Nano-NucleOTAC-mediated Z-DNA transformation disrupts mycobacterium tuberculosis biofilms and activates antibacterial immunity.

The biofilms of Mycobacterium Tuberculosis (Mtb) use extracellular DNA (eDNA) as a protective structural scaffold that shields resident bacteria from host immunity and antibiotics, promoting persistent infection. An FDA-approved DNase can partially hydrolyze eDNA but has limited affinity and nucleolytic activity against Z-form DNA (Z-DNA). After confirming the abundance of Z-DNA in Mtb biofilms, we develop Nano-NucleOTAC, a nano-nucleolysis targeting chimera that combines an eDNA-binding ligand (lactoferrin), a DNA intercalator (chloroquine) and DNase. Nano-NucleOTAC anchors to Z-DNA in Mtb H37Ra biofilms, enabling effective biofilm degradation via Z-DNA transformation and enzymatic cleavage. Following treatment, dispersed Mtb H37Ra clusters induce neutrophil extracellular trap (NET) formation and cytokine secretion (TNF, IL-1β, and IL-10) to recruit macrophages, while NET remodeling and nucleolysis enhance macrophage bactericidal effect vs. DNase alone. In mouse lung infection models, Nano-NucleOTAC improves lung drug retention, reduces pathology, and reactivates host immune responses. It also demonstrates high efficacy against Mtb strains of varying virulence (H37Ra, H37Rv) and non-tuberculous mycobacteria (Mycobacterium smegmatis). In clinical sputum samples, Nano-NucleOTAC potentiates rifampicin efficacy, and enhances neutrophil and macrophage antibacterial immunity in bronchoalveolar lavage fluid (BALF) and pus. This safe, versatile nucleic-acid-targeting strategy modulates bacterial biophysical barriers and host immunity to address biofilm-associated multidrug-resistant infections.