The minor groove topology of nucleic acid ligands plays a key role in nuclease resistance and targeting of HMGB1

Abstract

The architectural DNA‐binding protein High Mobility Group B1 (HMGB1) binds distorted double stranded DNA (e.g. kinked, cruciform) in the nucleus with extremely high affinity to modulate nuclear homeostasis. HMGB1 is a multifunctional protein that also plays a pivotal role outside of the nucleus. It is now clear that HMGB1 can also function as a Damage Molecular Pattern Molecule. In this capacity, HMGB1 can act as a friend or foe to: i)bolster innate immunity to ward off pathogens or ii)exacerbate unwanted immune responses that worsen disease states (e.g. lupus, rheumatoid arthritis). Research shows that cruciform DNA [four‐way junctions (4WJs)] and single strand DNA (ssDNA) can effectively bind/sequester HMGB1. The 4WJs and ssDNA investigated contain natural and phosphorothioate (PS) bonds. PS bonds are introduced to enhance nuclease resistance. Our prior study shows that intramolecular 4WJs composed of natural and PS bonds possess enhanced resistance against the endo‐ and exonucleases. We hypothesize that the increase in nuclease resistance is due to a combination of three factors: i)restricting access to the helical termini (end‐capping), ii)the presence of PS bonds and iii)altered minor groove topology. The objective of this study is to more stringently investigate the potential connection between minor groove topology and nuclease binding. Here, three nucleases are used that cleave double and ssDNA: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). The nucleases bind within the minor groove to initiate hydrolysis via a non‐specific (DNase I) or specific manner (Exo III and T5 Exo). We hypothesize that nucleases do not bind strongly to nucleic acid substrates with altered (likely wider) minor groove dimensions. As a result, nucleic acids with wider minor grooves tend to have enhanced nuclease resistance. Circular dichroism (CD) studies are used to investigate the minor groove topology of intramolecular 4WJs and ssDNA. The minor groove binder 4’,6‐diamidino‐2‐phenylindole (DAPI) is used to estimate the gross minor groove topology of each nucleic acid ligand. The data clearly shows the DNA substrates with altered minor groove dimensions, as reflected by weakened DAPI binding interactions, possess enhanced nuclease resistance. DNA substrates with enhanced nuclease resistance will be investigated further to target the DNA‐binding cytokine, HMGB1.