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Method Article
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.
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.
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 the nuclear translocation is necessary.
Previously, light-triggered translocation of proteins into the cell nucleus has been achieved using caged NLS7,8,9. NLS migrates into the cell nucleus by binding to cytoplasmic transport proteins6. In the reported methods, guest proteins bearing caged NLS are directly incorporated into the cytoplasm by microinjection8 or expressed in the target cells using a genetic code expansion technique9. Therefore, a method that can achieve both cellular uptake and photo-induced nuclear translocation is advantageous for practical applications.
Herein, we describe light-triggered nuclear translocation of guests in living cells using dendritic caged molecular glue (CagedGlue-R, Figure 1) tags. Water-soluble molecular glues10,11,12,13,14,15,16,17,18,19,20,21,22,23 bearing multiple Gu+ pendants have been previously developed, which tightly adhere to proteins11,12,13,14,15,16,17, nucleic acids18,19,20, phospholipid membranes21, and clay nanosheets22,23 through the formation of multiple salt bridges between their Gu+ pendants and oxyanionic groups on the targets. The Gu+ pendants of CagedGlue-R 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 stay in endosomes (Figure 2). Upon photoirradiation, the BANVOC groups of CagedGlue-R are detached to yield an uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which then facilitates the migration of the tagged guest into the cytoplasm followed by the cell nucleus (Figure 2). The CagedGlue-R tag can be uncaged by exposure to UV or two-photon near-infrared (NIR) light without serious phototoxicity. We demonstrate the spatiotemporally controlled nuclear delivery of macromolecular guests as well as small-molecule guests with CagedGlue-R tags, using quantum dots (QDs) and a fluorescent dye (nitrobenzoxadiazole; NBD), respectively, as examples.
Figure 1: Schematic structures of CagedGlue-R. The 9 guanidinium ion (Gu+) pendants of CagedGlue-R are protected by a butyrate-substituted nitroveratryloxycarbonyl (BANVOC) group. The BANVOC groups are cleaved by irradiation with UV or two-photon NIR light. The focal core of CagedGlue-R is functionalized with either nitrobenzoxadiazole (NBD) or dibenzocylooctyne (DBCO). Reprinted with permission from reference20. Please click here to view a larger version of this figure.
Figure 2: Schematic illustration of light-triggered nuclear translocation of guests conjugated with a CagedGlue-R tag. The guest/CagedGlue-R conjugate is taken up into living cells via endocytosis. Upon photoirradiation, the CagedGlue-R tag is uncaged to yield an UncagedGlue-R tag, which can facilitate endosomal escape of the tagged guest. Subsequently, the tagged guest migrates into the cell nucleus. Reprinted with permission from reference20. Please click here to view a larger version of this figure.
1. Preparation of Guests with CagedGlue-R Tags
Figure 3: Schematic illustration of the preparation of CagedGlue-QD. Please click here to view a larger version of this figure.
2. Preparation of Hep3B Cell Sample for Microscopic Observations
3. Observation of Nuclear Translocation of Small-molecule Guests Triggered by UV Light
4. Observation of Nuclear Translocation of Small-molecule Guests Triggered by Two-photon NIR Light
5. Observation of Nuclear Translocation of Macromolecular Guests Triggered by UV Light
6. Cell Viability Assay
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, th...
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...
The authors have nothing to disclose.
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).
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 |
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