Yale University

Innovir Laboratories, Inc.

Timothy W Nilsen

Thomas J Kindt

Two methodologies are provided: the first provides a means for rapidly and efficiently identifying essential and functional genes; and the second provides a means for obtaining biologically active nucleic molecules (ribozymes, EGSs, and antisense,) which can be used to inactivate functional genes. In the first method, a library of EGSs is prepared based on all possible known compositions. In a preferred embodiment, the EGSs are twelve or thirteen-mers for targeting bacterial RNAse to cleave a substrate. This library is added to the cells containing the genes to be screened, for example,,Those cells in which the EGS causes a loss of viability, or other phenotype, are identified. The EGS(s) responsible for the loss of viability are analyzed, and the resulting sequence information used to identify the gene within the known genomic sequences. In the second method, nucleotide molecules with optimal biological activity, for example, directing cleavage of a gene of interest by RNase P, are rapidly identified through the use of a vector including two reporter genes, the first in phase with the gene of interest, and the second as a control to verify that the vector is present in a cell or to aid in selection of cells containing the vector. Those cells where the gene of interest is cleaved by the functional oligonucleotide molecule can then be identified by reference to reporter gene 1. The responsible functional oligonucleotide molecules is then isolated and characterized.

Method for identifying essential or functional genes

Vectors and a method for the identification of affector RNA molecules, such as ribozymes, external guide sequences, anti-sense RNA, and triple helix-forming RNA, that inhibit expression of target RNA molecules are disclosed. The method identifies functional affector RNA molecules by screening or selecting for those RNA molecules that inhibit expression of a fusion transcript, which includes the sequence of an RNA molecule of interest, from a library of potential affector RNA molecules. The vectors include a reporter gene encoding the fusion transcript including the RNA molecule of interest and RNA encoding the reporter protein. The vectors also include a second reporter gene encoding a second reporter protein. Expression of the second reporter protein can be used both to detect transformation or transfection of the vector into cells and as a control for effects on the expression of the first reporter protein that are not due to inhibition of expression of the RNA molecule of interest. The vector also encodes an affector RNA molecule targeted to the RNA of interest. A key advantage of the disclosed method and vectors is the assessment of inhibition of expression of an RNA of interest in an in vivo setting which will be the same or similar to the setting where identified affector molecules will be used. Another advantage of the disclosed method is that all, or a substantial number of the accessible sites in the RNA of interest can be determined in one assay. Also disclosed are affector oligomers based on affector RNA molecules identified as inhibiting the expression of an RNA of interest. The disclosed method also allows direct comparison of the inhibitory activities of different affector RNA molecules directed to different target sites.

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Russell, OH, United States of America

Timothy W Nilsen