Use of inhibitors in reporter assays

Details Use of inhibitors in reporter assays Use of inhibitors in reporter assays

1998-04-28

2001-09-04

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S018000

Reexamination Certificate

active

06284461

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to compositions that inhibit reporter enzyme activity within a cell during enzymatic assays. These compositions can be used in methods that detect the activity of the reporter enzyme.
INTRODUCTION
A reporter gene assay measures the activity of a gene's promoter. It takes advantage of molecular biology techniques, which allow one to put heterologous genes under the control of any promoter and introduce the construct into the genome of a mammalian cell (see, Gorman et al., Mol. Cell Biol. 2:1044-1051 (1982); Alam et al., Anal. Biochem. 188:245-254 (1990)). Activation of the promoter induces the expression of the reporter gene, as well as, or instead of, the endogenous gene. By design, the reporter gene codes for a reporter protein that can easily be detected and measured. Commonly, the reporter protein is a reporter enzyme activity that converts a commercially available substrate into a product. This conversion can be conveniently followed by direct optical measurement and may allow for the quantification of the amount of reporter enzyme activity produced.
Reporter genes are commercially available on a variety of plasmids for the study of gene regulation in a large variety of organisms (see, Alam et al., supra, 1990). Promoters of interest can be inserted into multiple cloning sites provided for this purpose in front of the reporter gene on a plasmid (see, Rosenthal, Methods Enzymol. 152:704-720 (1987); Shiau et al., Gene 67:295-299 (1988)). Standard techniques are used to introduce these reporter genes into a cell type or whole organism (such as described in Sambrook et al. Molecular cloning, Cold Spring Harbor Laboratory Press (1989)). Resistance markers provided on a plasmid can then be used to select for successfully transfected cells.
Ease of use and the large signal amplification make this technique increasingly popular in the study of gene regulation. Every step in the cascade of DNA→RNA→Enzyme→Product amplifies the next one in the sequence. Generally, the further down in the cascade one measures, the more signal one obtains.
In a preferred reporter gene assay, the reporter gene, associated with or without a promoter, is transfected into cells, either transiently or stably. Activation of the reporter gene by, for examiner, the activation of a receptor, leads to a change in reporter enzyme activity levels via transcriptional and translational events. The amount of reporter activity enzyme present can be measured via its enzymatic action on a substrate. The substrate can be a small uncharged molecule that, when added to the extracellular solution, can penetrate the plasma membrane to encounter the reporter enzyme activity. A charged molecule can also be employed, but the charges can be masked by groups that will be cleaved by endogenous cellular enzymes (e.g., esters cleaved by cytoplasmic esterases).
Substrates that exhibit changes in their fluorescence spectra upon interaction with a reporter enzyme activity are particularly desirable due the sensitivity of assays that use such substrates. In some assays, the fluorogenic substrate is converted to a fluorescent product. Alternatively, the fluorescent substrate changes fluorescence properties upon conversion by the reporter enzyme activity. The product of such reporter enzyme activity should be very fluorescent to obtain maximal signal and very polar, to preferably stay inside the cell.
To achieve the high sensitivity in a reporter enzyme activity assay one has to maximize the amount of signal generated by a single reporter enzyme. An optimal reporter enzyme activity will convert 10
5
substrate molecules per second under saturating conditions (see, Stryer, Introduction to enzymes. In: Biochemistry, New York, W. H. Freeman and Co. (1981), pp. 103 to 134). Beta-lactamases will cleave about 10
3
molecules of their preferred substrates per second (Chang et al., Proc. Natl. Acad. Sci. USA 87:2823-2827 (1990)). Using a fluorogenic substrate one can obtain up to 10
6
photons per fluorescent product produced, depending on the type of dye used, when exciting with light of the appropriate wavelength. The signal terminates with the bleaching of the fluorophore (Tsien et al., Handbook of Biological Confocal Microscopy, ed: Pawley, J. B. Plenum Publishing Co (1990), pp. 169-178). These numbers illustrate the theoretical magnitude of signal obtainable in this type of measurement. In practice, a minute fraction of the photons generated will be detected, but this holds true for fluorescence, bioluminescence or chemiluminescence. A good fluorogenic substrate for a reporter enzyme activity should have a high turnover at the enzyme in addition to good optical properties such as high extinction and high fluorescence.
However, reporter genes can be expressed at low background levels even when the assay is designed not to have the reporter gene expressed. Under these circumstances, low levels of background reporter enzyme activity exist. Background reporter enzyme activity can cause the signal-to-noise ratio of the assay to suffer because de novo reporter enzyme activity (signal) is affected by background reporter enzyme activity (noise). The present invention solves these problems, and provides related benefits as well.
SUMMARY OF THE INVENTION
One aspect of the present invention is a method for increasing the signal-to-noise ratio of a reporter enzyme activity assay by contacting a sample comprising a membrane compartment containing a reporter enzyme activity with an inhibitor of the reporter enzyme activity, contacting the sample with a substrate for the reporter enzyme activity, and determining the activity of the reporter enzyme activity. Preferably, the inhibitor of the reporter enzyme activity is an irreversible inhibitor of the reporter enzyme and is membrane permeant. This method is particularly useful in intracellular assays where the inhibitor of the reporter enzyme activity is used to reduce background reporter enzyme activity.
Another aspect of the present invention is a homogeneous method for increasing the signal-to-noise ratio of a reporter enzyme activity assay by contacting a sample comprising a membrane compartment containing a reporter enzyme activity with an inhibitor of the reporter enzyme activity, contacting the sample with a substrate for the reporter enzyme activity, contacting the sample with a composition that degrades the inhibitor of the reporter enzyme activity, and determining the activity of the reporter enzyme activity.
A further aspect of the present invention is a method for increasing the dynamic range of a reporter enzyme activity assay by contacting a sample comprising a membrane compartment containing a reporter enzyme activity with an inhibitor of a reporter enzyme activity, contacting the sample with a substrate for the reporter enzyme activity, and determining reporter enzyme activity. This method is particularly useful for increasing the detection range of a reporter enzyme activity assay that has high background reporter enzyme activity and low substrate concentration.
Another aspect of the present invention is a method for extending the useful loading time or assay measurement time of a reporter enzyme activity assay by contacting a sample comprising a membrane compartment containing a reporter enzyme activity with an inhibitor of a reporter enzyme activity, contacting the sample with a substrate for the reporter enzyme activity, and determining reporter enzyme activity. This method is particularly useful for increasing the detection range of an assay that has high background reporter enzyme activity and low substrate concentration.
A further aspect of the present invention is a method for profiling the level of a reporter enzyme activity by contacting a sample comprising a membrane compartment containing a reporter enzyme activity with an inhibitor of a reporter enzyme activity, contacting the sample with a substrate for the reporter enzyme activity, and determining reporter enzyme activity.
Another aspect of the presen

Affiliated with

Also associated with

Rating

Use of inhibitors in reporter assays does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Use of inhibitors in reporter assays, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of inhibitors in reporter assays will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2523805