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

• Immobilized four-way junctions assembled from PNA and DNA strands contain mixed A- and B-form helices.
• The apparent level of A-form helical content of hybrid PNA–DNA four-way junctions is correlative with PNA content.
• The secondary structure of hybrid PNA–DNA junctions composed of single PNA strands is sensitive to Mg+2.
• The DNA-binding protein, HMGB1b, readily recognizes hybrid PNA–DNA 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.

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