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Modified Bis-pyrimidine Clamps for Triplex Formation and Their Use in SARS-CoV‑2 Detection.

Arnau Domínguez1,2,3, Raimundo Gargallo4, Carlos Cuestas-Ayllón2,5

  • 1Instituto de Quimica Avanzada de Cataluña (IQAC), Consejo Superior de Investigaciones Científicas (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain.

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|June 16, 2025
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Summary

Nucleic acid triple helices were modified to create bis-pyrimidine clamps for enhanced SARS-CoV-2 detection. These clamps offer a novel approach for biosensing and therapeutic applications, showing high sensitivity in detecting synthetic DNA targets.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Nucleic Acid Chemistry
  • Biosensing Technologies

Background:

  • Nucleic acid triple helices (triplexes) are crucial for gene regulation and diagnostics.
  • The coronavirus pandemic spurred the development of rapid SARS-CoV-2 detection methods like the Triplex Enhanced Nucleic Acid Detection Assay (TENADA).
  • Expanding detection capabilities requires novel triplex-forming structures targeting diverse viral sequences.

Purpose of the Study:

  • To explore triplex-forming bis-pyrimidine clamps targeting the SARS-CoV-2 ORF1a region.
  • To investigate the impact of 2 eal-sugar and 5-methylpyrimidine modifications on parallel triplex stability.
  • To evaluate the application of optimized clamps in a thermal lateral flow biosensing device.

Main Methods:

  • Design and synthesis of modified bis-pyrimidine clamps.
  • Assessment of triplex stability using Nuclear Magnetic Resonance (NMR) and biophysical techniques.
  • Integration of stable triplex clamps into a thermal lateral flow (TLF) sensing platform.

Main Results:

  • Modifications demonstrated distinct stabilizing effects on triplexes, influenced by their structural properties.
  • The study revealed novel insights into how modifications affect triplex stability through simultaneous Watson-Crick and Hoogsteen hydrogen bonding.
  • Optimized bis-pyrimidine clamps achieved low limits of detection (0.05–0.001 nM) for synthetic DNA targets in a TLF device.

Conclusions:

  • Modified bis-pyrimidine clamps are effective biosensing probes for nucleic acid detection.
  • Understanding modification strategies is key to advancing triplex-based clamps for biosensing and therapeutic uses.
  • This research expands the utility of triplexes in rapid diagnostic applications.