Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2...

Area of Science:

• Chemical Biology
• Nucleic Acid Chemistry
• Molecular Diagnostics

Background:

• Pseudo-complementary oligonucleotides are engineered to prevent self-hybridization while maintaining target binding.
• Previous work established a pseudo-complementary A:T base pair (U:D) crucial for double-stranded DNA (dsDNA) invasion.
• The G:C base pair is essential for nucleic acid structure and function, necessitating pseudo-complementary analogues for advanced applications.

Purpose of the Study:

• To develop pseudo-complementary analogues of the G:C base pair using peptide nucleic acids (PNA).
• To investigate the hybridization properties of these novel pseudo-complementary PNA:DNA systems.
• To demonstrate the utility of these systems for sensitive molecular detection, including pathogen identification.

Main Methods:

• Design and synthesis of PNA oligomers incorporating cationic phenoxazine analogues for pseudo-complementary G:C pairing.
• Evaluation of PNA homoduplex and PNA:DNA heteroduplex stability.
• Assessment of dsDNA invasion efficiency at physiological salt concentrations.
• Application in a lateral flow assay (LFA) for the detection of reverse transcription-recombinase polymerase amplification (RT-RPA) amplicons.

Main Results:

• Novel pseudo-complementary G:C PNA analogues were created, leveraging steric and electrostatic repulsion.
• These pseudo-complementary PNA oligomers preferentially hybridize with DNA over forming PNA homoduplexes.
• Efficient dsDNA invasion was achieved at physiological salt concentrations with low PNA equivalents (2-4).
• The system demonstrated high sensitivity for RT-RPA amplicon detection via LFA, enabling discrimination between SARS-CoV-2 strains based on single nucleotide differences.

Conclusions:

• Pseudo-complementary G:C PNA analogues effectively promote PNA:DNA hybridization over self-pairing.
• This strategy enables efficient dsDNA invasion, a key step for molecular detection.
• The developed method offers a sensitive and specific platform for pathogen detection, exemplified by SARS-CoV-2 strain discrimination.