De novo-designed ribozyme-controlled riboregulator for cell-free diagnostics

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Synthetic Biology

Background:

• Cell-free systems offer potential for nucleic acid assays but lack universal onsite diagnostics without preamplification.
• Existing methods often require preamplification, limiting rapid, field-deployable diagnostic capabilities.

Purpose of the Study:

• To develop a universal cell-free diagnostic platform, TRACKer (Target-Responsive non-preAmplification Cell-free diagnostic Kit), for detecting multiple respiratory viral RNAs.
• To enable rapid, preamplification-free nucleic acid detection in both laboratory and field settings.

Main Methods:

• TRACKer integrates three modules: ribozyme allostery for universal target recognition via strand displacement-mediated conformational switching, riboregulator activation for preamplification-free detection through reporter protein cascade expression, and an output module for adaptable reporter templates.
• Utilized strand displacement-mediated ribozyme conformational switching for target recognition.
• Employed cascade expression of reporter proteins for amplification-free detection.

Main Results:

• TRACKer enables rapid nucleic acid detection (under 70 minutes) without preamplification.
• Demonstrated attomolar sensitivity (1-10 aM) for detecting six different respiratory viruses.
• The system is robust and adaptable for diverse reporting scenarios, including luminescent quantification and lateral flow visualization.

Conclusions:

• TRACKer presents a robust, universal cell-free diagnostic platform for rapid, preamplification-free detection of respiratory viral RNAs.
• The developed platform offers a low-cost, scalable solution with potential for point-of-care diagnostics and broader applications in nucleic acid detection.