This invention introduces enzymatic bioluminescent beacons that generate bright, low-background light only upon binding their target sequence. A site-specific ligation strategy forms uniform 1 to 1 nanoluciferase–hairpin conjugates, enabling rapid, picomolar sensitivity detection with precise single-base discrimination in streamlined, equipment-light assays.
Background: Molecular diagnostics need fast, equipment-light assays with high specificity and sensitivity, yet current methods either require thermocyclers and long runtimes or rely on indirect signals with high probe loads that reduce accuracy. Heterogeneous bioluminescent conjugates further compromise reproducibility, limiting scalable, precise detection across clinical and field settings.
Technology Overview: E-beacons are stoichiometric 1 to 1 conjugates of nanoluciferase tethered at its C terminus to a quenched hairpin DNA probe. The hedgehog cholesterol ligase domain (HhC) enzymatically ligates nanoluciferase to a mono sterylated steramer hairpin, yielding uniform constructs. Target hybridization opens the hairpin, separates the quencher from nanoluciferase, and generates a turn on bioluminescent signal that is ATP independent, highly sensitive, and sequence specific, including single nucleotide variant discrimination.
Advantages: • Uniform 1 to 1 conjugation for high reproducibility and assay consistency • Ultra low background bioluminescence enabling picomolar sensitivity • Single nucleotide mismatch discrimination for precise genotyping • ATP independent mix and read format reducing equipment needs • Lower probe concentrations than fluorescent beacons for cost efficiency • Modular design adaptable to diverse nucleic acid targets and workflows • Rapid signal generation suitable for high throughput and point of care use
Applications: • Infectious disease diagnostics for rapid pathogen and variant detection • Oncology liquid biopsy assays for ctDNA and microRNA monitoring • Genetic testing and pharmacogenomics for SNP and mutation analysis • Research tools for gene editing validation and nucleic acid quantification • Environmental, food, and agricultural testing for pathogen surveillance
Intellectual Property Summary: • United States 63/355288 Provisional filed June 24 2022 status Converted July 18 2022 • United States 18/212537 Utility filed June 21 2023 published as US 2025/0146088 on May 8 2025 status Filed
Stage of Development: Lab Validation
Licensing Status: This technology is available for licensing.
Licensing Potential: Applicable to diagnostics developers, molecular assay platforms, and life science tool companies seeking high sensitivity, rapid, and equipment-light nucleic acid detection technologies.
Additional Information: Information available upon request.
Inventors: Brian Callahan