UV Resonance Raman Spectroscopic Marker Bands of Base Pair Formation During Nucleic Acids Assembly

The molecular assembly of nucleic acids (NA) governs the structure and function of DNA and RNA complexes, like the DNA double helix, DNAzymes or Ribozymes, via specific noncovalent NA-NA interactions. Here, we demonstrate the use of UV resonance Raman (UV RR) spectroscopy as a tool to inform about the structural details of the molecular assembly of NAs and their underlying interactions, with a particular focus on H-bonding between base pairs. Using experimental H/D exchange and hybrid DFT-based computational Raman spectra, we identify UV RR marker bands that involve vibrations of C=O, NH, and NH2 groups, which are relevant to H-bonding interactions in guanine-cytosine (G-C) and adenine-thymidine (A-T) Watson-Crick base pairs. Analysing peak shifts and changes in relative intensities of these marker bands that are consistent with computational spectra, we successfully follow the conformational assembly of NA complexes by recording their UV RR spectra during thermal hybridization to double helices. The thermal sigmoidal transitions show variation between different markers and are dependent on the length of the oligonucleotide strands, demonstrating the potential of UV RR as a chemically selective, label-free method to probe noncovalent interactions in NA assembly, and to inform on mechanistic details in complex DNA and RNA architectures.