Method of preparing self-circularized RNA

A method of preparing a circular ribonucleic acid (RNA), comprising:

• transcribing a vector to form a precursor RNA comprising the following elements operably connected to each other and arranged in tandem in a 5′ to 3′ orientation:
• a) a 5′ element,
• b) a 3′ Group I self-splicing intron fragment,
• c) none or an element comprising an internal ribosome entry site (IRES) and a protein coding region or an element comprising a noncoding region,
• d) a 5′ Group I self-splicing intron fragment, and
• e) a 3′ element;
• wherein the 5′ element and the 3′ element form a thermodynamically stable multiple way junction RNA structure with a Gibbs free energy (ΔG) from −190 kcal/mol to −9.0 kcal/mol, provided that the stable structure is not a duplex with at least 95% base pairing between the 5′ element and the 3′ element,
• wherein the 3′ Group I self-splicing intron fragment and the 5′ Group I self-splicing intron fragment form a self-cleaving and self-ligating RNA molecule, thereby generating the circular RNA,
• wherein the thermodynamically stable multiple way junction RNA structure comprises a 4-way junction (4WJ) comprising
• a first branch of the 4WJ domain is formed from a 5′ portion of a 4WJa sequence and a 3′ portion of a 4WJd sequence and comprises a first helical region,
• a second branch of the 4WJ domain is formed from a 3′ portion of the 4WJa sequence and a 5′ portion of a 4WJb sequence and comprises a second helical region, and
• a third branch of the 4WJ domain is formed from a 3′ portion of the 4WJb sequence and a 5′ portion of a 4WJc sequence and comprises a third helical region, and
• a fourth branch of the 4WJ domain is formed from a 3′ portion of the 4WJc sequence and a 5′ portion of the 4WJd sequence and comprises a fourth helical region,
• wherein each of said helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.