Name: spatiotemporally controlled nuclear translocation of guests in living cells using caged molecular glues as photoactivatable tags
Uploaded: 2019-01-17T13:00:00
Description: Watch this Scientific Journal Video about Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags at JoVE.com

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).

1. Preparation of Guests with CagedGlue-R Tags
<ol>
	<li>Prepare CagedGlue-NBD solution.
	<ol>
		<li>Synthesize CagedGlue-NBD (Figure 1) following the procedures previously described20.</li>
		<li>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.</li>
	</ol>
	<...

<ol>
	<li>Miller, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gene+therapy+comes+of+age.">Human gene therapy comes of age.</a> Nature. 357, 455-460 (1992).</li><li>Roth, J. A., Cristiano, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+for+Cancer:+What+Have+We+Done+and+Where+Are+We+Going.">Gene Therapy for Cancer: What Have We Done and Where Are We Going.</a> Journal of the National Cancer Institute. 89, 21-39 (1997).</li><li>Verma, I. M., Weitzman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GENE+THERAPY:+Twenty-First+Century+Medicine.">GENE THERAPY: Twenty-First Century Medicine.</a> Annual Review of Biochemistry. 74, 711-738 (2005).</li><li>Ragin, A. D., Morgan, R. A., Chmielewski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Import+Mediated+by+Nuclear+Localization+Signal+Peptide+Sequences.">Cellular Import Mediated by Nuclear Localization Signal Peptide Sequences.</a> Chemistry &amp; Biology. 9, 943-948 (2002).</li><li>Martin, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+protein+targeting+to+the+nucleolus.">Principles of protein targeting to the nucleolus.</a> Nucleus. 6, 314-325 (2015).</li><li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+nuclear+targeting+ability+of+nuclear+localization+signals.">Factors influencing the nuclear targeting ability of nuclear localization signals.</a> Journal of Drug Targeting. 24, 927-933 (2016).</li><li>Ventura, B. D., Kuhlman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Go+in!+Go+out!+Inducible+control+of+nuclear+localization.">Go in! Go out! Inducible control of nuclear localization.</a> Current Opinion in Chemical Biology. 34, 62-71 (2016).</li><li>Watai, Y., Sase, I., Shiono, H., Nakano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+nuclear+import+by+light-induced+activation+of+caged+nuclear+localization+signal+in+living+cells.">Regulation of nuclear import by light-induced activation of caged nuclear localization signal in living cells.</a> FEBS Letters. 488, 39-44 (2001).</li><li>Engelke, H., Chou, C., Uprety, R., Jess, P., Deiters, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Protein+Function+through+Optochemical+Translocation.">Control of Protein Function through Optochemical Translocation.</a> ACS Synthetic Biology. 3, 731-736 (2014).</li><li>Mogaki, R., Hashim, P. K., Okuro, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guanidinium-based+&amp;#34;molecular+glues&amp;#34;+for+modulation+of+biomolecular+functions.">Guanidinium-based &amp;#34;molecular glues&amp;#34; for modulation of biomolecular functions.</a> Chemical Society Reviews. 46, 6480-6491 (2017).</li><li>Okuro, K., Kinbara, K., Tsumoto, K., Ishii, N., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Glues+Carrying+Multiple+Guanidinium+Ion+Pendants+via+an+Oligoether+Spacer:+Stabilization+of+Microtubules+against+Depolymerization.">Molecular Glues Carrying Multiple Guanidinium Ion Pendants via an Oligoether Spacer: Stabilization of Microtubules against Depolymerization.</a> Journal of the American Chemical Society. 131, 1626-1627 (2009).</li><li>Okuro, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+Effects+of+a+Guanidinium+Ion+Appended+Dendritic+&amp;#34;Molecular+Glue&amp;#34;+on+the+ATP-Driven+Sliding+Motion+of+Actomyosin.">Adhesion Effects of a Guanidinium Ion Appended Dendritic &amp;#34;Molecular Glue&amp;#34; on the ATP-Driven Sliding Motion of Actomyosin.</a> Angewandte Chemie, International Edition. 48, 3030-3033 (2010).</li><li>Uchida, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoclickable+Dendritic+Molecular+Glue:+Noncovalent-to-Covalent+Photochemical+Transformation+of+Protein+Hybrids.">Photoclickable Dendritic Molecular Glue: Noncovalent-to-Covalent Photochemical Transformation of Protein Hybrids.</a> Journal of the American Chemical Society. 