DNA-based fluorescent probes of NOS2 activity in live brains

Area of Science:

• Immunology
• Molecular Biology
• Microbiology

Background:

• Innate immune cells utilize phagosomes to eliminate pathogens.
• Activation of inducible nitric oxide synthase (NOS2) by pathogen-associated molecular patterns (PAMPs) engaging Toll-like receptors (TLRs) is crucial for pathogen clearance.
• Some pathogens evade immune detection by hiding PAMPs from TLRs, complicating the study of immune responses.

Purpose of the Study:

• To develop a novel method for mapping nitric oxide (NO) activity within phagosomes and endosomes.
• To investigate PAMP-TLR interactions and their role in activating NOS2.
• To identify previously unrecognized PAMPs and their corresponding TLRs involved in innate immunity.

Main Methods:

• Design and synthesis of DNA-based probes capable of ratiometrically reporting NO levels.
• Molecular programming of probes to present specific PAMP stoichiometries.
• In vivo imaging of phagosomal NO production in microglia within live zebrafish brains.

Main Results:

• The developed DNA probes successfully mapped phagosomal and endosomal NO in real-time.
• Bacterial single-stranded RNA was identified as a PAMP that activates NOS2.
• This activation was mediated through the engagement of Toll-like receptor 7 (TLR-7).

Conclusions:

• The novel DNA probe technology enables precise mapping of NO production, revealing critical immune evasion strategies.
• Single-stranded RNA from bacteria serves as a PAMP activating TLR-7, leading to pathogen clearance.
• This platform offers a versatile tool for studying diverse PAMP-TLR interactions across various organisms.