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 tr iplex-forming oligonucleotides (TFOs) [d-psTTTCTTTNTTTTCTT; ps = thiop hosphate; N = A, G, C, T, 2'-deoxyinosine (I), 2'-deoxyxanthosine (X), 5-methyl-2'-deoxycytidine (m5C), or 5-bromo-2'-deoxyuridine (br5U)] t o investigate the Hoogsteen-like hydrogen bonding to the base in the t arget 34-mer strand ATGAGTGCCAA.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 p H 5.0. The triplexes with typical triplets of T.AT (51.3-degrees-C), b r5U.AT (52.4-degrees-C), C+.GC (66.7-degrees-C), and M5C+.GC (66.8-deg rees-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 th e N.TA triplet was also stabilized by I in the N position (40.7-degree s-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 de rivative. Results showed that the increased cleavage yields reflect th e higher thermal stability of the triplex formed in most cases, but a few exceptional cases existed. Especially, the G -containing TFO did n ot show the above correlation between thermal stability and cleavage y ield. The possibility of the binding of free Cu2+ ion to a G base or t he formation of the 8-oxo-G base could explain the results. The influe nce of reducing agents on cleavage efficiency was also examined.