RELATIVE SPECIFICITIES IN BINDING OF WATSON-CRICK BASE-PAIRS BY 3RD STRAND RESIDUES IN A DNA PYRIMIDINE TRIPLEX MOTIF

The specificity of binding of Watson - Crick base pairs by third stran d nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The ho st duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T 10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 du plexes with the Watson - Crick base pairs (XY) and 12 duplexes with mi smatch pairs (XZ), all of whose stabilities were compared. Two Z:XY co mbinations confirm the primary binding of AT and GC target pairs in ho mopurine . homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic str ength and pH, all of which were previously untested. The conclusions n ow drawn also differ substantially from those in previous studies. Thu s, by evaluating the depression in Tm due to base triplet mismatches s trictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combination s that destabilize qualify for inclusion in the third strand binding c ode for the pyrimidine triplex motif. Hence, none of the mismatch trip lets afford a general way of circumventing the requirement for homopur ine . homopyrimidine targets when third strands are predominated by py rimidines, as others have suggested. At the same time, the applicabili ty of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than ar e Watson - Crick duplexes to base pair mismatches.