Soluble rna polymerase protein and methods for the use thereof

An isolated polypeptide, characterized in that:

• (i) said polypeptide has sufficient RNA polymerase activity and is capable, when contacted with a nucleic acid template, of producing short complementary RNA copies of said template, which RNA copies are scattered throughout the entire template length and, optionally, capable of producing template-length complementary RNA copies;
• (ii) said polypeptide has enhanced solubility resulting in at least 3 times higher yield of the active polymerase, than in the case of polypeptide comprising the amino acid sequence of SEQ ID NO:
• 2 or encoded by the nucleic acid sequence comprising SEQ ID NO:
• 1;
• and
• (iii) said polypeptide is encoded by a nucleic acid sequence selected from the group of:
• (a) a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:
• 3;
• (b) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:
• 4;
• (c) a nucleic acid sequence, which differs from the nucleic acid sequence of (a) or (b) due to degeneracy of the genetic code;
• (d) a nucleic acid sequence hybridizing to the nucleic acid sequence of (a), (b) and/or (c) under stringent conditions;
• (e) a nucleic acid sequence encoding a polypeptide comprising the amino acids 709 to 1402 of SEQ ID NO:4 or any sequence longer than that up to the sequence comprising the amino acids 2 to 1402 of SEQ ID NO:
• 2;
• and
• (f) a nucleic acid sequence encoding an amino acid sequence, which shows at least 50% identity to the amino acid sequence SEQ ID NO:
• 2.

A method for studying nucleic acid secondary structure, preferably RNA secondary structure comprising the steps of:

• (a) providing nucleic acid target molecule, preferably RNA target molecule;
• (b) contacting said target molecule with an isolated RNA polymerase capable of producing short complementary RNA copies of the nucleic acid target as template, which RNA copies are scattered throughout the ssRNA regions of the template length and, optionally, template-length complementary RNA copies under conditions sufficient for RNA synthesis in a mixture additionally comprising radioactively or chemically labeled nucleotides, so that single-stranded elements of said target RNA are copied by the polymerase;
• (c) recovering and optionally purifying the newly produced labeled nucleic acid species from the reaction mixture;
• (d) using said labeled nucleic acid species as probes for microarray chip that comprises nucleic acid fragments of said target molecule;
• (e) interpreting data from the microarray analysis to deduce which parts of the target molecule are single-stranded;
• and optionally
• (f) building a model for the secondary or tertiary structure of the target molecule.

A method for studying nucleic acid-protein interactions, preferably RNA-protein interactions comprising the steps of:

• (a) providing a nucleic acid target and nucleic acid binding protein, preferably an RNA target and an RNA-binding protein;
• (b) contacting said target and the solution of said protein in an experimental mixture under conditions sufficient for target-protein interaction, and in a separate vessel, contacting said target with a control solution that lacks said protein.
• (c) contacting said experimental and control mixtures with an isolated RNA polymerase capable of producing short complementary RNA copies of said target as template, which RNA copies are scattered throughout the template length not covered by protein and, optionally, template-length complementary RNA copies under conditions sufficient for RNA synthesis;
• (d) recovering and optionally purifying the newly produced labeled nucleic acid species from both reaction mixtures;
• (e) using the two sets of labeled nucleic acid species as probes for two identical microarray chips that comprise nucleic acid fragments of the target;
• (f) interpreting data from the two microarray analyses to deduce which parts of the target molecule are accessible for the RNA synthesis;
• (g) comparing the two data sets to determine the difference between target in experimental and control mixtures;
• and optionally
• (h) interpreting the difference between the two data sets as a model for nucleic acid-protein interactions

A method for producing RNA trigger molecules to induce RNA interference in vivo or in vitro, comprising the steps of:

• (a) providing RNA or DNA template;
• (b) contacting said RNA or DNA template with an isolated RNA polymerase capable of producing short complementary RNA copies of said template, which are scattered throughout the entire template length and, optionally, template-length complementary RNA copies under conditions sufficient for RNA synthesis in a mixture comprising:
• nucleic acid template, said RNA polymerase, nucleoside triphosphates, and optionally pH buffer, ammonium acetate, PEG, Mg2+ ions, Mn2+ ions and/or non-ionic detergent;
• and
• (c) incubating the reaction mixture at temperature sufficient for RNA synthesis.