Isotope-edited variable temperature infrared spectroscopy for measuring transition temperatures of single A-T Watson-Crick base pairs in DNA duplexes

Abstract

Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2=O stretching vibration when it is within the DNA duplex via iso-topic substitutions at other atomic positions in the structure. After determining that this IR difference probe was localized enough to measure sub-molecular scale structures in high molecular weight complexes, we used this probe to develop a new isotope edited variable temperature infrared method to measure melting at various locations in a DNA structure. As an initial test of this “sub-molecular-scale thermometer”, we first applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair at the end of an A-T rich duplex is ~34.9±0.7˚C. This is slightly lower than the TmL of a single base pair at the middle position of the duplex (TmL ~35.6±0.2˚C), providing direct physical evidence for end fraying in A-T-rich DNA sequences.