TABLE 1.

Advantages and disadvantages of different ligation method.

Ligation method		Advantages	Disadvantages
Chemical ligation		• Only chemical reagents	• Low ligating efficiency
		• No biological components	• Biosafety concern
			• 2′, 5′-phosphodiester bonds
Enzymatic ligation	T4 DNA ligase	• Accurate	• Affected by significant RNA secondary structure
			• Affected by high percentage of Us
			• Low efficiency
			• Intermolecular end joining side reactions
	T4 RNA ligase 1	• High efficiency	• Low ligating efficiency for large RNA molecules
		• Synthesize as little as 6 to 8 nucleotides of circRNAs	• Affected by significant RNA secondary structure
			• Intermolecular end joining side reactions
	T4 RNA ligase 2	• More efficient for linear RNA precursor folding into a secondary structure with the ligation junction in a double-stranded region	• Low ligating efficiency for large RNA molecules
			• Intermolecular end joining side reactions
Ribozyme method	Group I intron self-splicing system	• Simple reaction condition and purification method	• Affected by significant RNA secondary structure
		• Can be used for RNA cyclization in vitro and in vivo
		• Can synthesize large circRNAs
	Group II intron self-splicing system	• Accurate ligation	• 2′, 5′-phosphodiester bonds
			• The mechanism remains unclear in vitro
	Hairpin ribozyme method	• High efficiency for small circRNAs	• Unstable
			• Exogenous HPR sequences