Programmable DOUBLE-GATED molecular switches enable extraction-free, direct zygosity resolution of single-nucleotide variants
Year: 2026
Type: conference-paper
Abstract: Accurate determination of allelic zygosity remains a major challenge in single-nucleotide polymorphism (SNP) detection. Most existing methods report only the presence or absence of a variant and often require complex workflows, long processing times, or careful interpretation of amplification signals. Here, we introduce the first double-gated, self-annealing loop primer (SALP)-based dual molecular-switch colorimetric loop-mediated isothermal amplification (LAMP) strategy for direct zygosity determination. We demonstrate the approach using the clinically (breast cancer and prostate cancer) relevant AKR1C4 rs17134592 SNP, where functional and clinical outcomes depend on allelic composition. Individuals with the heterozygous genotype (C/G) exhibit partial reduction in enzyme activity, whereas the homozygous mutant genotype (G/G) is associated with greater loss of function and increased disease severity. The assay comprises two reciprocal reactions that independently interrogate each allele: a mutant-selective F2 primer with its corresponding overlapping self-annealing loop primer (F2 M-SALP M) targets the mutant G allele, while a wild-type-selective pair (F2 W-SALP W) targets the wild-type C allele. In each reaction, allele specificity is enforced through two sequential molecular gates. The first gate resides at the 3′ terminus of the F2 primer, where the SNP-discriminating base is combined with a deliberate upstream mismatch to destabilize binding to the non-target allele. The second gate is introduced by the overlapping SALP, which incorporates an additional mismatch; its 3′ terminal nucleotides anneal to the F2-integrated region within the dumbbell intermediate, simultaneously interrogating both the SNP position and the engineered mismatch site. Together, these gates impose cumulative mismatch discrimination, generating at least two mismatches at the first gate and a more stringent three-mismatch barrier at the second gate against non-target amplification. To further enhance specificity and robustness, allele-discriminating regions of both F2 primers and SALPs were engineered using the LNA3 design principle, with strategically positioned locked nucleic acid (LNA) bases to increase binding affinity and mismatch sensitivity. Penultimate LNA modifications were additionally incorporated at primer termini to improve nuclease resistance and preserve performance in crude, extraction-free samples. The assay operates at a constant temperature (65 °C) and provides an intuitive colorimetric readout, where genotype is determined by a simple two-tube comparison, allowing unambiguous identification of homozygous and heterozygous states without instrumentation or curve analysis. We demonstrate the platform using the clinically relevant AKR1C4 rs17134592 SNP, achieving single-copy sensitivity, high specificity, and full concordance with Sanger sequencing across synthetic targets, cultured cells, and extraction-free crude lysates in human plasma. 4 The method eliminates the need for peptide nucleic acids or sample purification, substantially simplifying the workflow while maintaining analytical performance. Beyond this specific target, the underlying double-gated LNA design is generalizable and readily adaptable to diverse SNPs. This work establishes a scalable and accessible framework for precise zygosity determination, with broad applicability across clinical diagnostics, precision medicine, agriculture, environmental monitoring, and biosecurity, where rapid and reliable SNP analysis is critical.
Authors: Shaon, Md Akeruzzaman; id_orcid 0009-0007-3472-2916, Farhana, Fatema Zerin; id_orcid 0000-0001-8031-0620, Haque, Farjana; id_orcid 0000-0001-8297-2652, Bin Manjur, Omar Hamza; id_orcid 0009-0003-6358-5640, Tantu, Milkiyas Toru; id_orcid 0000-0001-8278-0343, Hanafy, Doaa; id_orcid 0000-0003-3590-9401, Shiddiky, Muhammad J A; id_orcid 0000-0003-4526-4109
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7220112972