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-TGAGTG-AGTAAAGAAARAAAAGAATGAGTGCCAA.d-TTGGCACTCATTCTTTTYTTTCTTTACT-CACTCA; 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 T(m) 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 P-32 -labeled DNA (34-mer) were carried out. The cleavage efficiencies were compared to the T(m) 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. The possibility of the binding of free Cu2+ ion to a G base or the formation of the 8-oxo-G base could explain the results. The influence of reducing agents on cleavage efficiency was also examined.