Table 2.

The advantage and disadvantage of the three circularization methods

	Advantage	Disadvantage
Chemical synthesis strategies	• No exogenous sequence introduction	• Low circularization efficiency for long linear precursor • Complicated design and synthesis procedures • Complementary template requirement • Low-throughput synthesis • Unknown immunogenicity
Enzymatic synthesis strategies	• No exogenous sequence introduction • Lowest immunogenicity	• Low circularization efficiency for long linear precursor • Complicated synthesis procedure • Complementary template requirement • Low-throughput synthesis
PIE system	• Simple synthesis procedure • Suitable for circularization of long linear precursor • Applicable to high-throughput synthesis • Low immunogenicity	• Exogenous sequence introduction (group I intron PIE system) • Contaminants present after synthesis