135, 4684-4687 (2013).</li><li>Garzoni, M., Okuro, K., Ishii, N., Aida, T., Pavan, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Shape+Effects+of+Molecular+Glue+on+Supramolecular+Tubulin+Assemblies.">Structure and Shape Effects of Molecular Glue on Supramolecular Tubulin Assemblies.</a> ACS Nano. 8, 904-914 (2014).</li><li>Mogaki, R., Okuro, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+glues+for+manipulating+enzymes:+trypsin+inhibition+by+benzamidine-conjugated+molecular+glues.">Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues.</a> Chemical Science. 6, 2802-2805 (2015).</li><li>Okuro, K., Sasaki, M., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boronic+Acid-Appended+Molecular+Glues+for+ATP-Responsive+Activity+Modulation+of+Enzymes.">Boronic Acid-Appended Molecular Glues for ATP-Responsive Activity Modulation of Enzymes.</a> Journal of the American Chemical Society. 138, 5527-5530 (2016).</li><li>Mogaki, R., Okuro, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+Photoswitch:+Selective+Photochemical+Modulation+of+Enzymes+under+Physiological+Conditions.">Adhesive Photoswitch: Selective Photochemical Modulation of Enzymes under Physiological Conditions.</a> Journal of the American Chemical Society. 139, 10072-10078 (2017).</li><li>Hashim, P. K., Okuro, K., Sasaki, S., Hoashi, Y., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductively+Cleavable+Nanocaplets+for+siRNA+Delivery+by+Template-Assisted+Oxidative+Polymerization.">Reductively Cleavable Nanocaplets for siRNA Delivery by Template-Assisted Oxidative Polymerization.</a> Journal of the American Chemical Society. 137, 15608-15611 (2015).</li><li>Hatano, J., Okuro, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+Bioorthogonal+1,3-Dipolar+Poly-cycloaddition+Promoted+by+Oxyanionic+Substrates+for+Spatiotemporal+Operation+of+Molecular+Glues.">Photoinduced Bioorthogonal 1,3-Dipolar Poly-cycloaddition Promoted by Oxyanionic Substrates for Spatiotemporal Operation of Molecular Glues.</a> Angewandte Chemie, International Edition. 55, 193-198 (2016).</li><li>Arisaka, A., Mogaki, R., Okuro, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caged+Molecular+Glues+as+Photoactivatable+Tags+for+Nuclear+Translocation+of+Guests+in+Living+Cells.">Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells.</a> Journal of the American Chemical Society. 140, 2687-2692 (2018).</li><li>Suzuki, Y., Okuro, K., Takeuchi, T., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction-Mediated+Dynamic+Disordering+of+Phospholipid+Membrane+by+Mechanical+Motions+of+Photoresponsive+Molecular+Glue:+Activation+of+Ion+Permeation.">Friction-Mediated Dynamic Disordering of Phospholipid Membrane by Mechanical Motions of Photoresponsive Molecular Glue: Activation of Ion Permeation.</a> Journal of the American Chemical Society. 134, 15273-15276 (2012).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-water-content+mouldable+hydrogels+by+mixing+clay+and+a+dendritic+molecular+binder.">High-water-content mouldable hydrogels by mixing clay and a dendritic molecular binder.</a> Nature. 463, 339-343 (2010).</li><li>Tamesue, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+versus+Dendritic+Molecular+Binders+for+Hydrogel+Network+Formation+with+Clay+Nanosheets:+Studies+with+ABA+Triblock+Copolyethers+Carrying+Guanidinium+Ion+Pendants.">Linear versus Dendritic Molecular Binders for Hydrogel Network Formation with Clay Nanosheets: Studies with ABA Triblock Copolyethers Carrying Guanidinium Ion Pendants.</a> Journal of the American Chemical Society. 135, 15650-15655 (2013).</li><li>Mohr, D., Frey, S., Fischer, T., G&#252;ttler, T., G&#246;rlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+passive+permeability+barrier+of+nuclear+pore+complexes.">Characterisation of the passive permeability barrier of nuclear pore complexes.</a> EMBO Journal. 28, 2541-2553 (2009).</li><li>Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry+and+Bioorthogonal+Reactions:+Unprecedented+Selectivity+in+the+Labeling+of+Biological+Molecules.">Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules.</a> Biochemistry. 48, 6571-6584 (2009).</li><li>Kl&#225;n, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoremovable+Protecting+Groups+in+Chemistry+and+Biology:+Reaction+Mechanisms+and+Efficacy.">Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy.</a> Chemical Reviews. 113, 119-191 (2013).</li></ol>

