Aurora: a fluorescent deoxyribozyme for high-throughput screening.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Fluorescence is crucial for molecular detection, visualization, and tracking.
• Existing fluorescent proteins and RNA aptamers have limitations in cost, synthesis, and stability.
• Functional DNA molecules offer potential advantages for sensitive and specific molecular detection.

Purpose of the Study:

• To develop a novel deoxyribozyme for generating robust and stable fluorescent signals.
• To create a sensitive assay for detecting ribonucleases using the deoxyribozyme.
• To utilize the deoxyribozyme in high-throughput screening for identifying novel enzyme inhibitors.

Main Methods:

• Engineering a deoxyribozyme capable of catalyzing a fluorescent reaction with a coumarin substrate.
• Developing a homogenous assay for quantifying ribonuclease activity.
• Implementing a high-throughput screening strategy to identify inhibitors of SARS-CoV-2 ribonuclease Nsp15.

Main Results:

• The novel deoxyribozyme rapidly and efficiently produces a stable fluorescent product.
• An engineered deoxyribozyme detected picomolar concentrations of ribonucleases in a homogenous assay.
• The deoxyribozyme was successfully used to screen for and identify novel inhibitors of SARS-CoV-2 Nsp15.

Conclusions:

• This work introduces a new functional DNA component for molecular detection applications.
• Catalytic DNA motifs can be effectively employed to address real-world challenges, such as drug discovery.
• The developed deoxyribozyme system offers a sensitive and stable alternative for biochemical assays and screening.