TARGETING PYRIMIDINE SINGLE STRANDS BY TRIPLEX FORMATION - STRUCTURALOPTIMIZATION OF BINDING

Recent reports describe a new strategy for the binding of single-stran ded pyrimidine sequences by triple helix formation. In this approach, a double-length purine-rich oligonucleotide binds a target strand, fol ding back to form an antiparallel pur pur pyr triple helix. We now des cribe a series of studies in which sequence and structural variations are made in such purine-rich ligands, in an effort to optimize binding properties. Comparison is made between the use of two separate strand s and the use of single two-domain ligands; the latter are found to bi nd more tightly and to aggregate less in media containing Na+ or K+. P lacement of mismatched bases in the target shows that sequence selecti vity of binding is as high as that for Watson-Crick duplex formation, Variation of the lengths and sequences of loops bridging the binding d omains demonstrates that dinucleotide loops composed of pyrimidines gi ve the highest stability. Oligoethylene glycol-derived loop replacemen ts are shown to give good binding affinity as well. The binding of an RNA target is shown to occur with the same affinity as the binding of DNA, In general, it is found that circular variants bind more tightly than do either separate strands or singly-linked ligands and unlike li near oligomers, the circular compounds do not aggregate to a large ext ent even in buffers containing 100 mM K+. Such structurally optimized ligands are useful in expanding the number of possible naturally-occur ring sequences which can be targeted by tripler formation.