Real-Space Spectral Determination of Short Single-Stranded DNA Sequence Structures.

Area of Science:

• Biophysics
• Molecular Biology
• Spectroscopy

Background:

• Analyzing flexible biomolecules like DNA is vital for understanding biological functions.
• Traditional methods struggle with small, disordered, or difficult-to-label/crystallize biomolecules.
• Single-molecule tip-enhanced Raman spectroscopy (TERS) offers label-free identification of DNA nucleobases.

Purpose of the Study:

• To demonstrate spectrally resolving individual nucleobases and their sequence structures in a single DNA molecule.
• To overcome challenges in achieving single-base resolution with TERS due to weak signals and DNA flexibility.
• To establish a proof-of-principle for sequencing flexible DNA at high resolution.

Main Methods:

• Development of subnanometer-resolved low-temperature tip-enhanced Raman spectroscopy (TERS).
• Application of TERS to analyze artificially designed short, single-stranded DNA molecules.
• TERS mapping of individual nucleobases to obtain spectral and structural information.

Main Results:

• Achieved spectrally resolved sequencing of individual nucleobases in real space within a single DNA strand.
• Demonstrated the capability to resolve sequence structures of DNA at single-base resolution.
• Obtained additional structural information, including molecular configurations and functional group locations, via TERS mapping.

Conclusions:

• The developed low-temperature TERS methodology enables label-free, high-resolution sequencing of flexible DNA.
• This technique provides insights into molecular configurations and functional group positions, aiding in tracking modifications and binding sites.
• This breakthrough advances the structural analysis of challenging biomolecules, paving the way for new biological discoveries.