Detailed study of sequence-specific DNA cleavage of triplex-forming oligonucleotides linked to 1,10-phenanthroline.
We introduced eight bases, including four base analogs, into 15-mer triplex-forming oligonucleotides (TFOs) [d-psTTTCTTTNTTTTCTT; ps = thiophosphate; N = A, G, C, T, 2'-deoxyinosine (I), 2'-deoxyxanthosine (X), 5-methyl-2'-deoxycytidine (m5C), or 5-bromo-2'-deoxyuridine(br5U)] to investigate the Hoogsteen-like hydrogen bonding to the base in the target 34-mer strand (d-TGAGTGAGTAAAGAAARAAAAGAATGAGTGCCAA.d-TTGGCACTCATTCTTTTYTTTCT TTACTCACTCA; RY = AT, GC, TA, or CG). We examined the thermal stability of 15-mer triplexes in buffer containing 100 mM sodium acetate and 1 M NaCl at pH 5.0. The triplexes with typical triplets of T.AT (51.3 degrees C), br5U.AT (52.4 degrees C), C+.GC (66.7 degrees C), and m5C+.GC (66.8 degrees C) at the central position showed relatively higher Tm values, as expected. The relatively high stability of the X.AT triplex (39.8 degrees C) was observed. Among the N.TA triplets, G.TA (44.8 degrees C) was thermally the most stable, and moreover, the data showed that the N.TA triplet was also stabilized by I in the N position (40.7 degrees C). Furthermore, the TFOs were converted to DNA-cleaving molecules by introducing a newly synthesized 1,10-phenanthroline (OP) derivative on the thiophosphate group at the 5' end. Cleavage reactions of the 32P-labeled DNA (34-mer) were carried out. The cleavage efficiencies were compared to the Tm values of triplexes with or without an OP derivative. Results showed that the increased cleavage yields reflect the higher thermal stability of the triplex formed in most cases, but a few exceptional cases existed. Especially, the G-containing TFO did not show the above correlation between thermal stability and cleavage yield.(ABSTRACT TRUNCATED AT 250 WORDS)
Shimizu
M
M
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Inoue
H
H
Ohtsuka
E
E
D015097:3-Mercaptopropionic Acid / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002614:Chelating Agents; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004355:Drug Stability; D005978:Glutathione / Q000494:pharmacology; D006358:Hot Temperature; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010618:Phenanthrolines / Q000378:metabolism*
D002614:Chelating Agents; D009838:Oligodeoxyribonucleotides; D010618:Phenanthrolines; D003300:Copper; D004247:DNA; D015097:3-Mercaptopropionic Acid; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid; D004229:Dithiothreitol; C025205:1,10-phenanthroline