A nucleic acid labeling chemistry reveals surface DNA on exosomes
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Chemical labeling of nucleic acids is essential to pinpoint the structure, localization, and function of RNA and DNA. Yet, reversible sequence-independent chemistries that can label native RNA and DNA remain poorly developed. Here we describe Reversible Uridine Nitrilium-mediated Addition (RUNA), a reversible covalent chemistry that selectively modifies uridine and thymidine residues via N3 deprotonation and reaction with a nitrilium ion intermediate generated from an aldehyde and an isonitrile. The reaction forms a stable N3 adduct that can be quantitatively reversed by hydrolysis. By labeling uridines and thymidines sequence-independently with reagents that are either membrane permeable or impermeable, we pinpointed the localization and function of DNA on exosomes. Although exosomes harbor nucleic acids, whether the latter are encapsulated in the exosome lumen or are surface-adhered is unknown. RUNA revealed that exosomal DNA is surface-exposed. The abundance of such surface-bound DNA increases upon DNA-damage accumulation in cancer cells that are treated with a PARP inhibitor. This surface-bound DNA drives exosome uptake by M2-polarized macrophages through scavenger receptors and triggers a shift toward an M1-like pro-inflammatory state. The selective labeling of surface DNA revealed an unexpected mechanism by which exosomes engage innate immune cells. RUNA is a versatile tool to analyze the nucleic acid content and functionality of extracellular vesicles in health and disease.
Significance Statement
Pinpointing the localization of RNA and DNA in cells and organelles is central to deriving insights into their biological functions in health and disease. We describe a new method, RUNA, for labeling nucleic acids that is sequence-independent and reversible. By varying RUNA reagents, we can distinguish between nucleic acids that are located either inside or outside of membrane compartments. Using RUNA, we showed that DNA is associated with the exterior surface of exosomes that are secreted by cancer cells. Further, the amount of exosomal DNA increases when the cancer cells are treated with an anti-cancer drug. This surface DNA promotes the uptake of exosomes by innate immune cells known as macrophages and modulates their inflammatory response.
