Thermostable Bioluminescent Intercalating Dyes for Real-Time, Integrated Nucleic Acid Amplification and Detection.

Area of Science:

• Molecular Biology
• Biotechnology
• Medical Diagnostics

Background:

• Point-of-care diagnostics require integrated nucleic acid amplification and detection.
• Existing luciferase-intercalating dye conjugates (LUMIDs) lack the thermostability for isothermal amplification.
• Limited thermostability hinders the integration of LUMIDs with sensitive amplification methods like loop-mediated isothermal amplification (LAMP).

Purpose of the Study:

• To re-engineer LUMIDs for enhanced thermostability and integration with LAMP.
• To develop a one-pot assay for real-time DNA amplification monitoring.
• To enable sensitive and rapid detection of human papillomavirus (HPV) subtypes.

Main Methods:

• Incorporated a thermostable NanoLuc luciferase variant and a DNA binding protein to maintain activity at LAMP temperatures (60°C-65°C).
• Introduced a caged luciferin substrate with esterase-controlled release for sustained bioluminescence.
• Combined LAMP with the re-engineered LUMIDs for one-pot assay development.

Main Results:

• Achieved stable luciferase activity and DNA binding at LAMP temperatures.
• Enabled one-pot LAMP-LUMID assays for real-time DNA amplification monitoring.
• Demonstrated attomolar sensitivity for detecting cancer-associated HPV subtypes within approximately 30 minutes.
• Validated assay performance in patient samples using a digital camera readout.

Conclusions:

• The enhanced LAMP-LUMID assay is a rapid, sensitive, and robust platform for molecular diagnostics.
• The assay demonstrates significant potential for point-of-care infectious disease detection, especially in low-resource settings.
• This integrated approach overcomes previous limitations of LUMID thermostability.