# Neutrophil Extracellular Traps are Free-Radical Generating DNAzymes

> **NIH NIH R21** · STANFORD UNIVERSITY · 2024 · $231,600

## Abstract

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.

## Key facts

- **NIH application ID:** 10888558
- **Project number:** 1R21AI178026-01A1
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** SAMUEL YANG
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $231,600
- **Award type:** 1
- **Project period:** 2024-03-06 → 2026-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10888558

## Citation

> US National Institutes of Health, RePORTER application 10888558, Neutrophil Extracellular Traps are Free-Radical Generating DNAzymes (1R21AI178026-01A1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10888558. Licensed CC0.

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