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.
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.
Kan
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Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205.
Callahan
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Trapane
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Miller
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Ts'o
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Huang
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DH
D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D003854:Deoxyribonucleotides / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009691:Nucleic Acid Denaturation; D011522:Protons; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D003854:Deoxyribonucleotides; D011522:Protons; D004247:DNA