Evidence for a DNA tripler in a recombination-like motif: I. Recognition of Watson-Crick base pairs by natural bases in a high-stability triplex

Data are presented on a tripler type with two parallel homologous strands f or which tripler formation is almost as strong as duplex formation at least for some sequences and even at pH 7 and 0.2 M NaCl. The evidence mainly re sts upon comparing thermodynamic properties of similar systems, A paperclip oligonucleotide d(A(12)C(4)T(12)C(4)A(12)) with two linkers C-4 obviously can form a triplex with parallel backfolded adenine strand regions, because the single melting transition of this complex splits in two transitions by introducing mismatches only in the third strand region. Respectively, a ha irpin duplex d(A(12)C(4)T(12)) and a single strand d(A(12)) form a tripler as a 1:1 complex in which the second adenine strand is parallel oriented to the homologous one in the Watson-Crick paired duplex. In this system the m elting temperature T-m of the tripler is practically the same as that of th e duplex d(A(12)).d(T-12), at least within a complex concentration range of 0.2-4.0 muM. The melting behaviour of complexes between tripler stabilizin g ligand BePI and the system hairpin duplex plus single strand supports the tripler model. Non-denaturing gel electrophoresis suggests the existence o f a tripler for a system in which five of the twelve A.T*A base triads are substituted by C.G*C base triads. The recognition between any substituted Watson-Crick base pair (X.Y) in the hairpin duplex d(A(4)XA(7)C(4)T(7)YT(4)) and the correspondingly replaced base (Z) in the third strand d(A(4)ZA(7)) is mutually selective. All triple xes with matching base substitutions (Z = X) have nearly the same stability (T-m values from 29 to 33.5 degreesC), whereas triplexes with non-matching substitutions (Z not equal X) show a clearly reduced stability (T-m values from 15 to 22 degreesC) at 2 muM equimolar oligonucleotide concentration. Most nucleic acid triple helices hitherto known are limited to homopurine-h omopyrimidine sequences in the target duplex. A stable tripler formation is demonstrated for inhomogeneous sequences tolerating at least 50% pyrimidin e content in the homologous strands. On the basis of the surprisingly simil ar thermodynamic parameters for duplex and tripler, and of the fact that th is tripler type seems to be more stable than many other natural DNA triplex es known, and an the basis of semiempirical and molecule mechanical calcula tions, we postulate bridging interactions of the third strand with the two other strands in the tripler according to the recombination motif, This tri pler, denoted by us 'recombination-like form', tolerates heterogeneous base sequences, Copyright (C) 2001 John Whey & Sons, Ltd.