Identification of Natural and Artificial Modifications in DNA at the Nanometer Level

ABSTRACT The modification of nucleobases in nucleic acids is a naturally occurring process that can affect various positions of the heteroaromatic ring systems. In addition, by the targeted introduction of an artificial substituent, these molecules can be used as markers for detecting specific regions in biomaterials. In the current study, short single‐ and double‐stranded DNA sequences are designed, and selected nucleobases are chemically modified. In a sequence containing all four nucleobases, a single thymine is modified with a side chain comprising internal and terminal carbon–carbon triple bonds. Following hybridization with the unmodified complementary sequence, the strand is examined with tip‐enhanced Raman scattering (TERS) under ambient conditions to identify the modified molecular site on the surface. The characteristic vibrational stretching modes of the alkyne groups do not overlap with those of nucleic acids and are therefore suitable for unambiguous identification. A modelling approach to the DNA structure is used to elucidate the observed band shifts and possible influence of the local environment, and the distance to the TERS probe is discussed. A second approach involves a simplified single‐stranded DNA sequence with noncomplementary nucleobases, including methylated cytosine, as a naturally occurring modification. TERS spectra are analyzed for the potential identification of the modified cytosine, despite the overlap of its vibrational modes with those of other nucleobases. The results demonstrate that TERS is highly efficacious in detecting individual modified nucleobases in DNA, enabling these markers to be traced on biomaterials with nanometer precision.