Novel (phenylethynyl)pyrene-LNA constructs for fluorescence SNP sensing in polymorphic nucleic acid targets

Area of Science:

• Biochemistry
• Molecular Biology
• Oligonucleotide Chemistry

Background:

• Single-nucleotide polymorphisms (SNPs) and mutations in nucleic acids are critical for disease diagnosis and treatment.
• Existing detection methods can be challenged by complex biological samples with low target concentrations or sequence variations.
• Locked nucleic acids (LNAs) offer enhanced hybridization properties for nucleic acid detection.

Purpose of the Study:

• To develop and validate novel fluorescent oligonucleotide probes for SNP detection.
• To assess the utility of these probes for identifying drug-resistance mutations in HIV-1.
• To establish diagnostic systems for homogeneous detection of clinically relevant mutations.

Main Methods:

• Synthesis of fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids.
• Demonstration of high-affinity hybridization and fluorescence response to single-base mismatches in DNA/RNA targets using dual-probe and doubly labeled probe formats.
• Development of diagnostic systems for homogeneous detection of HIV-1 protease gene mutations using clinical samples.

Main Results:

• Probes exhibited high-affinity hybridization to natural nucleic acid targets.
• Excellent fluorescence responses were observed for single-base mismatches in DNA/RNA.
• Two diagnostic systems successfully detected a drug-resistance mutation in HIV-1 protease cDNA and RNA from clinical samples.
• Detection was effective despite challenges including additional mutations, sequence variation, and low target concentration.

Conclusions:

• Novel fluorescent LNA probes are effective tools for detecting SNPs and clinically relevant mutations.
• The developed diagnostic systems offer a robust method for identifying drug-resistance mutations in HIV-1.
• These probes demonstrate significant potential for molecular diagnostics in complex biological samples.