Method for precisely preparing circular RNA with anabaena intron self-cleaving ribozyme

A method for precisely preparing a circular RNA with an Anabaena variabillis intron self-cleaving ribozyme, comprising:

• S1:
• analyzing a secondary structure of the Anabaena variabillis intron self-cleaving ribozyme, and screening out sequences in upstream and downstream exons that are closely related to the activity of the self-cleaving ribozyme;
• S2:
• selecting an artificial sequence containing the sequences screened out in the step S1, inverting original upstream and downstream intron sequences of the Anabaena variabillis, and then replacing an original exon sequence in the Anabaena variabillis intron self-cleaving ribozyme with the artificial sequence to form an Anabaena variabillis intron self-cleaving frame for preparing the circular RNA;
• S3:
• adding an 18-base reverse complementary sequence to each of the upstream and downstream of the Anabaena variabillis intron self-cleaving frame sequence obtained in the step S2, and then adding a T7 transcription promoter to both sides of the sequence and a restriction endonuclease cleavage site to both ends of the sequence to form a total linear RNA sequence, and finally introducing the total linear RNA sequence into a pUC19 skeleton vector to prepare a plasmid;
• S4:
• digesting the plasmid obtained in the step S3 with an endonuclease EcoRI to obtain a linearized plasmid, performing purification, obtaining a transcribed linear RNA through in vitro transcription and DNA template digestion, and performing purification again to obtain a purified transcribed linear RNA;
• and
• S5:
• cyclizing the purified transcribed linear RNA obtained in the step S4 in vitro and performing purification to obtain a purified cyclized RNA, and further performing tolerance testing and circular RNA cyclization interface verification to obtain the circular RNA, wherein the artificial sequence described in the step S2 is shown as SEQ ID NO. 2;
• the sequence of the prepared Anabaena variabillis intron self-cleaving frame is shown as SEQ ID NO. 3;
• the upstream 18-base sequence described in the step S3 is shown as SEQ ID NO. 4, and the downstream 18-base sequence is shown as SEQ ID NO. 5;
• the sequence of the T7 transcription promoter is shown as SEQ ID NO. 6;
• and the total linear RNA sequence is shown as SEQ ID NO. 7.