Abstract: A method of preparing a circular RNA includes transcribing a vector to form a precursor RNA, in which the vector includes the following elements operably connected to each other and arranged in the following sequence: a) a 5? element, b) a 3? Group I self-splicing intron fragment containing a 3? splice site dinucleotide, c) none or an element containing an internal ribosome entry site (IRES) and a protein coding region or an element containing a noncoding region, d) a 5? Group I self-splicing intron fragment containing a 5? splice site dinucleotide, and e) a 3? element, in which 5? element and 3? element form a stable structure with a Gibbs free energy (?G) 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 3? element, in which 3? Group I self-splicing intron fragment and 5? Group I self-splicing intron fragment form a self-cleaving and self-ligating RNA molecule, thereby generating circular RNA.

Abstract: A method for in vitro transcription of a DNA template into RNA includes providing a mixture containing a buffer substance, ribonucleoside triphosphates (NTPs), one or more magnesium salts in a concentration of from about 2 mM to about 60 mM, the DNA template, and a recombinant RNA polymerase, and incubating the reaction mixture at from about 25° C. to about 40° C. for from about 1 hour to about 12 hours thereby producing the RNA. A method for in vitro transcription includes providing a DNA template and a cap analogue that binds to ?1 and/or +1 nucleotides of promoter for in vitro transcription, thus producing more full length mRNAs, allowing for more flexibility on the choice of first mRNA base, and providing +2 position open for custom sequence.

Abstract: Provided is an automated system for preparing purified plasmid DNA in commercial scale.