Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells

We developed dendritic caged molecular glues (^CagedGlue-R) as tags for nucleus-targeted drug delivery, whose multiple guanidinium ion (Gu⁺) pendants are protected by an anionic photocleavable unit (butyrate-substituted nitroveratryloxycarbonyl; ^BANVOC). Negatively charged ^CagedGlue-R hardly binds to anionic biomolecules because of their electrostatic repulsion. However, upon exposure of ^CagedGlue-R to UV light or near-infrared (NIR) light, the ^BANVOC groups of ^CagedGlue-R are rapidly detached to yield an uncaged molecular glue (^UncagedGlue-R) that carries multiple Gu⁺ pendants. Because Gu⁺ forms a salt bridge with PO₄^–, ^UncagedGlue-R tightly adheres to anionic biomolecules such as DNA and phospholipids in cell membranes by a multivalent salt-bridge formation. When tagged with ^CagedGlue-R, guests can be taken up into living cells via endocytosis and hide in endosomes. However, when the ^CagedGlue-R tag is photochemically uncaged to form ^UncagedGlue-R, the guests escape from the endosome and migrate into the cytoplasm followed by the cell nucleus. We demonstrated that quantum dots (QDs) tagged with ^CagedGlue-R can be delivered efficiently to cell nuclei eventually by irradiation with light

Friction-Mediated Dynamic Disordering of Phospholipid Membrane by Mechanical Motions of Photoresponsive Molecular Glue: Activation of Ion Permeation

A water-soluble photoresponsive molecular glue, Azo-¹⁸Glue, consisting of a photochromic azobenzene core and two adhesive dendritic wedges with a total of 18 peripheral guanidinium ion (Gu⁺) pendants tightly adheres to the surface of a phospholipid membrane, even in buffer, via a multivalent salt-bridge formation with phosphate anions. A photomechanical motion of adhering Azo-¹⁸Glue possibly gives rise to dynamic structural disordering of the phospholipid membrane and activates transmembrane ion permeation. In sharp contrast, no activation of ion permeation results when poorly adhesive Azo-⁶Glue carrying only six Gu⁺ pendants is used in place of Azo-¹⁸Glue

Reductively Cleavable Nanocaplets for siRNA Delivery by Template-Assisted Oxidative Polymerization

A series of water-soluble telechelic dithiol monomers bearing multiple guanidinium ion (Gu⁺) units in their main chains were synthesized for packaging siRNA by template-assisted oxidative polymerization at their thiol termini. In the presence of siRNA, oxidative polymerization of ^TEGGu₄ affords a uniform-sized (7 ± 2 nm) nanocaplet containing siRNA (P^TEGGu₄⊃siRNA; P^TEGGu₄ = polymerized ^TEGGu₄). When this small conjugate is incubated with live cells, cellular uptake occurs, and the nanocaplet undergoes depolymerization in the reductive cytosolic environment to liberate the packaged siRNA. Consequently, gene expression in the live cells is suppressed

Structure and Shape Effects of Molecular Glue on Supramolecular Tubulin Assemblies

The possibility to arrange biological molecules into ordered nanostructures is an important issue in nano- and biotechnology. Nature offers a wide range of molecular “bricks” (<i>e.g.</i>, proteins, oligonucleotides, <i>etc</i>.) that spontaneously assemble into more complex hierarchical systems with unique functionalities. Such molecular building blocks can be also used for the construction of nanomaterials with peculiar properties (<i>e.g.</i>, DNA origami). In some cases, molecular glues able to bind biomolecules and to induce their assembly can be used to control the final structure and properties in a convenient way. Here we provide molecular-level description of how molecular glues designed to stick to the surface of microtubules (MTs) can control and transform the α/β-tubulin assembly upon temperature decreasing. By means of all-atom molecular dynamics (MD) simulations, we compared the adhesion to the MT surface of three molecular glues bearing the same guanidinium ion surface adhesive groups, but having different architecture, <i>i.e.</i>, linear or dendritic backbone. Our evidence demonstrates that the adhesive properties of the different molecular glues are dependent on the shape they assume in solution. In particular, adhesion data from our MD simulations explain how globular- or linear-like molecular glues respectively stabilize MTs or transform them into a well-defined array of α/β-tubulin rings at 15 °C, where MTs naturally depolymerize. The comprehension of the MT transformation mechanism provides a useful rationale for designing <i>ad hoc</i> molecular glues to obtain ordered protein nanostructures from different biological materials

Photoclickable Dendritic Molecular Glue: Noncovalent-to-Covalent Photochemical Transformation of Protein Hybrids

A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu⁺)/benzophenone (BP) pairs at its periphery (Glue^BP-FITC) serves as a “photoclickable molecular glue”. By multivalent salt-bridge formation between Gu⁺ ions and oxyanions, Glue^BP-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of “optical tweezers”

Reconstitution of microtubule into GTP-responsive nanocapsules

Nanocapsules that collapse in response to guanosine triphosphate (GTP) have the potential as drug carriers for efficiently curing diseases caused by cancer and RNA viruses because GTP is present at high levels in such diseased cells and tissues. However, known GTP-responsive carriers also respond to adenosine triphosphate (ATP), which is abundant in normal cells as well. Here, we report the elaborate reconstitution of microtubule into a nanocapsule that selectively responds to GTP. When the tubulin monomer from microtubule is incubated at 37 degrees C with a mixture of GTP (17 mol%) and nonhydrolysable GTP* (83 mol%), a tubulin nanosheet forms. Upon addition of photoreactive molecular glue to the resulting dispersion, the nanosheet is transformed into a nanocapsule. Cell death results when a doxorubicin-containing nanocapsule, after photochemically crosslinked for properly stabilizing its shell, is taken up into cancer cells that overexpress GTP.GTP-triggered release from drug carriers has huge potential in cancer therapy but current carriers suffers from off target release due to ATP also acting as a trigger. Here, the authors report on the development of a microtubule capsule which is engineered to be responsive to only GTP not ATP and demonstrate targeted drug delivery