Proton NMR and Optical Spectroscopic Studies on the DNA Triplex Formed by d-A-(G-A)₇-G and d-C-(T-C)₇-T

Abstract

Triplex and duplex formation of two deoxyribohexadecamers d-A-(G-A)-G (a) and d-C-(T-C)-T (b) have been studied by UV, CD, fluorescence, and proton NMR spectroscopy. Optical studies of a and b at dilute concentrations (microM range) yielded results similar to those seen for polymers of the same sequence, indicating that these hexadecamers have properties similar to the polymers in regard to triplex formation. The CD spectra of concentrated NMR samples (mM range) are similar to those observed at optical concentrations at both low and high pH, making possible a correlation between CD and NMR studies. In NMR spectra, two imido NH-N hydrogen bonded resonance envelopes at 12.6 and 13.7 ppm indicate that only the duplex conformation is present at pH greater than 7.7. Four new NH-N hydrogen-bonded resonance envelopes at 12.7, 13.5, 14.2, and 14.9 ppm are observed under acidic conditions (pH 5.6) and the two original NH-N resonances gradually disappear as the pH is lowered. Assignment of these four peaks to Watson-Crick G.C. Hoogsteen T.A Watson-Crick A.T, and Hoogsteen C+.G hydrogen-bonded imidos, respectively, confirm the formation of triple-stranded DNA NMR results also show that triplex is more stable than duplex at the same salt condition and that triplex melts to single strands directly without going through a duplex intermediate. However, in the melting studies, a structural change within the triple-stranded complex is evident at temperatures significantly below the major helix-to-coil transition. These studies demonstrate the feasibility of using NMR spectroscopy and oligonucleotide model compounds a and b for the study of DNA triplex formation.

DNA triple helices: Biological consequences and therapeutic potential

“…Physical studies of the triplex structure reveal that binding of the TFO induces structural distortions in the underlying duplex even though the Watson-Crick hydrogen bonding is preserved [34,35]. These features, along with facile chemistries to covalently couple DNA damaging agents to the TFOs, make triplexes attractive probes for directing site-specific DNA damage [36][37][38].…”

Triple Helical Polynucleotidic Structures: An FTIR Study Of The C+· G · C Triplet

“…However NMR studies recently showed that some variability existed in the sugar conformations of triple helixes containing deoxyriboses (24)(25)(26)(27). Vibrational spectroscopy is a powerful technique to investigate the secondary structures of nucleic acids (28,29).…”

DNA triplex formation of oligonucleotide analogs consisting of linker groups and octamer segments that have opposite sugar-phosphate backbone polarities

“…Interestingly, the sugar-phosphate backbone polarity of the third strand is parallel to that of the homopurine strand (Arnott et al, 1976;Moser & Dervan, Triplex Formation by Opposite Polarity Oligonucleotides 1987;Santos et al, 1989, Rajagopal & Feigon, 1989a. Circular dichroism (CD) spectroscopy has been useful for monitoring triplex formation in polymer and oligomer systems Gray et al, 1987;Xodo et al, 1990;Callahan et al, 1991;Ono et al, 1991). The generation of a large negative band in the short-wavelength region (<230 nm)…”

“…In addition, ethidium bromide is a wellknown DNA binding dye whose fluorescence emission intensity is enhanced dramatically when intercalating into a DNA duplex. As this emission is reduced by the binding of a third strand to an existing duplex, ethidium bromide has been used as a probe to study triplex formation in DNA (Lee et al, , 1984Morgan et al, 1979;Xodo et al, 1990;Kan et al, 1991;Scaria & Shafer, 1991).…”

“…These bands are not observed in the spectra of the duplexes or in those of the third strands. The presence of a negative band below 230 nm has been shown to be indicative of triplex formation (Lee et al, 1984, Callahan et al, 1991Kan et al, 1991).…”