DNA triplex formed by d-A-(G-A)7-G and d-mC-(T-mC)7-T in aqueous solution at neutral pH.
Evidence from UV spectroscopic melting experiments indicated that the DNA oligonucleotide 5'-d-A-(G-A)7-G-3' (1), a repeating AG sequence found in the human genome, and its complement 5'-d-mC-(T-mC)7-T-3' (mC: 5-methyl-C) (2), can form both a triplex (with a Tm = 44 degrees C) and a duplex (with a Tm = 69 degrees C) around physiological pH (7.2) in micromolar concentration solution with 0.1 M NaCl. In addition, the triplex can be detected at a pH as high as 8.4 (Tm = 27 degrees C). The stability of the triplex formed by 1 and 2, as monitored by UV melting experiments, is found to increase as the pH is lowered from 8.4 (Tm = 27 degrees C) to 6 (Tm = 79 degrees C). However, the stability of the duplex, formed by 1 and 2, is found to be unchanged (the Tm is approximately 69 degrees C) in the same pH range. There is no triplex, as observed by UV with the oligonucleotides having identical sequences, when the regular cytosine base was used [d-C-(T-C)7-T, (3)] at a pH > 6 in the same concentration range. The stoichiometric ratio of the triplex formed with 1 and 2 is also found to be 1:2 by means of a UV mixing titration study. This result suggests that the conformation of the triplex of 1 and 2 also involves T.A.T and mC+.G.mC base triads. The formation of a triplex by 1 and 2 can be observed by native gel electrophoresis in submicromolar conditions with magnesium ion present. The results of this study strongly support the theory that replacement of regular cytidine nucleotides by 5-methylcytosine nucleotides facilitates the formation of the DNA triplex at physiological pH. This could thus be used an an anti-gene probe via the formation of triplex under the conditions described.
Lin
S B
SB
Department of Clinical Pathology, College of Medicine, National Taiwan University, Taipei, ROC.
Kao
C F
CF
Lee
S C
SC
Kan
L S
LS
D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid
D009838:Oligodeoxyribonucleotides