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

[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.[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.[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 miniaturized formats has led to the development of high de...

AI Technical Summary

Benefits of technology

[0081] Optical probes for detecting protein tyro sine phosphatase activity according to the present invention are designed by incorporating the desired phosphorylation motif into a peptide, for example like those in (Table 2), or other such motifs developed now or in the future, and either enzymatically or chemically phosphorylating the appropriate amino acid. Dephosphorylation of the tyrosine residue in such optical probes by a tyrosine directed protein phosphatase activity modulates the rate of optical probe hydrolysis by chymotrypsin compared to the phosphorylated optical probe.

[0172] For example arrays of kinase or phosphatase optical probes may be used to rapidly profile the selectivity of a test chemical with respect to its ability to inhibit related kinases or phosphatases. Such arrays may be located within a microtiter plate, or as a printed array, for example as disclosed in U.S. Pat. Nos., 4,216,245 issued Aug. 5, 1980 to Johnson, 5,721,435 issued Feb. 24, 1998 to Troll, and 5,601,980 issued Feb. 11, 1997 issued to Gordon et al. Such a system provides the ability to rapidly profile large numbers of kinases or phosphatases in the presence or absence of a test chemical in order to profile in a simple, miniaturized high throughput format the selectivity of a candidate modulator.

Problems solved by technology

These estimates are imprecise due to the large number of sequence database entries that are different splice forms or duplicates of the same PTP sequence.

Images