Nuclease resistance and protein recognition properties of DNA and hybrid PNA-DNA four-way junctions.

Nucleic acids possess unique biochemical features that make them ideal candidates to inhibit "difficult to target" proteins. The limited stability of nucleic acids in vivo presents a major obstacle to their development as drugs. Here, immobile four-way junctions (4WJs) are used to target the DNA-binding cytokine, High Mobility Group B1. Hybrid 4WJs composed of DNA and peptide nucleic acids (PNA) are investigated. PNA possess enhanced nuclease stability vs. DNA. 4WJs are incubated with Exonuclease III and DNase I. The nuclease assays show that 4WJs containing multiple PNAs possess significantly higher stability. Circular dichroism assays are used to probe the groove topology of 4WJs with the minor groove binder, DAPI. The CD data indicates that multi-PNA 4WJs possess altered minor groove dimensions that reduces DAPI binding affinity. Logic suggests that the minor groove of multi-PNA hybrids possess significant perturbations to the topology and local electrostatic environment that prevents proper binding/recognition by nucleases and thus enhances stability.

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions.

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The Tm values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5'→3' (T5 Exo) and 3'→5' direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.

Characterization of the structural and protein recognition properties of hybrid PNA-DNA four-way junctions.

The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA-DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg(+2). For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg(+2). The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1 °C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b.