Optical probes and assays

Abstract

This invention provides an optical probe useful as an optical probe or sensor of post translational type modifications, such as phosphorylation. The invention comprises a polypeptide moiety, which contains a recognition motif for a post translational type activity and a protease site, which is coupled to a probe moiety. Modification of the polypeptide, by the post translational type activity, results in a modulation of the rate at which a protease cleaves the polypeptide which is sensed by a measurable change in at least one optical property of the optical probe upon cleavage. The present invention also includes a recombinant nucleic acid molecule that encodes an optical probe and a vector and host cell or library of cells that include the recombinant nucleic acid molecule. The optical probe can be used in methods to determine whether a sample, including a cell or a sample from an organism, contains a post-translational type modification activity. Such methods can also be used to determine whether a test chemical modulates the activity of a modifying activity, and thus can be used to identify therapeutic compositions. The identification of such therapeutic compositions can be automated using a system that includes an optical probe.

Description

RELATED APPLICATIONS[0001]This application is a continuation of application U.S. Ser. No. 09 / 306,542, filed May 5, 1999, now allowed, the entire contents of which are hereby incorporated by reference herein.FIELD OF THE INVENTION[0002]The present invention relates generally to the fields of chemistry and biology. More particularly, the present invention relates to optical probes for post translational type modification activities, such as phosphorylation, and methods for their use.INTRODUCTION[0003]Systems and methods for rapidly identifying chemicals with biological activity in samples, especially small liquid samples, is of particular relevance to the agrochemical and pharmaceutical fields. Various strategies are typically used to reduce processing times and associated costs of screening large numbers of chemical entities, including simplified assay design, automation, robotics and miniaturization of sample size. The advent of high throughput analysis and increasing use of miniatu...

AI Technical Summary

Problems solved by technology

Higher density plates enable faster analysis and handling of large sample or chemical libraries, such as in automated screening systems, but place considerable constraints on the assays that can be successfully employed within them.

In spite of their great potential importance however, there are few existing methods of measuring such activities that are homogenous, non radioactive and sensitive enough to accurately and reproducible work in high throughput, or ultra high throughput screening systems.

However, current methods of measuring protein kinases, have many disadvantages, which prevents or hampers the ability to rapidly screen for drugs using miniaturized automated formats of many thousands of compounds.

Furthermore, this kinase assay approach requires purification of the target protein, and final radioactive incorporation into target proteins is usually very low giving the assay poor sensitivity.

In high throughput screening operations, this approach requires large amounts of radioactivity, which can be an environmental and health hazard.

Alternative kinase assay methods, such as those based on phosphorylation-specific antibodies using ELISA-type approaches, involve the difficulty of producing antibodies that distinguish between phosphorylated and non-phosphorylated proteins.

Furthermore, most kinase measurements have the requirement for cell lysis, multiple incubations, and washing stages are time consuming, complex to automate, and potentially susceptible to artifacts.

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