native group I intron-containing genes; several splicing intermediates are produced as byproducts
PIE system with group II introns	It can be used to mimic an endogenous circRNA sequence precisely without any extraneous sequence; can be applied both in vivo and in vitro	Limited characterization
Hairpin ribozymes	Can generate high yields of small circRNAs (∼50–150 nucleotides) with sequence homogeneity	CircRNAs contain ribozyme sequences and cognate cleavage sites with the potential for ribozymatic activity