SUNY at Albany
June 19-23, 2001
UV Resonance Raman Spectroscopy Study of a DNA Triple Helix
UV Resonance Raman spectroscopy (UVRR) is used to investigate an intramolecular DNA triple helix, which is labeled with 15N at the adenine in the fifth position in the sequence: d(5Õ-AGAGAGAA-CCCC-TTCTCTCT-TTT-TCTCTCTT-3Õ). The labeled adenine is in a TA*T triad where it forms a Watson-Crick base pair with a thymine and hydrogen bonds in a Hoogsteen configuration with the thymine in the third strand. In this triad the adenine forms hydrogen bonds at the N1, N7 and C6-NH2 positions. As a consequence of these H-bonds, the vibrational bands associated with these positions are expected to shift in frequency.
In order to unravel the relatively complex vibrational spectrum of the triplex, we have been studying the adenine analog, 9-methyladenine (9-MeA). In this study we compare the UVRR spectra of 9-MeA with the frequency of the vibrational modes obtained from ab initio calculations. To compare calculations and UVRR spectra, the following studies were undertaken in our lab: i) A UV Resonance Raman (UVRR) pH titration (pH 7.0-1.0) of 9-MeA was performed and a mode at 1693 cm-1 is observed at low pH. This mode was observed previously at pH<5 in dAMP and was assigned to a C6=N stretching vibration. ii) Ab initio calculations of the normal modes of 9-MeA, both neutral and singly-protonated, were performed using Gaussian98 at the B3LYP level of theory with a 6-31G* basis set. Ab initio calculations show the molecular displacements that comprise a particular vibration, and provide insight into the functional groups that are contributing to the vibration. The calculated modes correlated well with the observed spectra. iii) The 9-MeA spectra and calculated modes are also compared with spectra of adenine which has been 15N labeled at the C6-NH2 position. The UVRR spectra and structure of the DNA triple helix is discussed within the context of these mode assignments.References and Footnotes
Eliana Tsukroff and Ishita Mukerji*
Department of Chemistry and *Molecular Biology and Biochemistry Department, Molecular Biophysics Program, Wesleyan University, Middletown, CT 06459-0175