Modified Bis-pyrimidine Clamps for Triplex Formation and Their Usein SARS-CoV‑2 Detection

The formation of nucleic acid triple helices (“triplexes”) is an area of great interest due to their potential role in thenatural and artificial regulation of gene expression or for use in analytical, diagnostic, or synthetic methods. During the coronaviruspandemic, a large search for novel methods for the detection of SARS-CoV-2 was undertaken. Based on triplex affinity capture andusing polypurine reverse-Hoogsteen hairpins, a method known as Triplex Enhanced Nucleic Acid Detection Assay (TENADA) wasdeveloped for the rapid detection of SARS-CoV-2 without the need for polymerase chain reaction (PCR) amplification. In this work,to expand the targeting scope of this method, we explored triplex-forming bis-pyrimidine clamps targeting a polypurine sequence inthe ORF1a region of SARS-CoV-2. To enhance parallel triplex stability, 2′-sugar and 5-methylpyrimidine modifications wereincorporated into both strands of the clamps, and their effect on the triplexes formed was assessed via NMR and other biophysicalmethods. The results revealed distinct stabilizing effects of the modifications, influenced by their size, sugar puckering, and capacityto form short contacts with neighboring residues. The dual ability of clamps to simultaneously form Watson−Crick and Hoogsteenhydrogen bonds offers a novel perspective on the effect of modifications on triplex stability, previously unexplored with triplexformingoligonucleotides (TFOs). Finally, the bis-pyrimidine clamps that formed the most stable parallel triplexes were applied in athermal lateral flow (TLF) sensing device, demonstrating their potential as biosensing probes. These clamps effectively detected thesynthetic DNA target with limits of detection (LoDs) ranging from 0.05 to 0.001 nM. Understanding the best modification strategiesand their impact on the triplex structure will advance the development of clamps as biosensing and therapeutic agents.