Detection of single nucleotide variants in nucleic acid biomarkers using Raman spectroscopy

Single nucleotide variants (SNVs), including single-nucleotide polymorphisms, are key biomarkers for numerous human diseases, including cancer, and their accurate detection at single loci remains a major diagnostic challenge. Imaging-based approaches for in situ nucleic acid analysis further require highly specific, non-fluorescent readout strategies. Here, we report a Raman spectroscopy-based method for single-nucleobase discrimination using alkyne-tagged oligonucleotides. Ab initio calculations predict, and Raman measurements confirm, that the alkyne stretching vibrations of 5-ethynyluracil is highly sensitive to hydrogen-bonding interactions with its complementary nucleobase. Incorporation of this moiety as 5-ethynyl-2'-deoxyuridine into oligonucleotide probes enables detection of specific nucleobases within target DNA strands by high-resolution Raman spectroscopy. Distinct Raman shifts allow discrimination of single nucleotide variants, including the clinically relevant BRAF V600E mutation. These results demonstrate that alkyne-tagged base discriminating probes provide a robust Raman readout for SNV detection at single nucleotide resolution, establishing a foundation for future nucleic acid diagnostics and imaging applications.