Quantitative surface-enhanced Raman spectroscopy of single bases in oligodeoxynucleotides.

Area of Science:

• Biophysical Chemistry
• Spectroscopy
• Molecular Biology

Background:

• Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for molecular detection.
• Understanding SERS signal contributions from individual DNA bases is crucial for sequence analysis.
• Secondary structure can influence spectroscopic signals from nucleic acids.

Purpose of the Study:

• To determine if SERS signals from single-stranded DNA (ss-DNA) are additive based on constituent nucleobases.
• To investigate the influence of DNA sequence and secondary structure on SERS spectra.
• To assess the potential of SERS for detecting DNA modifications.

Main Methods:

• Acquisition of SERS spectra from unmodified ss-DNA sequences using silver colloid.
• Systematic variation of synthetic oligodeoxynucleotides by adding/substituting nucleobases.
• Thermal pre-treatment to eliminate secondary structure effects.
• Multivariate analysis of spectral data.

Main Results:

• SERS difference spectra could identify added nucleobases, matching reference spectra.
• A minor end effect (30%) was observed for nucleobases near the 3' terminus.
• Sequence substitutions led to spectral variations attributed to secondary structure changes.
• Thermal pre-treatment normalized spectra, indicating additive contributions from bases.
• Multivariate analysis showed 99% of variance explained by base identity, not position.

Conclusions:

• SERS spectra of ss-DNA are predominantly the sum of signals from individual nucleobases, with minimal positional information.
• Secondary structure significantly impacts SERS spectra, but can be mitigated by thermal pre-treatment.
• SERS is highly suitable for detecting post-transcriptional modifications in DNA due to its sensitivity to base identity.