It is proposed to develop an approach for constructing enzyme-based sensors for detecting biomolecules. The sensor is an enzyme modified by site-directed mutagenesis to create a binding site specific for a target biomolecule. A library of engineered enzyme sensors is constructed to serve as a repository of potential binders. The binding site is positioned in the immediate vicinity of the enzyme's active site such that the active site is allosterically affected by the binding of the biomolecule to the binding site. Binding is detected in a chemical reaction catalyzed by the enzyme using a chromogenic or fluorogenic substrate. Implementation will be straightforward and as simple as adding the biomolecule of interest, or a sample that contains it, to the enzyme sensor and observing the outcome of the reaction (i.e., color or fluorescence).
Public Health Relevance Statement: Narrative The project will result in creation of a general approach to obtain enzyme-based sensors capable of detecting the presence of biomolecules and automatically signaling their presence. The approach can be used in research and development, molecular diagnostic testing and drug discovery. The ease of testing allows the method to be applied broadly.
Project Terms: Active Sites; Affect; Affinity; Alkaline Phosphatase; Allosteric Regulation; Amino Acids; Antibodies; Bacteriophage M13; Bacteriophages; base; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Biotin; Bovine Serum Albumin; Capsid Proteins; chemical reaction; Chromogenic Substrates; Color; Computer-Aided Design; Contractor; design; Development; DNA Sequence; drug discovery; Electroporation; Engineering; enzyme activity; enzyme structure; Enzymes; Escherichia coli; Exhibits; experimental study; Fluorescein; Fluorescein-5-isothiocyanate; Fluorescence; Fluorogenic Substrate; gene synthesis; Genes; Goals; Grant; Heterogeneity; HIV Antibodies; Human; Incubated; Individual; Influenza Hemagglutinin; interest; Isothiocyanates; Libraries; Literature; Location; Measures; Methodology; Methods; Molecular; Molecular Diagnostic Testing; mutant; Mutate; Nucleic Acids; Oligonucleotides; Outcome; Paper; particle; Peptides; Phage Display; Phase; Plasmid Cloning Vector; Positioning Attribute; Process; Property; Protein p53; Proteins; Reaction; repository; research and development; Sampling; screening; sensor; Signal Transduction; Site-Directed Mutagenesis; small molecule; suicide substrates; System; Temperature; Testing; three dimensional structure; Time; time use; Tumor Antigens; vector; Virion; Work
