Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-04
2002-11-05
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S069100
Reexamination Certificate
active
06475733
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to biosensors that are useful in detecting chemical compounds of interest. Such biosensors are receptors such as G-protein coupled receptors, tyrosine kinase receptors, and/or ion channels, selected via mutagenesis. More specifically, the biosensors of the invention are highly specific and highly sensitive in detecting low levels of the chemical compounds of interest.
BACKGROUND OF THE INVENTION
Current methods for detecting chemical compounds of interest that offer the greatest sensitivities, such as mass spectrometry and chromatography, require cumbersome fragile equipment that need regular maintenance and calibration. These conventional assays are also limited by the specificity of the methods, the possibility of false positive detection of structurally-related compounds and the speed of chemical detection.
Natural biosensors are intricate biological systems that have evolved over billions of years to discriminate between chemical structures, to sense small numbers of molecules and to register a response in less than a second through amplification of the signal within the cell. These natural biosensors work through protein receptors.
The most common example of such highly-discriminating sensors are the olfactory receptors which are members of the G-protein coupled receptor (GPCR) superfamily (Buck, L. and Axel, R. (1991) Cell 65:175-87). The nose is the most sophisticated chemical sensor ever devised. In less than a second a nose can detect and distinguish between vast numbers of chemicals. Nature's unrelenting application of the evolutionary paradigm—selective pressure for survival of the fittest—has honed this instrument to perfection. For example, salmon use biosensors to return to their specific birth streams and a male moth, using one to three highly specific pheromone receptors can track and locate a single female several miles away. Other animals have developed the ability to distinguish thousands of distinct molecules using a complement of approximately 1,000 receptors. Dogs are routinely used for detecting explosives, illicit substances and for locating victims buried in the rubble of natural or man-made disasters.
Besides their contribution to olfaction, the importance of GPCRs to higher organisms including humans can be noted in the fact that 2,500 of the roughly 100,000 genes encoded in the human genome are for GPCRs (including the 1,000 for olfaction). An immense range of structurally diverse ligands are detected by the GPCRs. In addition to thousands of odorants comprised of naturally occurring and synthetic chemicals, GRCR ligands include structures from sugars (sucrose) to lipids (prostaglandins, leukotrienes) to peptides (from dipeptides—Nutrasweet —to proteins of 10 kD) to ions (calcium) to small aromatic molecules (melatonin, catecholamines, etc.) and even photons. This diversity of known ligands suggests that the range of chemical structures that can be detected by suitably-evolved receptors is unrestricted.
Perhaps not surprisingly, when small molecules activate GPCRs, they appear to do so by binding to amino acids located deep in their transmembrane regions. The panoply of GPCRs seen today have broadly similar structural motifs. For example, the seven membrane spanning regions of bacteriorhodopsin define an elliptical pocket (Roper, D., Jacoby, E., et al. (1994) Journal of Receptor Research 14:167-86). It is within this well that the photosensitive retinal (ligand) lies. Retinal is covalently attached to a lysine on transmembrane domain seven. This protein, along with its relatives halorhodopsin and sensory rhodopsin, comprise an ancient class of bacterial proteins that respond to photons by pumping protons and chloride ions, and by activation of a second protein respectively. All modem GPCRs apparently share the overall structure of these photoresponsive molecules including both the seven transmembrane regions and the corresponding intracellular and extracellular domains.
Small ligands, such as the retinal of rhodopsins, bind to GPCRs within wells defined by the GPCR's seven transmembrane spanning domains. This has been carefully delineated in a few cases such as the one described for the &bgr;
2
adrenergic receptor (Strader, C. D., et al., (1989)
Amer. J. Resp. Cell
&
Molec. Biol.
1: 81-85).
Although the constitution of the seven transmembrane domains of GPCRs is limited by requirement for overall hydrophobicity, the range of amino acid variation within the transmembrane regions, from receptor to receptor, varies greatly. In all cases there is an overall pattern of hydrophobic and hydrophilic amino acids as required by the alpha helical nature of the sequences. For the most part, hydrophobic amino acids are required for the face of each transmembrane domain that faces outward towards the lipid bilayer. The amino acids facing inward show greater variability. Not surprisingly, receptors with the same ligand, such as the &bgr;1-3 receptors have greater sequence homology to each other than to disparate receptors such as those for olfaction or gastrin releasing peptide (GRP). As with an antibody selected following immunization with a particular compound, there is no clear a priori correlation between the structure of the ligand, in terms its physico-chemical properties, and the general structural features of the receptor.
The ~1,000 olfactory receptors, taken together, recognize over 10,000 different chemicals including many synthetic, non naturally occurring ones such as numerous odorous organic molecules developed by the chemical industry. Different GPCRs such as the dopamine 1 and 2 receptors share the same ligand, yet the two receptors are only somewhat related. Meanwhile, one receptor may be activated by more than one ligand with varying degrees of similarity. Both the number and diversity (or alternatively the degree of focus) of the set of chemicals used to drive the selection of the set of receptors to be used in a sensor influence the range and specificity of the final sensor.
There is a need for highly specific and highly sensitive sensors that detect, a range of chemical compounds.
There is a need for sensors that detect, within a short period of time, a range of chemical compounds.
There is an additional need for standard analytical methods to monitor products for authenticity, or compliance to standards.
SUMMARY OF THE INVENTION
The invention provides novel methods for identifying mutated receptors, novel methods for testing a sample for the presence of a ligand, novel methods for generating and identifying a fingerprint for a ligand and novel detectors for identifying the presence of a ligand which binds to a cell surface receptor. The invention also relates to methods for analyzing products based upon the presence of ligands in such products that are constituents of the products. These methods allow for providing a ‘signature’ for the product, enabling authentication and monitoring of products for safety, security purposes, fraud and quality control. Other aspects of the invention will be readily apparent to those of ordinary skill in the art from a reading of the detailed description of the invention.
According to one aspect of the invention, a method is provided for identifying a mutated receptor that binds the ligand. First there is obtained or there is generated a plurality of nucleic acids that code for a plurality of mutated receptors. The plurality of nucleic acids then are introduced into a plurality of cells. It is preferred that the cells in their natural state do not generate a signal when contacted with the ligand. There are different nucleic acids in different of the plurality of cells. The plurality of cells then are contacted with the ligand. An intracellular signal in a cell, generated by a ligand binding to one of the plurality of mutated receptors, is detected. The signal is indicative of the presence of a mutated receptor that binds the ligand, when the mutated receptor is selected from the group consisting of mutated G protein coupled receptors, tyrosine kinas
Horlick Kenneth R.
Lerner Pharmaceuticals, Inc.
Wolf Greenfield & Sacks P.C.
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