Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-13
2003-04-08
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S018000, C435S091200, C536S024300, C536S024310, C536S024320, C536S024330, C536S023100
Reexamination Certificate
active
06544746
ABSTRACT:
SEQUENCE LISTING
A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f).
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to methods of detecting and quantifying specific proteins, in particular sequence-specific DNA binding proteins, by changes in luminescence signal intensity or changes in color due to the processing of a calorimetric substrate. The invention is used in any application where the detection or quantification of DNA binding activity of a DNA binding protein is desired.
2. Description of the Related Art
The ability to detect and quantify specific protein molecules is of great importance in basic research and in clinical applications. Determination of the level of a specific protein is one of the most useful and important experimental procedures in biomedical research and molecular diagnostics. Cellular levels of specific proteins are commonly used as diagnostic markers for many diseases.
Protein-nucleic acid interactions are an extremely important and physiologically relevant type of macromolecular contact found in the cell. Many proteins that play an important role in regulating many cellular processes possess natural sequence-specific DNA binding activity. These proteins include transcription factors, chromatin remodeling factors and DNA maintenance enzymes. For a review of DNA binding proteins, see Benjamin Lewin,
Genes VII
, Oxford University Press, New York, 2000, which is herein incorporated by reference.
Transcription factors bind to specific cognate DNA elements, which include promoters, enhancers and silencer elements. They may be activators, repressors or both, depending on the cellular context, whose levels are important for regulation of gene expression. Thus, many of these proteins are important in disease development and disease diagnosis. For example, several transcription factors, which when overexpressed or inappropriately expressed, are oncogenes. These oncogenic transcription factors include myc, myb, fos, jun, rel and erb. Another cancer related transcription factor, p53, is involved in development of many cancers (Ko, L. L., and Prives,
C. Genes Dev
. 10, 1054-1072, 1996).
Chromatin remodeling factors are also important for the regulation of gene expression. Generally, regions of highly condensed chromatin, called heterochromatin, contain genes which are not actively transcribed, whereas regions of loose or non-condensed chromatin, called euchromatin, contain genes that are actively transcribed. During cellular differentiation, cancerous transformation and normal physiological homeostasis, chromatin may be remodeled. That is, some chromosomal regions become inaccessible to transcription factors and RNA polymerase, whereas other regions become accessible. Several DNA binding factors are involved in this dynamic process including nucleosome proteins (e.g., histones), histone acetyltransferases, histone deacetylases, DNA methyltransferases, nucleoplasmins, HMG proteins, repressor complex proteins, polycomb-related factors and trithorax-related factors.
DNA maintenance enzymes are DNA binding proteins necessary for the repair of damaged DNA, faithful replication of DNA and exchange of genetic information during recombination. Several types of cancer and other disease syndromes are the result of defective DNA maintenance enzymes. For example, Xeroderma pigmentosum, a horrific genetic disease whereby the sufferer is predisposed to skin cancer, is due to defective nucleotide-excision repair enzymes. Hereditary non-polyposis colorectal cancer is caused in large part by defective mismatch repair enzymes. Some forms of hereditary breast cancers are due to defective homologous recombination enzymes. For a review of genome maintenance systems and their role in cancer, see Hoeijmakers, J. H. J., Nature 411, 366-374, 2001, which is herein incorporated by reference. Thus, there is a significant interest in convenient and accurate methods for detecting, monitoring and/or quantifying DNA binding activity of DNA binding proteins.
The most common approaches taken to detect proteins exhibiting sequence-specific DNA binding activity are gel shift assays and various DNA footprinting assays (Fried, M.G., and Crothers, D. M.
Nucleic Acids Res
. 9, 6505-6525, 1981; Galas, D. J., and Schmitz, A.
Nucleic Acid Res
. 5, 3157-3170, 1978). These methods are laborious and time-consuming procedures, which typically involve the use of dangerous and expensive radioisotopes. Furthermore, these methods are not generally adaptable to high-throughput assay formats. Different fluorescence based methodologies for detecting and studying DNA binding proteins have been developed to overcome the deficiencies of gel shift and DNA footprinting assays.
Detection of molecules by fluorescence has several important advantages compared to alternative detection methods. Fluorescence provides an unmatched sensitivity of detection, as demonstrated by the detection of single molecules using fluorescence (Weiss, S.
Science
283, 1676-1683, 1999). Detection of fluorescence, changes in fluorescence intensity or changes in emission spectra can be easily achieved by the selection of specific wavelengths of excitation and emission. Fluorescence provides a real-time signal allowing real-time monitoring of processes and real-time cellular imaging by microscopy (see Lakowicz, J. R.
Principles of Fluorescence Spectroscopy
, Kluwer Academic/Plenum Press, New York, 1999, which is herein incorporated by reference). Additionally, well-established methods and instrumentation for high-throughput detection of fluorescence signals exist in the art.
Current methods for detecting DNA binding proteins in solution using fluorescence rely on one of the following phenomena: (i) a change in the fluorescence intensity of a fluorochrome (also called a fluorophore or a fluorescent probe or label), which is present either on the protein or on the DNA, as a result of the perturbation of the microenvironment of the probe upon protein-DNA complex formation; (ii) a change of fluorescence polarization of the fluorochrome, which is present either on the protein or on the DNA, as a result of an increase in the molecular size of the protein-DNA complex relative to the unbound DNA or protein molecules; and (iii) resonance energy transfer between one fluorochrome present in DNA and another fluorochrome present in a protein as a result the proximity between DNA and the protein in protein-DNA complex. For a review on methods of detecting fluorescence signal detection, see Hill, J. J., and Royer, C. A.
Methods in Enzymol
. 278, 390-416, 1997, which is herein incorporated by reference.
In the first group of methods (group i), the change in the fluorescence signal is the result of a change in the microenvironment of the fluorescence probe which occurs upon the formation of a protein-DNA complex. Since the generation of the change in the fluorescence signal relies on the unpredictable chance that the formation of a protein-DNA complex will in fact change the environment of the fluorescence probe significantly enough to provide a measurable change in fluorescence, this approach is not generally applicable in that it will work in some cases but not in others. The outcome of such an assay depends on the nature of the protein, DNA sequence, the length of DNA fragment, identity of the fluorescence probe used, and the method of attachment of the fluorescence probe to DNA. Therefore, it is essentially impossible to predict when this method will or will not work since the mechanisms of the changes of fluorescence intensity due to the change in probe environment are not well understood. Examples of the application of this idea to the detection of protein-DNA complexes using fluorochromes attached to the protein or the DNA can be found in the following technical literature, which are
Chakrabarti Arun Kr.
Jones W. Gary
St. Louis University
Thompson & Coburn LLP
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