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

Abstract

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 5ʹ→3ʹ (T5 Exo) and 3ʹ→5ʹ direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.

Dumbbell-Shaped Antisense Oligonucleotide Prodrugs Showed Improved Antinuclease Stability and Anticancer Efficacy

“…These results indicate that ASONs modified with the hairpin structure at both ends to form the dumbbell-shaped conformation have greatly improved stability against enzymatic degradation, since both ends of the strand can be “locked” by the recessive hairpin stem helix, thus effectively avoiding hydrolytic cleavage by exonucleases in vivo . The mechanism is mainly related to repulsive forces or steric hindrance between the ASON conformation and the nuclease, that is, the end intramolecular base-pairing and loop domain may reduce conformational entropy and eliminate nuclease binding to free oligonucleotide ends. − …”

Influence of an Intrinsically Disordered Region on Protein Domains Revealed by NMR-Based Electrostatic Potential Measurements

“…This suggests that the D/E repeats interacting with the Δ30 variant remain as a random coil, which is also supported by 13 C α chemical shifts (Figure S8). To examine competition between D/E repeats and DNA, we used a four-way junction DNA (32 base pairs) named J1, which exhibits high affinities for the full-length HMGB1 , and the Δ30 variant (the binding affinity data are shown in Figure S9). When unlabeled J1 was added to a solution of the complex of 15 N DERT30 and the unlabeled Δ30 variant, the signals from 15 N-labeled DERT30 moved toward the chemical shifts for the free state (Figure A, middle).…”

“…Due to the cations condensed around the negatively charged macromolecule, the total Na + concentration in the solution is higher than the Na + concentration in the original buffer, and the slope in the plot corresponds to the number of condensed cations ΔN cation . 21 base pairs) named J1, 26 which exhibits high affinities for the full-length HMGB1 8,27 and the Δ30 variant (the binding affinity data are shown in Figure S9). When unlabeled J1 was added to a solution of the complex of 15 N DERT30 and the unlabeled Δ30 variant, the signals from 15 N-labeled DERT30 moved toward the chemical shifts for the free state (Figure 4A, middle).…”