DNA triple helix formation at target sites containing several pyrimidine interruptions: Stabilization by protonated cytosine or 5-(1-propargylamino)dU

DNase I footprinting has been used to study the formation of parallel tripl exes at oligopurine target sequences which are interrupted by pyrimidines a t regular intervals. TA interruptions are targeted with third strand oligon ucleotides containing guanine, generating G.TA triplets, while CG base pair s are targeted with thymine, forming T.CG triplets. We have attempted to op timize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third stran d thymines with the positively charged analogue 5-(1-propargylamino)dU (U-P ). For the target sequence (AAAT)(5)AA, in which pyrimidines are positioned at every fourth residue, tripler formation with TG-containing oligonucleot ides is only detected in the presence of a tripler-binding ligand, though s table triplexes were detected at the target site (AAAAAT)(3)AAAA. Triplex s tability at targets containing pyrimidines at every fourth residue is incre ased by introducing guanines into the duplex repeat unit using the targets (AGAT)(5)AA and (ATGA)(5)AA. In contrast, placing C+.GC triplets on the 5'- side of G.TA, using the target (AGTA)(5)TT, produces complexes of lower sta bility. We have attempted further to increase the stability of these comple xes by using the positively charged thymine base analogue UP, and have show n that (TU(P)TG)(5)TT forms a more stable complex with target (AAAT)(5)AA t han the unmodified third strand, generating a footprint in the absence of a tripler-binding ligand. Tripler formation at (AGTA)(5)AA is improved by us ing the modified oligonucleotide (TCGU(P))(5)TT, generating a complex in wh ich the charged triplets C+.GC and U-P.AT alternate with uncharged triplets . In contrast, placing U-P.AT triplets adjacent to C+.GC, using the third s trand oligonucleotide (U(P)CGT)(5)TT, reduces tripler formation, while the third strand with both substitutions, (U(P)CGU(P))(5)TT, produces a complex with intermediate stability. It appears that, although adjacent U-P.AT tri plets form stable triplexes, placing U-P.AT adjacent to C+.GC is unfavorabl e. Similar results were obtained with fragments containing CG inversions wi thin the oligopurine tract, though triplexes at (AAAAAC)(3)AA were only det ected in the presence of a tripler-binding ligand. Placing C+.GC on the 5'- side of T.CG triplets also reduces tripler formation, while a 3'-C+.GC prod uces complexes with increased stability.