Characterization of the secondary structure and protein binding affinity of an intramolecular DNA four‐way junction.

Abstract

Oligodeoxynucleotides (ODN) offer great promise as antigene and antisense reagents due to their high binding specificity, straight‐forward synthesis and low toxicity. Research shows that ODNs can also target proteins such as 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 redox sensitive damage associated molecular pattern (DAMP) molecule. And several studies link deleterious HMGB1 inflammatory signaling with an array of diseases and immune disorders such as: atherosclerosis, cystic fibrosis, lupus and sepsis. Hence, HMGB1 is gathering a great deal of attention as a biomarker and therapeutic target. We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. In order to enhance the stability of 4WJs for ex vivo studies, the helical termini of a DNA junction are modified to generate the intramolecular construct, i‐J1. The secondary structure, thermostability and protein binding affinity of i‐J1 are investigated vs. a control 4WJ (J1) and the intramolecular 4WJ, J4. Non‐denaturing gel electrophoresis assays show that i‐J1 possesses a global structural conformation that is analogous to the control 4WJ. Circular dichroism (CD) studies show that the secondary structure of i‐J1 is composed of B‐ and A‐form DNA helices. Moreover, the melting temperature (Tm) of i‐J1 is significantly higher than J1. The Tm of i‐J1 is ~ 30°C higher than J1. Finally, electrophoretic mobility shift assays (EMSAs) show that the HMG protein, HMGB1b, binds i‐J1 with nearly identical affinity to J1. The data clearly shows that the junction lattice of a DNA four‐way junction can be altered to enhance conformational stability and retain protein binding affinity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.