Parallel Triple Helix Research Articles

Stabilization of triple helical DNA by a benzopyridoquinoxaline intercalator.

Biophysical, footprinting, and chemical probing experiments are described which characterize the triple helix-stabilizing effects of a benzo[f]pyridoquinoxaline derivative BfPQ-4,3 structurally related to the previously reported benzo[f]pyridoindole compound BePI [Mergny et al. (1992) Science 256, 1681-1684]. Two parallel triple helix model systems have been investigated; one in which the third strand matched perfectly a 27 base pair purine-pyrimidine motif in target DNA and another in which the third strand was one nucleotide longer, i.e., a 28-mer. In the latter system, the pairing of the (Y)28 third strand to the (Y.R)27 target induces the formation of a bulge containing at least one unpaired base, which can be evidenced by chemical probing experiments with osmium tetroxide. BPQ, which uinwinds a duplex DNA by 17 degrees as judged by viscometric experiments and otherwise behaves as a typical nonspecific intercalculating drug, promotes the formation of Y.R.Y parallel triple helix containing both T.A.T and C.G.C+ triplets. Both DNase I and MPE.FeII footprinting experiments concur that triplex formation with the target (Y.R)27 sequence can be detected in the presence of BPQ at about 10-fold lower oligonucleotide concentrations than are required to produce an equivalent footprint in the absence of the drug. In addition, BPQ will promote binding to the polypurine-polypyrimidine target sequence by the longer mismatched oligonucleotide, providing significant stabilization of the parallel bulge-containing(Y.R)27,(Y)28 triplex with nearly the same efficiency as the bulge-free (Y.R)27.(Y)28 triplex. Thus in vivo BPQ might enhance the formation of both undesired and desired DNA triplexes. By performing an MPE*FeII probing reaction with a 5'-32 P-labeled oligonucleotide third strand, we have obtained evidence that BPQ is actually bound to the triplex region and may distort in a sequence-specific fashion.

Footprinting Studies on Ligands which Stabilize DNA Triplexes: Effects on Stringency within a Parallel Triple Helix

We have examined the effect of four triplex-binding ligands on the interaction of the oligodeoxynucleotides T8NT8 (N = A, G, C, T) with DNA fragments containing the sequences A8XA8.T8YT8 (X = G, C, T; Y = C, G, A) by DNase I footprinting. The ligands form a series of quinoline derivatives with an alkylamine chain in the 4-position and different aryl substituents in the 2-position. By themselves these compounds do not alter DNase I digestion of the DNA duplexes at concentrations up to 100 microM. At a concentration of 10 microM they potentiate triplex formation, lowering the concentration of oligonucleotide required to produce a clear footprint by as much as 100-fold. As well as stabilizing triplexes which consist of well-characterized DNA triplets, they also promote the formation of complexes which contain central triplet mismatches. This reduction in the stringency of triple helix formation may be used to broaden the range of triplex target sequences and enable recognition at sites which contain short regions for which there are no good triplet matches.

An energetic evaluation of a “Smith” collagen microfibril model

An energy minimized three-dimensional structure of a collagen microfibril template was constructed based on the five-stranded model of Smith (1968), using molecular modeling methods and Kollman force fields (Weiner and Kollman, 1981). For this model, individual molecules were constructed with three identical polypeptide chains [Gly-Pro-Pro)n, (Gly-Prop-Hyp)n, or (Gly-Ala-Ala)n, where n = 4, 12, and 16) coiled into a right-handed triple-helical structure. The axial distance between adjacent amino acid residues is about 0.29 nm per polypeptide chain, and the pitch of each chain is approximately 3.3 residues. The microfibril model consists of five parallel triple helices packed so that a left-handed superhelical twist exists. The structural characteristics of the computed microfibril are consistent with those obtained for collagen by X-ray diffraction and electron microscopy. The energy minimized Smith microfibril model for (Gly-Pro-Pro)12 has an axial length of about 10.2 nm (for a 36 amino acid residue chain), which gives an estimated D-spacing (234 amino acids per chain) of approximately 66.2 nm. Studies of the microfibril models (Gly-Pro-Pro)12, (Gly-Pro-Hyp)12, and (Gly-Ala-Ala)12 show that nonbonded van der Waals interactions are important for microfibril formation, while electrostatic interactions contribute to the stability of the microfibril structure and determine the specificity by which collagen molecules pack within the microfibril.