The 1720 cm -1 signal should also contain a certain amount of contributions from the N-H bending, as the N-H bending vibration is known to couple to the carbonyl stretching in amide bonds. Therefore, we site-specifically incorporated doubly labeled (1,3-15 N) T and performed isotope-editing IR to deconvolute the degree to which N-H bending contributes, in addition to the TC2=O and TC4=O oscillators (Figure ). We observed multiple signals for the TN3-H in the edited spectra and found several signals of equal intensities in the edited spectra (Figure ). This major signal for this isotope at 1720 cm -1 is assigned to N-H bending, with a ~20% contribution to the mode when normalized as a total area summed over the other available T isotopes. These combined isotopic substitution studies indicate T 12 C2=O ought to be localized enough to the 12 C2=O atoms for our signal to be quite sensitive to water penetration at one part of the duplex upon heating (Figure ) and thus be a measurement of local transition temperatures in a sequence's structure. As a proof-of-concept, we performed nucleobase isotope editing experiments as a function of temperature on a low melting A-T rich sequence, (dA-dT)9, to test if the difference signal observed upon TC2=O isotope editing at a specific location in a DNA duplex yields an observable transition curve (Figure ). We determined the global melting temperature of this duplex under high salt concentrations and low singlestranded DNA concentrations using VT-UV-visible spectroscopy (Figure ). We found that both salt and DNA concentration affect the global melting temperature to a certain degree that is aligned with previous studies and established calculations (Table ). We then determine the global melting temperature of this duplex under low sodium concentrations (40 mM) and high singlestranded DNA concentrations (~7 mM) by using VT-IR with the assumption of the two-state model and found that the global melting temperature of this duplex is ~35.6±0.3˚C. We then applied nucleobase (TC2=O) isotope editing and variable temperature IR to monitor local base pair melting. If our signal is sensitive to local melting as we predicted from our physical model, we ought to get different transition temperatures when we measure at various locations in the duplex, as ends of DNA sequences are known to fray. We thus inserted our TC2=O probe at the end and middle positions of our sequence. We found that when we place our probe at the end position, the temperature at which half of the single base pair at position 2 is ruptured is ~34.9±0.7˚C which is expected to be lower than the that measured for the duplex's global Tm. Placing our probe in the middle at position 5 gives a TmL of ~35.6±0.2˚C, higher than the end position and identical to the global Tm. This observation indicates that base pairs at the end position of the duplex rupture at slightly lower temperatures than the base pairs at the middle position, consistent with the end fraying hypothesis in short duplexes. With this result, we have provided physical evidence that our T 13 C2 isotope IR probe can serve as a sub-molecular-scale thermometer to measure the local melting transitions of DNA structural motifs containing thymine.