Energetics of a stable intramolecular DNA triple helix formation.
We have designed and synthesized by conventional chemical techniques a 38mer oligonucleotide consisting of a 5'd(Pu)10d(C)4d(Py)10d(T)4d(Py)10(3') sequence. This oligonucleotide assumes a randomly coiled conformation at pH 12. At pH 8.0 a hairpin helix forms between its 5' purine decamer sequence and the consecutive pyrimidine decamer leaving the second pyrimidine decamer as a dangling disordered 3' extension. On reducing the pH to 4.5 this second pyrimidine decamer folds back onto the major groove of the hairpin helix resulting in an intramolecular triple-stranded stem-loop structure. We have used a variety of biochemical (gel mobility, P1 nuclease digestion) and biophysical (ultraviolet light and circular dichroism spectroscopy, fluorimetry, microcalorimetry) techniques to characterize the different conformers, their stability and the folding pathway into an intramolecular triple helix. The thermodynamic properties of this intramolecular triple strand in 100 mM-Na+ are: tm, 71 degrees C; delta HvH, 119.4(+/- 11.9) kcal mol-1; delta Hcal, 121.9 (+/- 6.1) kcal mol-1 at pH 4.5; those of the hairpin are: tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.9(+/- 3.5) kcal mol-1 at pH 8.0. At intermediate pH values, the triplex to coil transition breaks up into its component triplex to hairpin and hairpin to coil transitions with thermodynamic properties: tm, 41 degrees C; delta HvH, 58.7(+/- 4.2) kcal mol-1; delta Hcal, 39.8(+/- 2.0) kcal mol-1; and tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.6(+/- 3.5) kcal mol-1 at pH 6.7.
Völker
J
J
Department of Biochemistry, University of Cape Town, Rondebosch, Republic of South Africa.
Botes
D P
DP
Lindsey
G G
GG
Klump
H H
HH
D001483:Base Sequence; D002151:Calorimetry; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases