Neutrophil Extracellular Traps are Free-Radical Generating DNAzymes

PROJECT SUMMARY / ABSTRACT Neutrophils are the first line of host defense against invading pathogens that trap and kill pathogens through a process called NETosis. Upon stimulation by the pathogen, neutrophils extrude decondensed chromatin in the form of DNA fibers that trap invading cells and forms a scaffold to harbor associated proteins and antimicrobial peptides. Bactericidal activity has been attributed to the action of these associated proteins and peptides through oxidative stress resulting from the free radicals they generate. DNA however, the largest component of NETs, is only believed to offer the trapping scaffold despite a suicidal path a neutrophil undergoes to extrude the DNA. DNA is known to form non-canonical secondary structures like the i-motif, triple helices, and G-quadruplexes (G4). G4 is formed by the stacking of planar square structures of four guanine bases associated by Hoogsteen hydrogen binds. These secondary structures are concentrated around transcription start sites in promoter regions and genome-wide high-resolution sequencing has detected >700,000 G4s in the human genome. G4s are known to sequester free hemin to protect the cell from hemin toxicity and this complex (G4/H) subsequently regulates gene expression. In vitro, G4/H has been characterized as a DNAzyme that mimics peroxidases to decompose to hydrogen peroxide to produce hydroxyl radicals. Hydroxyl radicals are known to be most reactive and toxic to cells in vivo. While G4/H activity is well understood in vitro and the G4 is known to sequester free hemin within cells, the existence of G4 structures in the decondensed chromatin of NETs or the peroxidase like activity of G4/H in vivo is unknown. We propose to discern the existence of G4/H complexes in NETs using antibodies specific to hemin and G4 using multiple approaches like colocalization of immunofluorescence and ChIP-seq on NETs specific DNA pulled with myeloperoxidase specific antibodies from whole blood of healthy individuals after IL-8 or bacterial stimulation and patients with COVID-19. We propose to characterize the enzymatic activity of the G4/H DNAzyme in NETs and demonstrate the local concentrated effect of free radicals generated on trapped bacteria and prove that this phenomenon occurs naturally ex vivo. Finally, we propose to exhibit the biological outcomes from the G4/H DNAzyme generated free radicals by testing bactericidal activity, host cell injury, and posttranslational modifications of NETs associated proteins giving rise to autoimmune disorders like systemic lupus erythematosus.