Rapid genotyping using pyrene-perylene locked nucleic acid complexes

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Fluorescence Resonance Energy Transfer (FRET) is a powerful tool for studying molecular interactions.
• Developing sensitive and specific assays for nucleic acid detection, particularly for single-nucleotide polymorphisms (SNPs), remains a challenge.

Purpose of the Study:

• To develop a novel FRET-based assay for the detection of single-stranded DNA and RNA.
• To enable sensitive and accurate detection of single-nucleotide polymorphisms (SNPs) in nucleic acid targets.

Main Methods:

• Utilized novel pyrene-perylene FRET pairs attached to locked nucleic acid (LNA)/DNA probes.
• Employed ratiometric emission upon target binding with three combinations of fluorescent LNA/DNA reporter strands.
• Leveraged the dipole-dipole orientation parameter for SNP sensing via FRET.

Main Results:

• Achieved SNP detection with a large Stokes shift (115 nm) and high fluorescence quantum yields.
• Demonstrated a low limit of target detection (< 5 nM).
• Successfully performed rapid and accurate genotyping of HIV Pol cDNA and RNA fragments.

Conclusions:

• The novel pyrene-perylene FRET pairs offer significant advantages for nucleic acid detection and SNP sensing.
• The developed assay shows potential for broad applications in molecular imaging and clinical diagnostics.
• This approach advances FRET-based SNP detection with enhanced sensitivity and specificity.