A reduction-triggered fluorescence probe for sensing nucleic acids.

Area of Science:

• Chemical Biology
• Molecular Diagnostics
• Biochemistry

Background:

• Oligonucleotide detection is crucial for molecular diagnostics.
• Existing methods often require enzymes or harsh conditions, limiting their applicability.
• Development of novel, sensitive, and stable probes is needed.

Purpose of the Study:

• To develop a novel reduction-triggered fluorescence probe for oligonucleotide sensing.
• To create a probe with high signal-to-background ratio and stability.
• To demonstrate the probe's utility in detecting nucleic acids under biological conditions.

Main Methods:

• Synthesis of a new fluorogenic compound derived from Rhodamine with an azide group.
• Activation of the probe via reduction of the azide group by reagents like dithio-1,4-threitol or triphenylphosphine.
• Design of a two-strand DNA probe system where one strand carries the fluorogenic compound and the other carries the reducing agent.

Main Results:

• Achieved a 2100-fold enhancement in fluorescence signal intensity (signal/background ratio).
• Demonstrated enzyme-free and reagent-free automatic reaction under biological conditions, yielding results within 10-20 minutes.
• Proved probe stability across a range of pH conditions (pH 5-10) and high temperatures (90°C) with minimal background signal.
• Successfully detected oligonucleotides at the single nucleotide level in solution and endogenous RNA in bacterial cells.

Conclusions:

• The developed reduction-triggered fluorescence probe offers a sensitive, rapid, and stable method for oligonucleotide detection.
• This probe system is suitable for diverse biological applications, including single nucleotide detection and bacterial RNA sensing.
• The probe's robustness and efficiency pave the way for advanced molecular diagnostic tools.