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.

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Oligodeoxynucleotides (ODN) offer great promise as therapeutics due to their high binding specificity, low toxicity and ease of synthesis. Hence, it is not surprising that ODNs are being developed as antigene and antisense reagents. ODNs can also target proteins to control gene expression. One strategy uses double stranded DNA as “decoy” sequences that are directed against transcription factors such as NF‐kB and specificity protein 1. More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, cruciform and bent DNA are directed against the damage‐associated molecular pattern molecule, High Mobility Group B1 (HMGB1). We hypothesize that immobilized DNA four‐way junctions (4WJs) can target HMGB1. To enhance the stability of immobilized 4WJs for ex vivo studies, the helical termini are modified to generate the intramolecular constructs. Phosphorothioate (PS) bonds are inserted into the 4WJ lattice to enhance nuclease resistance. In this study, the nuclease resistance of five intramolecular 4WJs and the DNA control, J1 are evaluated. J1 is a classic immobilized 4WJ that is composed of four asymmetric strands of DNA. Three nucleases are used: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I is an endonuclease that cleaves dsDNA and ssDNA in a largely non‐specific manner. Exo III has expanded functionally. Here, we focus on the 3′®5′ exonuclease activity of the enzyme. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction. The nuclease protection data shows that each intramolecular 4WJ has significantly higher nuclease resistance values than J1 against DNase I and Exo III. Circular dichroism studies are being developed to investigate the minor groove binding properties of J1 vs. the intramolecular 4WJs. We hypothesize that perturbations within the minor groove of intramolecular 4WJs may enhance the nuclease resistance of these constructs vs. J1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Research shows that cruciform and bent DNA can target the DNA‐binding cytokine, High Mobility Group B1 (HMGB1). HMGB1 was initially classified as a highly abundant architectural nuclear protein. It is now clear that HMGB1 also functions as a damage associated molecular pattern molecule (DAMP). And several studies link deleterious HMGB1 proinflammatory signaling with an array of diseases and immune disorders such as: atherosclerosis, cystic fibrosis, lupus and sepsis. Hence, HMGB1 garners a great deal of attention as a disease biomarker and therapeutic target. We hypothesize that immobilized DNA four‐way junctions (4WJs) can target HMGB1. To enhance the stability of 4WJs for ex vivo studies, the helical termini are modified to generate intramolecular constructs. Phosphorothioate (PS) bonds are inserted into the intramolecular junction lattice to enhance nuclease resistance. In this study, the secondary structure of five intramolecular 4WJs are investigated vs. the DNA control, J1. J1 is an immobilized 4WJ that is composed of four asymmetric strands of DNA. Circular dichroism (CD) studies show that the secondary structure of each intramolecular 4WJ (DNA and PS constructs) is composed of B‐ and A‐form helices. Whereas, the DNA control J1 is devoid of A‐form helices. The melting temperature (Tm) of each intramolecular 4WJ is significantly higher than J1. The Tm values for the intramolecular 4WJs are 25° – 30°C higher than J1. Electrophoretic mobility shift assays show that HMG proteins bind intramolecular 4WJs with similar affinity to J1. The data clearly shows that the lattice of an immobilized DNA 4WJ can be redesigned to enhance conformational stability and maintain: i) native secondary structure and ii) HMG binding affinity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.