SBIR-STTR Award

We describe an integrated approach for rapid discovery of small, structurally defined nucleic acid "probe" sequences that bind with high affinity and selectivity to a target of interest. Such targets are proteins, bio-terror agents, pathogenic organisms and viruses. The probes will be engineered into proprietary AlloSwitchTM indicators that respond rapidly to the presence of the target, giving rise to a fluorescent or luminescent signal. In this phase I project we propose to rapidly screen and isolate high-affinity combining probes (c-probes). We will use high throughput methods to isolate at least the known probe sequences specific for alpha-thrombin and platelet derived growth factor (PDGF) from a defined library containing billions of DNA sequences. Alternative c-probes for thrombin and PDGF will be sought in libraries that have a predefined secondary structure. In phase II, the approach will be expanded to include a wider variety of structural motifs and new targets. The targets for discovery of probes and creation of AlloSwitches will include proteins involved in clotting and thrombosis, and growth factors involved in wound healing, angiogenesis, and proliferative diseases. AlloSwitch based assays will assist in the discovery of small molecules that can be used for therapies to regulate the interactions of these proteins in blood. This stands in contrast to therapies based on aptamers or antibodies, which are difficult to protect from hydrolysis by enzymes in plasma. Establishing a highly efficient and rapid screening approach for short DNA/RNA probe discovery will accelerate the commercialization of our biosensor technology in other fields.

Public Health Relevance:
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We describe an integrated approach for High Throughput Screening of AptamersTM (HTSATM, patent pending) to discover small, structurally defined nucleic acid aptamer sequences that bind with high affinity and selectivity to a target of interest. Such targets are proteins, bio-terror agents, pathogenic organisms and viruses. These aptamers will be engineered into the [QOUTA]probe[QOUTA] sequence of our patented AlloSwitchTM indicators that re- spond rapidly to the presence of the target, giving rise to a fluorescent or luminescent signal. In phase I we demonstrated that it is feasible to rapidly screen and isolate high-affinity HT-aptamers. We have used HTSA to isolate previously known aptamers with nanomolar affinity for human athrombin (from DNA hairpin loops) and for human coagulation factor IXa (from RNA with concatenated hairpin and internal loops), and an unprece- dented aptamer with apparent specificity for hexose sugars (from DNA hairpins) with affinity in the top third of known aptamers for small molecules. Thus, the feasibility of HTSA has been demonstrated. In phase II, the approach will be expanded to include a wider variety of structural motifs and new targets. The targets for discovery of probes and creation of AlloSwitches will include proteins involved in clotting and thrombosis, and growth factors involved in wound healing, angiogenesis, and proliferative diseases. AlloSwitch based assays will assist in the discovery of small molecules that can be used for therapies to regulate the interactions of these proteins in blood. This stands in contrast to therapies based directly on aptamers or antibodies, which are difficult to protect from hydrolysis by enzymes in plasma and to formulate as pharmaceuticals in pills. This project will also accelerate the commercialization of our biosensor technology in other fields.

Public Health Relevance:
We present an integrated approach for rapid discovery of small, structurally defined nucleic acid [QOUTA]HT- aptamer[QOUTA] sequences that bind with high affinity and selectivity to proteins and other targets. The approach will be used to create sensors to discover small molecules as candidates for drugs against selected protein targets involved in wound healing and thrombosis. Beyond this project, discovery of high affinity HT-aptamers will be used in detecting waterborne pathogens, biological warfare agents, and diagnostic applications with costs ex- pected to be reduced by a factor of ten or more compared to antibody-based reagents.

Thesaurus Terms:
Affinity; Antibodies; Aptamer Technology; Assay; Bacteria; Base Sequence; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biologic Warfare; Biological Assay; Biological Warfare; Biosensing Technics; Biosensing Techniques; Biosensor; Blood; Blood Coagulation Factor Ix, Activated; Blood Plasma; Cell Communication And Signaling; Cell Signaling; Cell Surface; Clotting; Coagulation; Coagulation Factor Ixa; Coagulation Process; Contracting Opportunities; Contracts; Custom; Dna; Dna Library; Dna Bank; Deoxyribonucleic Acid; Detection; Diagnostic; Disease; Disorder; Drugs; Engineering; Engineerings; Enzymes; Evolution; Factor Ix, Activated; Factor Ixa; Food Safety; Gfac; Gene Probes, Rna; Gene Products, Rna; Goals; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; Hexoses; High Throughput Assay; Human; Human, General; Hydrolysis; Image; In Vitro; Intracellular Communication And Signaling; Investigators; Legal Patent; Length; Libraries; Licensing; Man (Taxonomy); Man, Modern; Marketing; Measures; Medication; Methods; Microarray Analysis; Microarray-Based Analysis; Molecular Biology, Nucleic Acid Sequencing; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Monitor; Nucleic Acids; Nucleic Acid Sequencing; Nucleotide Sequence; Nucleotides; Organism; Patents; Pharmaceutic Preparations; Pharmaceutical Agent; Pharmaceutical Preparations; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Phase; Pill; Plasma; Proteins; Rna; Rna Probes; Rna Sequences; Rna, Non-Polyadenylated; Randomized; Reagent; Research Personnel; Researchers; Resistance; Reticuloendothelial System, Blood; Reticuloendothelial System, Serum, Plasma; Ribonucleic Acid; Sbir; Sbirs (R43/44); Sampling; Screening Procedure; Serum, Plasma; Services; Signal Transduction; Signal Transduction Systems; Signaling; Small Business Innovation Research; Small Business Innovation Research Grant; Specificity; Structure; Surface Plasmon Resonance; Technology; Therapeutic; Thrombosis; Virus; Viruses, General; Wound Healing; Wound Repair; Angiogenesis; Aptamer; Base; Biological Signal Transduction; Biosensing; Biowarfare; Commercial Application; Commercialization; Conformation; Conformational State; Cost; Cross Reactivity; Design; Designing; Disease/Disorder; Drug Candidate; Drug Discovery; Drug/Agent; Enzyme Activity; Gene Product; High Throughput Screening; Imaging; Inhibitor; Inhibitor/Antagonist; Interest; Living System; Luminescence; Member; Microarray Technology; Next Generation; Nuclease; Nucleic Acid Sequence; Pathogen; Pill (Pharmacologic); Public Health Relevance; Randomisation; Randomization; Randomly Assigned; Resistant; Response; Screening; Screenings; Sensor; Sensor (Biological); Single Molecule; Small Molecule; Stem; Sugar; Synthetic Dna; Synthetic Construct; Tissue Repair; Water Quality; Waterborne