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Abstract 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 residuesviaN3 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 using reagents that are either membrane permeable or impermeable, we demonstrate 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 exosomes display DNA on their outer surface. The abundance of such surface DNA increases upon DNA-damage accumulation in cancer cells that are treated with a PARP inhibitor. This surface 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 StatementPinpointing 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 outer surface of exosomes that are secreted by cancer cells. Further, the amount of surface 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.
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Synthetic Genes For Dynamic Regulation Of DNA‐Based Receptors
Abstract We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA‐based receptors: a triplex‐forming DNA complex, an ATP‐binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling.
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- Award ID(s):
- 2107483
- PAR ID:
- 10545540
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Angewandte Chemie
- Volume:
- 136
- Issue:
- 17
- ISSN:
- 0044-8249
- Subject(s) / Keyword(s):
- DNA nanotechnology, gene networks, receptors, modeling
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/ange.202319382
