Recombinant rna production
AssigneesConsejo Superior de Investigaciones Cientificas, Universitat Politècnica de València
InventorsJosé-Antonio DARÒS, Verónica ARAGONÉS, María-Teresa CORDERO
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Abstract
This invention relates to the production of RNA by co-expressing a tRNA ligase and a chimeric RNA molecule comprising a target RNA and a plant viroid scaffold, such as Eggplant latent viroid, in a host cell. This co-expression causes the production of large amounts of the chimeric RNA molecule in the host cells and may be useful for example in the production of RNA aptamers and other RNA molecules.
Claims41
A method of RNA production comprising;
- expressing in a host cell;
- a nucleic acid encoding a chimeric RNA molecule comprising a target RNA and a plant viroid scaffold;
- and,
- a nucleic acid encoding a tRNA ligase.
A method according to any one of the preceding claims comprising isolating and/or purifying the chimeric RNA molecule from the host cell.
A method according to any one of the preceding claims wherein the host cell is a prokaryotic cell.
A method according to any one of the preceding claims wherein the nucleic acids are heterologous to the host cell.
A method according to any one of the preceding claims wherein the target RNA is inserted within the plant viroid scaffold in the chimeric RNA molecule.
A method according to any one of the preceding claims wherein the chimeric RNA molecule produced by the host cells is monomeric.
A method according to any one of the preceding claims wherein the target RNA is 5 to 1000 ribonucleotide bases in length.
A method according to any one of the preceding claims wherein the target RNA is an RNA aptamer.
A method according to any one of the preceding claims wherein the plant viroid scaffold comprises all or part of a plant viroid.
A method according to any one of the preceding claims wherein the plant viroid is an Avsunviroidae viroid.
A method according to any one of the preceding claims wherein the plant viroid is Avocado sunblotch viroid (ASBVd), Peach latent mosaic viroid (PLMVd), Chrysanthemum chlorotic mottle viroid (CChMVd) or Eggplant latent viroid (ELVd
A method according to any one of the preceding claims wherein the plant viroid scaffold comprises a nucleotide sequence having at least 60% identity to one or more of;
- the sequence of bases 1 to 55, 142 to 278 and 311 to 311 of SEQ ID NO:
- 1;
- the sequence of bases 1 to 55, 117 to 213, and 311 to 333 of SEQ ID NO:
- 1;
- and the sequences of any one of SEQ ID NOS:
- 1 to 4.
A method according to any one of the preceding claims wherein the tRNA ligase is a plant tRNA ligase.
A method according to any one of the preceding claims wherein the tRNA ligase comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO:
- 5.
A method according to any one of the preceding claims wherein the tRNA ligase is constitutively expressed in the cell
A method according to any one of the preceding claims wherein the nucleic acids are contained in expression vectors.
A method according to any one of the preceding claims wherein the method comprises introducing the nucleic acids or expression vectors into the host cell.
An isolated nucleic acid encoding a chimeric RNA molecule chimeric RNA molecule comprising a target RNA and a plant viroid scaffold.
A vector comprising a nucleic acid sequence encoding a plant viroid scaffold, said nucleic acid sequence comprising a cloning site for insertion of a heterologous nucleotide sequence encoding a target RNA into the nucleic acid sequence.
A set of vectors comprising a first vector comprising an isolated nucleic acid encoding a chimeric RNA molecule and a second vector comprising a nucleic acid encoding a tRNA ligase.
A host cell that expresses;
- a chimeric RNA molecule comprising a target RNA and a plant viroid scaffold, and;
- a tRNA ligase.
A system for the production of RNA comprising;
- a host cell,
- a nucleic acid encoding a chimeric RNA molecule comprising a target RNA and a plant viroid scaffold, and
- a nucleic acid encoding a tRNA ligase.
A kit for the production of RNA comprising;
- a nucleic acid encoding a chimeric RNA molecule comprising a target RNA and a plant viroid scaffold;
- or
- a nucleic acid encoding a plant viroid scaffold,
- said nucleic acid comprising a cloning site for insertion of a target RNA into the plant viroid scaffold.
A kit according to claim 40 further comprising a nucleic acid encoding a tRNA ligase.
Dependencies are inferred from the claim text.