The modeling, synthesis, and characterization of oligomers containing 2-aminoquinazolin-5-yl 2'-deoxynucleotide residues are reported. The 2-aminoquinazoline residues sequence specifically bind via Hoogsteen base pairing as a third strand in the center of the major groove at T:A base pair Watson-Crick duplex sequences. Evidence for the formation of a sequence specific three-stranded structure is based on thermal denaturation UV-vis and fluorescence studies. The novel 2-aminoquinazoline C-nucleotide is a component of a system designed to overcome the homopurine requirement for triple helix structures.

Here, we describe the 1.6-A X-ray structure of the DDD (Dickerson-Drew dodecamer), which has been covalently modified by the tethering of four cationic charges. This modified version of the DDD, called here the DDD(4+), is composed of [d(CGCGAAXXCGCG)](2), where X is effectively a thymine residue linked at the 5 position to an n-propyl-amine. The structure was determined from crystals soaked with thallium(I), which has been broadly used as a mimic of K(+) in X-ray diffraction experiments aimed at determining positions of cations adjacent to nucleic acids. Three of the tethered cations are directed radially out from the DNA. The radially directed tethered cations do not appear to induce structural changes or to displace counterions. One of the tethered cations is directed in the 3' direction, toward a phosphate group near one end of the duplex. This tethered cation appears to interact electrostatically with the DNA. This interaction is accompanied by changes in helical parameters rise, roll, and twist and by a displacement of the backbone relative to a control oligonucleotide. In addition, these interactions appear to be associated with displacement of counterions from the major groove of the DNA.

A new strategy to form stable and sequence-specific triple helical DNA structures at mixed purine/pyrimidine sequences using a combination of four C-glycosides (TRIPsides) has been described [Li et al. (2003) J. Am. Chem. Soc. 125, 2084]. The partial realization of the approach is demonstrated by incorporating two of the four TRIPsides into oligomers that can potentially fold into intramolecular triplexes that contain one or two major groove crossovers of the purine Hoogsteen H-bond information. Using temperature-dependent electronic and fluorescence spectroscopy and differential scanning calorimetry, it is demonstrated that stable triplexes form at physiological conditions at non-homopurine targets. In addition, triplexes using the TRIPsides form in a highly sequence specific manner.

Stable DNA triple-helical structures are normally restricted to homopurine sequences. We have described a system of four heterocyclic bases (TRIPsides) that, when incorporated into oligomers (oligoTRIPs), can recognize and bind in the major groove to any native sequence of DNA [Li et al., J. Am. Chem. Soc. 2003, 125, 2084]. To date, we have reported on triplex-forming oligomers composed of two of these TRIPsides, i.e., antiTA and antiGC, and their ability to form intramolecular triplexes at mixed purine/pyrimidine sequences. In the present study, we describe the synthesis and characterization of the antiCG TRIPside and its use in conjunction with antiTA and antiGC to form sequence-specific intra- and/or intermolecular triplex structures at mixed purine/pyrimidine sequences that require as many as four major groove crossovers.

We have investigated the unfolding thermodynamics for incorporating cationic side chains in the Dickerson-Drew dodecamer duplex. Incorporation of two 3-aminopropyl-2'-deoxyuridine residues (one on each self-complementary strand) lowers the stability of the duplex. This reduction is driven by unfavorable heat contributions due to the removal of electrostricted water and higher exposure of polar and nonpolar atomic groups that immobilize structural water. These cationic chains effectively remove counterions from the major groove, neutralizing some negatively charged phosphates. The overall results are consistent with the NMR solution of the modified duplex that showed a small bend at each modified site.