Cyclic RNA and protein production method
The present invention provides a cyclic RNA preferable for carrying out rotary protein translation in which translation domains other than that of the target protein are sufficiently short and translation efficiency is high, and a method for producing protein that uses this cyclic RNA as template. More specifically, the present invention provides a cyclic RNA that encodes a protein, has a full-length number of bases that is equal to or greater than 102 and is a multiple of 3, has at least one start codon, does not have a stop codon in the same reading frame as the start codon, and does not contain an internal ribosome entry site (IRES). In addition, the present invention provides a method for producing protein in a eukaryotic cell expression system that consists of using the aforementioned cyclic RNA as template and expressing a protein encoded by that cyclic RNA.
1. A method for producing protein in eukaryotic cells or in a eukaryotic cell expression system, comprising:
introducing a cyclic RNA into eukaryotic cells, or adding the cyclic RNA to a eukaryotic cell-free expression system; and
expressing a protein encoded by the cyclic RNA using the cyclic RNA in the eukaryotic cells or eukaryotic cell-free expression system,
wherein the cyclic RNA encodes a protein, has a full-length number of bases that is equal to or greater than 102, and is a multiple of 3, has at least one start codon, does not have a stop codon in the same reading frame as the start codon, and does not contain an internal ribosome entry site (IRES).
2. The method for producing protein according to claim 1 , wherein the eukaryotic cells are mammalian cells, and the eukaryotic cell-free expression system is a mammalian cell-free expression system.
3. A method for producing protein in a prokaryotic cell or in a prokaryotic cell expression system, comprising:
introducing a cyclic RNA into prokaryotic cells, or adding the cyclic RNA to a prokaryotic cell-free expression system; and
expressing a protein encoded by the cyclic RNA using the cyclic RNA in the prokaryotic cells or the prokaryotic cell-free expression system,
wherein the cyclic RNA encodes a protein, has a full-length number of bases that is from 102 to 561 and is a multiple of 3, has at least one ribosome binding site recognized by ribosomes derived from prokaryotic cells, has a plurality of start codons, with only one of the plurality of start codons within 1 to 20 bases downstream from the ribosome binding site, and does not have a stop codon in the same reading frame as the start codon.
4. The method according to claim 3 , wherein the cyclic RNA does not contain an internal ribosome entry site (IRES).
5. The method according to claim 3 , wherein the cyclic RNA has an Shine-Dalgarno (SD) sequence as the ribosome binding site.
6. The method according to claim 3 , wherein the cyclic RNA has an open reading frame comprising the start codon within 1 to 20 bases downstream from the ribosome binding site, and has no other reading frame which comprises a start codon.
7. The method according to claim 3 , wherein the cyclic RNA comprises a stop codon in a different reading frame from the start codon.
8. The method according to claim 3 , wherein the protein comprises long chain repeated regions of peptide sequences defined by the cyclic RNA.
9. The method according to claim 3 , wherein the cyclic RNA comprises a domain encoding a protease cleavage site.
10. The method according to claim 1 , wherein the cyclic RNA has a Kozak sequence upstream from the start codon.
11. The method according to claim 1 , wherein the cyclic RNA has an open reading frame comprising the start codon, and has no other reading frame which comprises a start codon.
12. The method according to claim 1 , wherein the cyclic RNA comprises a stop codon in a different reading frame from the start codon.
13. The method according to claim 1 , wherein the protein comprises long chain repeated regions of peptide sequences defined by the cyclic RNA.
14. The method according to claim 1 , wherein the cyclic RNA comprises a domain encoding a protease cleavage site.
15. The method according to claim 1 , wherein the cyclic RNA comprises a full-length number of bases that is less than or equal to 561 bases.