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

Area of Science:

• Biochemistry
• Spectroscopy
• Genetics

Background:

• Single nucleotide variants (SNVs) are crucial biomarkers for diseases like cancer.
• Accurate detection of SNVs at the single loci level is a significant diagnostic challenge.
• Existing in situ nucleic acid analysis methods require specific, non-fluorescent readout strategies.

Purpose of the Study:

• To develop a Raman spectroscopy-based method for single-nucleobase discrimination.
• To utilize alkyne-tagged oligonucleotides for enhanced nucleic acid analysis.
• To establish a foundation for future nucleic acid diagnostics and imaging.

Main Methods:

• Ab initio calculations and Raman spectroscopy were employed.
• Alkyne-tagged oligonucleotides, specifically 5-ethynyl-2'-deoxyuridine, were synthesized and utilized.
• The sensitivity of alkyne stretching vibrations to hydrogen-bonding interactions was investigated.

Main Results:

• The alkyne stretching vibrations of 5-ethynyluracil demonstrated high sensitivity to hydrogen-bonding with complementary bases.
• Raman spectroscopy enabled the detection of specific nucleobases within target DNA strands.
• Distinct Raman shifts allowed for the discrimination of single nucleotide variants, including the BRAF V600E mutation.

Conclusions:

• Alkyne-tagged base-discriminating probes offer a robust Raman readout for SNV detection at single nucleotide resolution.
• This method provides a foundation for advanced nucleic acid diagnostics.
• The technique holds promise for future in situ nucleic acid imaging applications.