A Spinach molecular beacon triggered by strand displacement

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Aptamer Engineering

Background:

• The Spinach RNA aptamer is a fluorescent reporter but lacks sequence-specific activation.
• Existing methods for aptamer activation can be complex or lack specificity.

Purpose of the Study:

• To engineer a sequence-dependent activation mechanism for the Spinach RNA aptamer.
• To develop a novel method for specific nucleic acid detection and real-time amplicon monitoring.

Main Methods:

• Re-engineering the Spinach aptamer (Spinach.ST) with extended ends for inactive conformation.
• Utilizing toehold-initiated strand displacement with trigger oligonucleotides for activation.
• Applying primer design for hairpin formation to adapt target sequences.

Main Results:

• Spinach.ST demonstrates sequence-specific activation and binds the DFHBI fluorophore upon trigger hybridization.
• The system achieves high specificity, discriminating against single-nucleotide mismatches.
• Real-time detection of RNA amplicons from DNA/RNA targets using in vitro transcribed Spinach.ST was achieved.

Conclusions:

• Engineered Spinach.ST enables sequence-specific RNA aptamer activation and detection.
• This method offers a versatile platform for specific nucleic acid detection and amplicon monitoring.
• Potential exists for adapting Spinach reporters to non-nucleic acid analytes.