Identification and use of effectors and allosteric molecules for the alteration of gene expression

A method of selecting an effector, comprising the steps of:

• a) providing an allosteric control module suitable for use in the modulation of gene expression;
• b) contacting said allosteric control module with one or more effectors;
• and
• c) determining whether or not the interaction of said allosteric control module and an effector results in an alteration of the catalytic activity of said allosteric control module.

A method of determining whether a molecule not previously known to be an effector may be used in combination with an allosteric control module to specifically alter the expression of a gene of interest which comprises:

• (a) contacting a sample which contains a predefined number of eucaryotic cells with the molecule to be tested, each cell comprising a DNA construct encoding,
• i) an allosteric control module, and
• ii) a reporter gene that produces a detectable signal, coupled to, and under the control of, a promoter,
• under conditions wherein the molecule if capable of acting as a modulator of the gene of interest, causes a detectable signal to be produced by the reporter gene;
• (b) quantitatively determining the amount of the signal produced in (a);
• (c) comparing the amount of signal determined in (b) with the amount of signal produced and detected in the absence of any molecule being tested or with the amount of signal produced and detected upon contacting the sample in (a) with other molecules, thereby identifying the test molecule as an effector which causes a change in the amount of detectable signal produced by the reporter gene, and thereby determining whether the test molecule specifically alters expression of the gene of interest.

A process for preparing an allosteric control module for the regulation of gene expression, which comprises:

• (a) screening a random nucleic acid library to select an aptamer binding to a selected effector molecule;
• and
• (b) preparing a nucleic acid comprising a sequence for the selected aptamer and a sequence encoding a protein of interest;
• wherein the gene encoding the protein of interest is not expressed when the effector molecule binds to the sequence for the selected aptamer.