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° 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.

本研究的目的是评估分支 DNA 四向连接的结构和蛋白质识别特征，以探索这些分子的治疗潜力。经典的固定 DNA 4WJ，J1，用作设计新型分子内连接的基质，包括天然和硫代磷酸酯键。在这里，我们将 H2 型微型发夹插入 J1 臂的螺旋末端，以生成四个新的分子内四通接头。插入发夹以减少末端磨损并有效消除潜在的核酸酶结合位点。我们将 J1 的结构和蛋白质识别特征与四个分子内四通连接进行比较：i -J1、i -J1(PS1)、i -J1(PS2) 和i-J1（PS3）。圆二色性研究表明，每个分子内 4WJ 的二级结构主要由 B 型螺旋组成。热展开研究表明，分子内四通结明显比 J1 更稳定。Tm _{_}发夹式四通接头的值比对照 J1 高 25.2° 至 32.2°C。关于蛋白质识别，凝胶位移测定表明 DNA 结合蛋白 HMGBb1 和 HMGB1 以与对照 J1 相似的亲和力水平结合发夹四向连接。为了评估核酸酶抗性，将四向连接与 DNase I、核酸外切酶 III (Exo III) 和 T5 核酸外切酶 (T5 Exo) 一起孵育。这些酶探测非特异性 (DNase I) 和 5ʹ→3ʹ (T5 Exo) 和 3ʹ→5ʹ 方向 (Exo III) 发生的核酸切割。核酸酶消化测定清楚地表明，分子内四向连接比对照 J1 具有显着更高的核酸酶抗性。