| CPC C12N 15/11 (2013.01) [A61K 48/0058 (2013.01); G16B 30/10 (2019.02); C12N 2310/532 (2013.01)] | 14 Claims |
|
1. A recombinant nucleic acid molecule for preparing a circular RNA in vitro, the recombinant nucleic acid molecule being selected from any one of (i) to (ii):
(i) the recombinant nucleic acid molecule comprising elements arranged in the following order in the 5′ to 3′ direction:
an intron fragment II, a truncated fragment II of a coding element, a translation initiation element, a truncated fragment I of a coding element, and an intron fragment I,
wherein the truncated fragment II is directly joined with intron fragment II without any intervening sequences, and truncated fragment I is directly joined with intron fragment I without any intervening sequences,
wherein a 3′ end of the truncated fragment I of the coding element comprises a ribozyme recognition site I that consists of a first predetermined number of nucleotides located at the 3′ end of the truncated fragment I of the coding element,
a 5′ end of the truncated fragment II of the coding element comprises a ribozyme recognition site II that consists of a second predetermined number of nucleotides located at the 5′ end of the truncated fragment II of the coding element,
wherein the nucleotide sequence of the truncated fragment I of the coding element and the nucleotide sequence of the truncated fragment II of the coding element form a coding element sequence encoding at least one target polypeptide in the 5′ to 3′ direction; the nucleotide sequence of the truncated fragment I of the coding element corresponds to a partial sequence close to the 5′ direction in the coding element sequence; and the nucleotide sequence of the truncated fragment II of the coding element corresponds to a remaining partial sequence close to the 3′ direction in the coding element sequence,
wherein the nucleotide sequence of the intron fragment I and the nucleotide sequence of the intron fragment II form an intron sequence in the 5′ to 3′ direction; the nucleotide sequence of the intron fragment I comprises a partial sequence close to the 5′ direction in the intron sequence; and the nucleotide sequence of the intron fragment II comprises a remaining partial sequence close to the 3′ direction in the intron sequence, wherein the coding element comprises a coding sequence that is generated by codon-optimization matching the coding sequence to a natural exon sequence ligated to the 5′ end of the intron fragment I and/or a natural exon sequence ligated to the 3′ end of the intron fragment II, wherein the coding sequence that is generated by codon-optimization is located within the ribozyme recognition site I and/or the ribozyme recognition site II, wherein a sum of the first predetermined number and the second predetermined number is not equal to 3y, wherein y≥1 and y is an integer; or
(ii) the recombinant nucleic acid molecule comprising elements arranged in the following order in the 5′ to 3′ direction:
an intron fragment III, a truncated fragment IV of a coding element, a translation initiation element, a truncated fragment III of a coding element, and an intron fragment IV,
wherein the truncated fragment IV is directly joined with intron fragment III without any intervening sequences, and truncated fragment III is directly joined with intron fragment IV without any intervening sequences,
wherein a 3′ end of the truncated fragment III of the coding element comprises a ribozyme recognition site IV that consists of a second predetermined number of nucleotides located at the 3′ end of the truncated fragment III of coding element,
a 5′ end of the truncated fragment IV of the coding element comprises a ribozyme recognition site III that consists of a first predetermined number of nucleotides located at the 5′ end of the truncated fragment IV of coding element,
wherein the nucleotide sequence of the truncated fragment III of the coding element and the nucleotide sequence of the truncated fragment IV of the coding element form a coding element sequence encoding at least one target polypeptide in the 5′ to 3′ direction; the nucleotide sequence of the truncated fragment III of the coding element corresponds to a partial sequence close to the 5′ direction in the coding element sequence; and the nucleotide sequence of the truncated fragment IV of the coding element corresponds to a remaining partial sequence close to the 3′ direction in the coding element sequence,
wherein the sequence of the intron fragment III is a reverse sequence or a reverse complementary sequence of the nucleotide sequence of the intron fragment I, and the sequence of intron fragment IV is a reverse sequence or a reverse complementary sequence of the nucleotide sequence of the intron fragment II; the sequence of the ribozyme recognition site III is a reverse sequence of the nucleotide sequence of the ribozyme recognition site I, and the sequence of the ribozyme recognition site IV is a reverse sequence of the nucleotide sequence of the ribozyme recognition site II,
wherein the coding element comprises a coding sequence that is generated by codon-optimization matching the coding sequence to a natural exon sequence ligated to the 5′ end of the intron fragment I and/or a natural exon sequence ligated to the 3′ end of the intron fragment II,
wherein the coding sequence that is generated by codon-optimization is located within the ribozyme recognition site III and/or the ribozyme recognition site IV, wherein a sum of the first predetermined number and the second predetermined number is not equal to 3y, wherein y≥1 and y is an integer.
|