US 12,247,202 B2
Circular RNAS and preparation methods thereof
Dongsheng Dai, Suzhou (CN); Lynn Shan, Suzhou (CN); and Zilin Dai, Suzhou (CN)
Assigned to EXCLCIRC (SUZHOU) BIOMEDICAL CO., LTD., Suzhou (CN)
Filed by EXCLCIRC (SUZHOU) BIOMEDICAL CO., LTD., Jiangsu (CN)
Filed on Sep. 9, 2023, as Appl. No. 18/464,200.
Claims priority of application No. 202211105849.6 (CN), filed on Sep. 10, 2022; and application No. 202310557196.3 (CN), filed on May 17, 2023.
Prior Publication US 2024/0093185 A1, Mar. 21, 2024
Int. Cl. C12N 15/11 (2006.01); C07K 14/195 (2006.01); C12N 9/22 (2006.01); C12N 15/63 (2006.01)
CPC C12N 15/11 (2013.01) [C07K 14/195 (2013.01); C12N 9/22 (2013.01); C12N 15/63 (2013.01)] 6 Claims
 
1. A DNA molecule for making a circular RNA, comprising elements operably connected and arranged, from a 5′ to 3′ direction, in the following order:
(a) a full-length intron having the nucleotide sequence of SEQ ID NO: 1;
(b) an E2 fragment which includes a downstream exon of the full-length intron, the E2 fragment having the nucleotide sequence of SEQ ID NO: 2;
(c) an internal ribosome entry site (IRES) fragment that is from Coxsackie virus B3 (CVB3);
(d) a target DNA sequence that encodes a target peptide; and
(e) an E1 fragment which includes an upstream exon of the full-length intron, the E1 fragment having the nucleotide sequence of SEQ ID NO: 8; wherein:
the full-length intron, the downstream exon, and the upstream exon are from a pre-tRNALeu gene of genus Anabaena;
3′ end of the E1 fragment is configured to produce a hydroxyl group in an in vitro transcription reaction, wherein the hydroxyl group is formed by using a restriction endonuclease cleavage when preparing an in vitro transcription template with the DNA molecule, followed by the in vitro transcription reaction, and the in vitro transcription template includes the full-length intron, the E2 fragment, the target DNA sequence, and the E1 fragment;
the hydroxyl group is capable of initiating splicing in a one-step transesterification reaction at a splice site between RNA fragments transcribed from the full-length intron and the E2 fragment in a linear RNA that is produced from the DNA molecule in the in vitro transcription reaction, such that the linear RNA is configured to self-circularize to produce the circular RNA; and
the DNA molecule does not include any intron sequence at the 3′ end of the E1 fragment.