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

20 December 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

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.

Supplementary materials

Title
Description
Actions
Title
Supporting methods and results
Description
Additional experimental details, methods, experimental setup, and calculations were included in the supporting document.
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.