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 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58631/58631fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58631/58631fig2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58631/58631fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Azide-PEG4-NHS ester</td>
			<td>Click Chemistry Tools</td>
			<td>AZ103</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-dot 655 ITK</td>
			<td>Invitrogen</td>
			<td>Q21521MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Regenerated cellulose membrane (MWCO 3,500)</td>
			<td>NIPPON Genetics</td>
			<td>TOR-3K</td>
			<td></td>
		</tr>
		<tr>
			<td>Regenerated cellulose membrane (MWCO 25,000)</td>
			<td>Harvard Apparatus</td>
			<td>7425-RC25K</td>
			<td></td>
		</tr>
		<tr>
			<td>Hep3B Cells</td>
			<td>ATCC</td>
			<td>HB-8064</td>
			<td></td>
		</tr>
		<tr>
			<td>8-chambered glass substrate</td>
			<td>Nunc</td>
			<td>155411JP</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>Nunc</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#39;s minimal essential medium (EMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10370-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GE Healthcare</td>
			<td>SH30406.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline (D-PBS)</td>
			<td>Wako Pure Chemical Industries</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Red</td>
			<td>Lonza Walkersville</td>
			<td>PA-3015</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Dojindo</td>
			<td>H342</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8</td>
			<td>Dojindo</td>
			<td>CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl-Zeiss</td>
			<td>LSM 510</td>
			<td>Equipped with two-photon excitation laser (Mai Tai laser, Spectra-Physics)</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Xenon light source</td>
			<td>Asahi Spectra</td>
			<td>LAX-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax Paradigm</td>
			<td></td>
		</tr>
	</tbody>
</table>

spatiotemporally controlled nuclear translocation of guests in living cells using caged molecular glues as photoactivatable tags

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.

Before photoirradiation, Hep3B cells incubated with CagedGlue-NBD exhibited punctate fluorescence emission from their interior (&#955;ext = 488 nm; Figures 4A and 4C, green). An analogous micrograph was obtained upon excitation at 543 nm for red-fluorescent dye (Figures&#160;4B and 4C, red), indicating that CagedGlue-NBD localized in the endosomes. Accordingly, th...

Watch this Scientific Journal Video about Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags at JoVE.com

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

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 ...

