Influence of sequence-dependent cytosine protonation and methylation on DNA triplex stability.
To investigate cytosine protonation and its influence on the sequence-dependent thermal stability of DNA triplexes in detail, we have employed homo- and heteronuclear NMR experiments on specifically (15)N-labeled oligodeoxynucleotides that were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. These experiments reveal that cytosines in central positions of the triplex are significantly protonated even at neutral pH. However, semiprotonation points for individual cytosine bases as determined from pH-dependent measurements show considerable differences depending on their position. Thus, protonation is disfavored for adjacent cytosines or for cytosines at the triplex termini, resulting in a smaller contribution to the overall free energy of the triple helical system. In contrast, protonation of the base upon substitution of 5-methylcytosine for cytosine in the triplex third strand is only affected to a minor extent, and triplex stabilization by the methyl substituent is shown to primarily arise from stacking energies and/or hydrophobic effects.
Leitner
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D
Institut für Chemie der Freien Universität Berlin, Takustrasse 3, und Institut für Biochemie der Freien Universität Berlin, Fabeckstrasse 36A, D-14195 Berlin, Germany.
Schröder
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W
Weisz
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K
D044503:5-Methylcytosine; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D006863:Hydrogen-Ion Concentration; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D011522:Protons
D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D011522:Protons; C051802:triplex DNA; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA