This protocol describes light-triggered nuclear translocation of guests in living cells using caged molecular glue tags. This method is promising for site-selective nuclear-targeting drug delivery.
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Method Article
Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags
In This Article
Summary
Abstract
The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases. Here, we demonstrate light-triggered nuclear translocation of guests using caged molecular glue (CagedGlue-R) tags, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Guests tagged with CagedGlue-R are taken up into living cells via endocytosis and remain in endosomes. However, upon photoirradiation, CagedGlue-R is converted into uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which facilitates the endosomal escape and subsequent nuclear translocation of the guests. This method is promising for site-selective nuclear-targeting drug delivery, since the tagged guests can migrate into the cytoplasm followed by the cell nucleus only when photoirradiated. CagedGlue-R tags can deliver macromolecular guests such as quantum dots (QDs) as well as small-molecule guests. CagedGlue-R tags can be uncaged with not only UV light but also two-photon near-infrared (NIR) light, which can deeply penetrate into tissue.
Introduction
The cell nucleus, which carries genetic information, is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases including cancer and genetic disorders1,2,3. For nuclear delivery of drugs, conjugation of peptide tags such as nuclear localization signals (NLS)4,5,6 has been widely investigated. However, in order to reduce undesired side effects, spatiotemporal control of t....
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Protocol
1. Preparation of Guests with CagedGlue-R Tags
- Prepare CagedGlue-NBD solution.
- Synthesize CagedGlue-NBD (Figure 1) following the procedures previously described20.
- Prepare a stock solution of CagedGlue-NBD (10 mM) in dry dimethyl sulfoxide (DMSO).
Note: Store the stock solution in dark. The solution can be diluted with aqueous buffers or cell culture media upon usage.
- Prepare CagedGlue-QD solution.
- Synthesize CagedGlue-dibenzocylooctyne (CagedGlue-DBCO) (
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Results
Before photoirradiation, Hep3B cells incubated with CagedGlue-NBD exhibited punctate fluorescence emission from their interior (λext = 488 nm; Figures 4A and 4C, green). An analogous micrograph was obtained upon excitation at 543 nm for red-fluorescent dye (Figures 4B and 4C, red), indicating that CagedGlue-NBD localized in the endosomes. Accordingly, the fluoresc.......
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Discussion
Previous investigations of light-triggered translocation of proteins into the cell nucleus have been achieved using caged NLS7,8,9. As mentioned earlier, these methods require additional techniques to incorporate the NLS-tagged proteins into the cytoplasm. In contrast, our CagedGlue-R tag enables not only photo-induced nuclear translocation but also cellular uptake of the guests. This feature of the CagedGl.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We acknowledge the Center for NanoBio Integration, the University of Tokyo. This work was supported by Grant-in-Aid for Young Scientists (B) (26810046) to K.O. and partially supported by Grant-in-Aid for Specially Promoted Research (25000005) to T.A. R.M. thanks the Research Fellowships of Japan Society for the Promotion of Science (JSPS) for Young Scientists and the Program for Leading Graduate Schools (GPLLI).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Azide-PEG4-NHS ester | Click Chemistry Tools | AZ103 | |
| Q-dot 655 ITK | Invitrogen | Q21521MP | |
| Regenerated cellulose membrane (MWCO 3,500) | NIPPON Genetics | TOR-3K | |
| Regenerated cellulose membrane (MWCO 25,000) | Harvard Apparatus | 7425-RC25K | |
| Hep3B Cells | ATCC | HB-8064 | |
| 8-chambered glass substrate | Nunc | 155411JP | |
| 96-well culture plate | Nunc | 167008 | |
| Eagle's minimal essential medium (EMEM) | Thermo Fisher Scientific | 10370-021 | |
| Fetal bovine serum (FBS) | GE Healthcare | SH30406.02 | |
| Dulbecco's phosphate buffer saline (D-PBS) | Wako Pure Chemical Industries | 045-29795 | |
| LysoTracker Red | Lonza Walkersville | PA-3015 | |
| Hoechst 33342 | Dojindo | H342 | |
| Cell Counting Kit-8 | Dojindo | CK04 | |
| Confocal laser scanning microscope | Carl-Zeiss | LSM 510 | Equipped with two-photon excitation laser (Mai Tai laser, Spectra-Physics) |
| Confocal laser scanning microscope | Leica | TCS SP8 | |
| Xenon light source | Asahi Spectra | LAX-102 | |
| Microplate reader | Molecular Devices | SpectraMax Paradigm |
References
- Miller, A. D. Human gene therapy comes of age. Nature. 357, 455-460 (1992).
- Roth, J. A., Cristiano, R. J. Gene Therapy for Cancer: What Have We Done and Where Are We Going. Journal of the National Cancer Institute. 89, 21-39 (1997).
- Verma, I. M., Weitzman, M....
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