Real-Space Spectral\nDetermination of Short Single-Stranded\nDNA Sequence Structures

Resolving\nthe sequence and structure of flexible biomolecules\nsuch\nas DNA is crucial to understanding their biological mechanisms and\nfunctions. Traditional structural biology methods remain challenging\nfor the analysis of small and disordered biomolecules, especially\nthose that are difficult to label or crystallize. Recent development\nof single-molecule tip-enhanced Raman spectroscopy (TERS) offers a\nlabel-free approach to identifying nucleobases in a single DNA chain.\nHowever, a clear demonstration of sequencing both spatially and spectrally\nat single-base resolution is still elusive due to the challenges caused\nby weak Raman signals and the flexibility of DNA molecules. Here,\nwe report a proof-of-principle demonstration to this end, spectrally\nresolving in real space individual nucleobases and their sequence\nstructures within a short, single-stranded DNA molecule artificially\ndesigned. This breakthrough is achieved through the development of\nsubnanometer-resolved low-temperature TERS methodology for such thermally\nunstable flexible biomolecules. Further TERS mapping over individual\nnucleobases provides additional structural information about the molecular\nconfigurations and even the locations of functional groups, offering\na way to track modification types and binding sites in biomolecules.