Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis

a technology of molecular interactions and sensitivity adjustment, applied in the field of adjustment of sensitivity, genetic molecular interaction systems, including proteinprotein interaction systems for detection and analysis, can solve the problems of incomplete inability or prolonged time required to elucidate important biologically relevant interactions, failure to detect relevant biological interactions, and current methods of selection are limited to "all or nothing"

Inactive Publication Date: 2004-09-16
HYBRIGEN
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  • Description
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AI Technical Summary

Problems solved by technology

This creates problems related to both the detection of numerous biologically irrelevant interactions, as well as a failure to detect relevant biological interactions.
The consequences of this problem may be either a complete inability or prolonged time required to elucidate important biologically relevant interactions, cellular pathways, and potentially related modulatory agents and drugs.
The current methods of selection are limited to an "all or nothing" auxotrophic nutrient, antibiotic selection or other means of affecting survival (Fields and Song, 1989; Gyuris et al., 1993; Bai and Elledge, 1997).
However, the introduction of genetic selection introduced a new and severely limiting aspect to the in vivo genetic molecular detection systems.
Systems are designed to exclude these interactions because, if systems are too sensitive, they will detect too much background.
However, if the system is not sensitive at all, important interactions will be missed.
This control is useful and exerts its effects by modulating reporter activity, but it does not provide for the continuous adjustability of the sensitivity of a two-hybrid protein interaction system.
Thus, the Gyuris system further demonstrates the limitation of the prior art: it is either on or off, above or below the same detection threshold set by the reporters chosen when the system was constructed.
Thus, the third molecule will interfere with the ability of the bait and prey to interact.

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  • Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis
  • Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis

Examples

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example 1

Interaction Hybrid System Adjusted to Give Variable Quantitative Reporter Output without Modifying the Reporter System

[0054] As noted above, the Brent lab has shown that a given set of two-hybrid protein interactors yield a uniform quantitative reporter output directly proportional to their strength of interaction (Estojak et al., 1995). Utilizing the novel adjustable yeast interaction hybrid system (IHS), introduced and described as a more preferred embodiment in the paragraphs above, three sets of proteins pairs previously demonstrated to interact in a two-hybrid system are demonstrated to give variable levels of reporter output when expressed at different relative concentrations. The level of expression of the first hybrid protein containing the bait is proportional to the concentration of estradiol, and the level of the second hybrid protein containing the prey derived from a library is proportional to dexamethasone concentration (Kralli et al., 1995; Gaido et al., 1997 (FIGS. 4...

example 2

Interaction Hybrid System Adjusted to Low Sensitivity for Promiscuous Bait

[0058] When low sensitivity is desired, as in the screening of the mammalian baits IRAK kinase, its Drosophila homolog Pelle kinase, or its plant kinase homologues, high expression of the bait hybrid protein is achieved using 10.sup.-9 M estradiol (for example, as in Table 2, below). Low relative expression of the library / prey hybrid proteins is achieved only at 10.sup.-7 M dexamethasone (for example, as in Table 2, below). The excess of bait hybrid protein decreases the background expression of weak and irrelevant interactions common to these kinases (refer to FIG. 6). This enables the successful selection, screening and discovery of their respective interactions with activators and scaffolds from a random cDNA library. By muting the background signal, many irrelevant interactions are reduced or eliminated which otherwise would interfere with timely and cost-effective analysis of these screening results.

2TABL...

example 3

Interaction Hybrid System Adjusted to High Sensitivity for Poor Quality Bait

[0060] If high sensitivity is desired, as in the screening of the mammalian baits Interleukin-1 receptor, or its Drosophila homolog Toll, or its plant or mammalian homologues, minimal expression of the bait hybrid protein is achieved using 10.sup.-9M estradiol. High relative expression of the library / prey hybrid protein is achieved using 10.sup.-5M dexamethasone. The excess of library / prey hybrid protein apparently drives the weak interaction equilibrium toward forming heterodimers; more of the limiting bait hybrid proteins are occupied at a given time by interaction with the abundant library / prey hybrid proteins (FIG. 6). The system thereby detects the weak (high Kd) interaction of these receptors with their cytoskeletal adapter and cytoplasmic proteins.

3TABLE 3 High sensitivity Screen with toll Receptor intracellular domain as bait Total False Bait Estradiol Dexamethasone Positives Positives Toll 10.sup.-9...

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Abstract

A method for detecting interactions between first and second interacting molecules a variable sensitivity. This variable sensitivity may be obtained by providing for the overexpression of either a bait hybrid protein containing a DNA binding domain (desensitization) or a prey hybrid protein containing the DNA activation domain for a reporter gene (enhanced sensitivity). The use of exogenous activators of one or the other according to the needs of a particular system is readily accomplished.

Description

RELATED PATENT APPLICATION[0001] This Patent Application is a Continuation of U.S. patent application Ser. No. 09 / 680,738 filed on Oct. 6, 2000, which claims priority to U.S. Provisional Application Serial No. 60 / 158,079 filed on Oct. 7, 1999 and incorporated by reference herein.BACKGROUND OF THE PRESENT INVENTION[0002] Genetically-based interaction systems are commonly used in scientific research and in commercial and therapeutic applications derived from that research. Current genetically-based interaction systems are severely limited by a fixed level of interaction sensitivity which is either completely "on" or completely "off" (Fields and Song, 1989; Bartel et al., 1993; Gyuris et al., 1993; Mendelsohn and Brent, 1994; Phizicky and Fields, 1995; Bai and Elledge, 1997; Brachmann and Boeke, 1997; Finley and Brent, 1997; Young, 1998). This creates problems related to both the detection of numerous biologically irrelevant interactions, as well as a failure to detect relevant biologi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/10C12N15/74C12Q1/00C12Q1/02G01N33/74
CPCC12N15/1055G01N33/743C12Q1/02
Inventor EDWARDS, DAVID N.LEON, ARLENERANNEY, DAVID F.
Owner HYBRIGEN
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