Method for adding cap structures to RNA using immobilized enzymes
The present invention relates to an immobilized capping enzyme, preferably an immobilized Vaccinia virus capping enzyme. Furthermore, the present invention relates to an immobilized cap-specific nucleoside 2′-O-methyltransferase, preferably an immobilized Vaccinia virus cap-specific nucleoside 2′-O-methyltransferase. Moreover, the present invention relates to a method for immobilizing said enzymes and to a method of using said enzymes for the addition of a 5′-cap structure to RNAs. Moreover, the present invention relates to an enzyme reactor for performing the capping reaction using said immobilized enzymes and the subsequent separation of the 5′-capped RNA product. In addition, the present invention relates to a kit comprising the capping enzyme and/or the cap-specific nucleoside 2′-O-methyltransferase.
1. A method for producing capped ribonucleic acid (RNA) molecules, comprising:
i) contacting a viral capping enzyme being immobilized onto a solid support with RNA molecules, a nucleotide, and a methyl donor under conditions suitable for forming a 5′-cap0 structure, wherein the viral capping enzyme comprises a heterodimer of a catalytic polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 1 having RNA triphosphatase (TPase), guanylyltransferase (GTase) and methyltransferase (MTase) activity, and a regulatory polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the viral capping enzyme is immobilized onto the solid support by covalent binding, wherein the covalent binding is a disulfide bridge or a thioether bond; and
ii) isolating the capped RNA molecules by filtration, chromatography, or centrifugation.
2. The method of claim 1 , wherein the viral capping enzyme is immobilized by covalent binding to a thiol-activated solid support, haloacetyl functionalized solid support, pyridyl disulfide-functionalized solid support, maleimide-activated solid support, epoxy-activated solid support, or a mixture thereof.
3. The method of claim 1 , wherein the viral capping enzyme is immobilized via a thiol group of at least one cysteine residue and wherein at least one cysteine residue of the viral capping enzyme is substituted with a different amino acid.
4. The method of claim 1 , wherein the solid support comprises a material selected from the group consisting of agarose, silica, magnetic beads, methacrylate beads, and nanoparticles.
5. The method of claim 1 , wherein the viral capping enzyme comprises an amino acid sequence at least 95% identical to SEQ ID NO:1.
6. The method of claim 1 , wherein the viral capping enzyme comprises at least one newly introduced cysteine residue compared to a wild-type viral capping enzyme.
7. The method of claim 6 , wherein the newly introduced cysteine residue is attached to the C terminus of the viral capping enzyme.
8. The method of claim 7 , wherein the newly introduced cysteine residue is attached to the C terminus of the viral capping enzyme via a linker.
9. The method of claim 1 , wherein the viral capping enzyme comprises only one cysteine residue or is mutated to comprise only one cysteine residue.
10. The method according to claim 1 , further comprising a step of iii) converting the cap0 structure into a cap1 structure by contacting the RNA comprising a 5′-cap0 structure with a cap-specific nucleoside 2′-O-methyltransferase and a methyl donor.
11. The method according to claim 10 , wherein the cap-specific nucleoside 2′-O-methyltransferase is immobilized onto a solid support.
12. The method according to claim 11 , wherein the cap-specific nucleoside 2′-O-methyltransferase comprises the amino acid sequence according to any one of SEQ ID NOs: 3, 11, 12, 128-160, and 328-360 or a functional variant thereof having at least 90% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 3, 11, 12, 128-160, and 328-360.
13. The method of claim 8 , wherein the linker is selected from the group consisting of SEQ ID NOs: 15-39.
14. The method of claim 1 , wherein the methyl donor is S-adenosylmethionine.
15. The method of claim 1 , further comprising:
iii) washing the viral capping enzyme immobilized onto the solid support;
iv) contacting the viral capping enzyme immobilized onto the solid support with RNA molecules, a nucleotide, and a methyl donor under conditions suitable for forming a 5′-cap0 structure; and
v) isolating the capped RNA molecules by filtration, chromatography, or centrifugation.