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 T-m values of the hairpin four-way junctions are 25.2 degrees to 32.2 degrees 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 5MODIFIER LETTER PRIME -> 3MODIFIER LETTER PRIME (T5 Exo) and 3MODIFIER LETTER PRIME -> 5MODIFIER LETTER PRIME direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.

Automated summary

This study aims to analyze the structure and protein recognition features of branched DNA four-way junctions, with a focus on the therapeutic potential of these molecules. The results show that inserting hairpins into the junctions can increase stability and nuclease resistance, while maintaining similar protein binding affinity as the control.