Book of Abstracts: Albany 2003
June 17-21 2003
Structure and Energetics of a DNA Triple Helix
Investigated by UV Resonance Raman Spectroscopy
The presence of triple helical DNA has been known since the earliest studies of DNA structure (reviewed in (1)). In the past decade interest in triplex DNA has reemerged due to interest in the potential use of nucleic acids as therapeutics (2-4), and because of evidence that alternative (i.e. non-B-DNA) structures may have specific functional roles in vivo (5, 6). In triple helical DNA a third DNA strand binds in the major groove of the duplex, forming Hoogsteen base pairs with the central purine strand. The third strand binds to a particular duplex sequence with high specificity. Because of the potential uses for triplex DNA, understanding how the third strand recognizes and binds to the duplex, and what contributes to the stability of the molecule is important.
Our lab is studying the 31-mer intramolecular triple helix d(5'-AGAGAGAA-CCCC-TTCTCTCT-TTT-TCTCTCTT-3'). This oligonucleotide has a mirror repeat sequence similar to S1-hypersensitive sites found in the promoter region of several genes (7). It had been hypothesized that these mirror repeats were able to form triple-stranded DNA, and the lab of Dr. Juli Feigon presented NMR structural data showing this to be true (8). Subsequently, this triplex molecule was characterized by the lab of Dr. Irina Russu. Russu lab members reported base pair opening rates and the internal dynamics of an adenine amino group (9, 10).
In our lab, UV absorption and UV Resonance Raman (UVRR) spectra as a function of temperature were obtained. We compare the triplex with the related hairpin d(5'-AGAGAGAA-CCCC-TTCTCTCT). The frequency and intensity of the vibrational modes measured by UVRR spectroscopy as a function of temperature, reveal changes in molecular conformation and in stacking of the DNA bases. The molecules were probed using several different excitation wavelengths to differentially enhance the signal from the various bases in the molecule. The molecule was probed at 260, 240, 220, and 210 nm that preferentially enhances the signal of adenosines, guanines, protonated cytosines and ribose-associated modes, respectively.
Vibrational modes of adenosine do not exhibit melting behavior until about 50 °C, consistent with their placement in the center of the base triads, while frequencies associated with cytidines and thymidines melt at lower temperatures indicating increased flexibility, presumably in the loop regions. In addition, a 15N label at the exocyclic amino group of the A5 position, reports on the local structure of a triad in the center of the molecule. Finally, Tm?s, as determined by UVRR, are in good agreement with those determined by UV-Vis absorption spectroscopy.
Department of Chemistry
1Molecular Biology and Biochemistry Department
Molecular Biophysics Program
Middletown, CT 06459-0175
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