A label-free biosensor for DNA detection based on ligand-responsive G-quadruplex formation.

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Accurate detection of specific single-stranded DNA sequences is crucial for diagnostics and research.
• Existing methods for nucleic acid detection can be complex, time-consuming, or require chemical modifications.

Purpose of the Study:

• To develop a facile, label-free assay for sensitive and selective detection of specific single-stranded DNA sequences.
• To utilize molecular beacons (MBs) and N-methyl mesoporphyrin IX (NMM) for a novel fluorescence-based detection system.

Main Methods:

• A label-free assay employing molecular beacons (MBs) and the fluorescent dye N-methyl mesoporphyrin IX (NMM).
• Demonstration using a reverse transcription oligonucleotide sequence from human immunodeficiency virus (HIV) RNA as a model target DNA.
• Monitoring fluorescence changes of NMM based on G-quadruplex formation induced by target DNA binding to MBs.

Main Results:

• In the absence of target DNA, MBs remained in a stem-closed form, resulting in low NMM fluorescence.
• Upon target DNA presence, MBs transitioned to an open form, promoting G-quadruplex formation and significantly enhancing NMM fluorescence.
• The assay demonstrated a detection limit of 1.4 nM and was completed in under 30 minutes.

Conclusions:

• A simple, rapid, and label-free biosensor was successfully developed for specific single-stranded DNA detection.
• The G-quadruplex-NMM fluorescence enhancement mechanism provides a sensitive and selective detection strategy.
• This approach eliminates the need for sophisticated techniques or DNA modifications, offering broad applicability for nucleic acid detection.