This method can help answer key question in the area of drug delivery, such as how to deliver a drug to a specific cellular-granular. The main advantage of this technique is that nuclear translocation of gas can be induced by light. Demonstrating the procedure will be Rina Mogaki and Akio Arisaka the grad students from my group.To begin prepare the quantum dot linked cage molecular glue by synthesizing caged molecular glue linked dibenzocyclooctyne as described in more detail elsewhere. Prepare a 10 millimolar stock solution of the caged molecular glue linked dibenzocyclooctyne in dry DMSO. Then prepare the quantum dot linked caged molecular glue by first making aseye functionalized quantum dots.To accomplish this add 100 microliters of 125 micromolar aseye PEG4 NHS ester in DMF to 400 microliters of a DMF solution containing 500 nanomolar of quantum dots that are coated with amine functionalized peg. Stir the mixture for one hour at room temperature. Place the resulting solution in a regenerated cellulose membrane and dialyze it for 24 hours against 800 ml of DMF.Next dilute the stock solution of caged molecular glue linked dibenzocyclooctyne to 50 micromolar with DMF and add 200 microliters of it to the post dialysis solution. Stir the mixture for three hours at room temperature. Then dialyze the resulting mixture for 24 hours against 800 ml of fresh DMF using a regenerated cellulose membrane with the molecular weight cut off of 25000.After 24 hours dilute the resulting solution to 200 nanomolar with DMF. Maintain human hepatocellular carcinoma HEP3 B-cells in Eagle's minimal essential medium containing 10%FBS under standard culture conditions. One day before the experiment seed 5000 HEP3 B-cells in 200 microliters of culture medium into each well of a 8 chambered glass slide.Then cover the slide and place it into an incubator for 24 hours. The next day remove the culture medium and rinse the cells twice with 100 microliters of pre-warmed PBS. To each well of the 8 chambered slide add 200 microliters of FBS free medium containing 10 micromolar of a fluorescently dyed caged molecular glue.After incubating for 3 hours remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. To visualize the endosomes add 200 microliters of culture media containing 100 nanomolar of an additional red fluorescent dye such as LysoTracker Red. Incubate the resulting cell sample for 20 minutes.Then remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. Afterwards return the cells to 200 microliters of the culture medium. To induce nuclear translocation of the fluorescently dyed caged molecular glue place the cells under a 100 watt xenon light source equipped with a 365 nanometer bandpass filter.Expose the cells to UV light for 2 minutes. During this time keep the control cell sample without UV exposure in the dark. The lid of the glass substrates can be taken off for efficient UV exposure however take caution as prolonged exposure to UV light may cause cell death.To visualize the nuclei add one microliter of Hoeschst stain to the culture medium. Incubate the resulting cell sample for 10 minutes. Then place the sample on the stage of a confocal laser scanning microscope.Record the micrographs for the fluorescently dyed caged molecular glue, the LysoTracker Red, and the nuclear stain. As previously shown, seed 5000 human hepatocellular carcinoma HEP3 B-cells into each well of an 8 well chamber slide and culture under standard conditions for 24 hours. Supply the cell samples with 200 microliters of FBS free culture medium containing the fluorescently dyed caged molecular glue.Incubate the resulting cell sample for 3 hours. Then remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. Return the cells to 200 microliters of culture medium.Then subject to sample confocal laser scanning microscopy and record the micrographs as described in the accompanying text protocol. As previously shown seed 5000 human hepatocellular carcinoma HEP3 B-cells into each well of an 8 well chamber slide and culture under standard conditions for 24 hours. Supply the cell samples with 200 microliters of FBS free culture medium containing 10 nanomolar of the quantum dot linked caged molecular glue.Incubate the resulting cell sample for 3 hours. Then remove the medium and rinse the cell sample twice with 100 microliters of PBS. After rinsing the cells return them to 200 microliters of culture medium.Then place the plate under a 100 watt xenon light source equipped with a 365 nanometer bandpass filter. Expose the cell sample to UV light for 2 minutes keeping the control samples in the dark. To visualize the nuclei add 1 microliter of Hoechst stain to the culture medium.Incubate the resulting cell sample for 10 minutes. Then image the cells using a confocal laser scanning microscope. Seed the human hepatocellular carcinoma HEP3 B-cells the day before the experiment at 5000 cells per well into a 96 well culture plate.Then cover the cells with 200 microliters of culture media and then place the plate in an incubator for 24 hours. The next day remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. Then add 200 microliters of FBS free culture medium containing the fluorescently dyed caged molecular glue at concentrations ranging from 0.1 to 100 micromolar.Incubate the plate for 3 hours and then place a plate under the UV light source. Expose the cell sample to UV light for 2 minutes keeping the control samples in the dark. Following UV exposure at 10 microliters of the Cell Counting Kit-8 reagent to the culture medium and incubate the resulting cell sample for 2 hours.Then read the absorption of the samples at 450 nanometers using a microplate reader. Before photo radiation HEP3 B-cells incubated with fluorescently dyed caged molecular glue exhibits punctate fluorescence emission from the interior an analogous micrograph showing the LysoTracker Red indicates that fluorescently dyed caged molecular glue was localized in the endosomes. Accordingly the fluorescence emission assignable to the fluorescently dyed caged molecular glue was observed outside the cell nucleus prior to UV radiation.After UV radiation the fluorescent suggests that fluorescently dyed caged molecular glue was uncaged and migrated into the cytoplasm and the cell nucleus. Such nuclear translation of the fluorescently dyed caged molecular glue can be induced site selectively by 2-photon near infrared light. The fluorescently dyed caged molecular glue in non irradiated areas did not escape from the endosomes and remained as punctate fluorescence.The dendritic caged molecular glue tags can also deliver macromolecular guests such as quantum dots into the cell nucleus. These conjugates can be taken up into the cells and after UV exposure for 2 minutes the fluorescence emission of the quantum dots can be seen within the nucleus. After this development this technique paved the way for researchers in the field of gene therapy to explore low side effect treatment.

Research

JoVE Journal

Chemistry

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

Laser-heating and Radiance Spectrometry for the Study of Nuclear Materials in Conditions Simulating a Nuclear Power Plant Accident

Major and severe accidents have occurred three times in nuclear power plants (NPPs), at Three Mile Island (USA, 1979), Chernobyl (former USSR, 1986) and ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, ...

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Herein is presented a general strategy to perform reactions under mild to moderate CO2 pressures with dry ice. This technique obviates the need for ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...