Eline Bartolami

Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters†

We report herein the implementation of a dynamic covalent chemistry approach to the generation of multivalent clusters for DNA recognition. We show that biomolecular clusters can be expressed in situ by a programmed self-assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titration calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA-binding clusters. Furthermore, we show that a chemical effector can be used to trigger DNA release through component exchange reactions.

Strained Cyclic Disulfides Enable Cellular Uptake by Reacting with the Transferrin Receptor

In this study, we demonstrate that appendage of a single asparagusic acid residue (AspA tag) is sufficient to ensure efficient cellular uptake and intracellular distribution of fully unprotected peptides. We apply this new delivery method to induce apoptotic response in cancer cells using long (up to 20mer) BH3 domain peptides. Moreover, to understand the molecular mechanism of the cellular uptake, we perform chemical proteomics experiments and identify the direct molecular targets of the asparagusic acid tag. Our findings document covalent bond formation between the asparagusic acid moiety and the cysteines 556 and 558 on the surface of the transferrin receptor resulting in subsequent endocytic uptake of the payload. We believe that the small size, low cellular toxicity and the efficient transferrin receptor-mediated uptake render the AspA tag highly attractive for various life science applications.

Efficient Delivery of Quantum Dots into the Cytosol of Cells Using Cell-Penetrating Poly(disulfide)s

Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo to investigate single molecule dynamics is impaired by the absence of an efficient way to chemically deliver them into the cytosol of cells. Indeed, current methods (using cell-penetrating peptides for instance) provide very low yields: QDs stay at the plasma membrane or are trapped in endosomes. Here, we introduce a technology based on cell-penetrating poly(disulfide)s that solves this problem: we deliver about 70 QDs per cell, and 90% appear to freely diffuse in the cytosol. Furthermore, these QDs can be functionalized, carrying GFP or anti-GFP nanobodies for instance. Our technology thus paves the way toward single molecule imaging in cells and living animals, allowing to probe biophysical properties of the cytosol.

Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension

The disulfide dihedral angle in epidithiodiketopiperazines (ETPs) is near 0°. Application of this highest possible ring tension to strain-promoted thiol-mediated uptake results in efficient delivery to the cytosol and nucleus. Compared to the previous best asparagusic acid (AspA), ring-opening disulfide exchange with ETPs occurs more efficiently even with nonactivated thiols, and the resulting thiols exchange rapidly with nonactivated disulfides. ETP-mediated cellular uptake is more than 20 times more efficient compared to AspA, occurs without endosomal capture, depends on temperature, and is “unstoppable” by inhibitors of endocytosis and conventional thiol-mediated uptake, including siRNA against the transferrin receptor. These results suggest that ETP-mediated uptake not only maximizes delivery to the cytosol and nucleus but also opens the door to a new multitarget hopping mode of action.

A metal-free synthetic approach to peptide-based iminosugar clusters as novel multivalent glycosidase inhibitors

Multivalent bioconjugates represent emerging tools for enzyme inhibition. In particular, iminosugar clusters have recently shown promising results for the inhibition of glycosidases. However, most of them are prepared by copper-mediated click reactions. We report herein a metal-free approach based on oxime ligation for preparing iminosugar clusters using cyclic and linear tetra-aldehyde peptide scaffolds and oxyamine iminosugars (40–70% yield). Glycosidase inhibition assays revealed the superiority of the clusters made of the linear scaffold, displaying up to a 77-fold increase of relative potency per iminosugar. Thus, this metal-free approach provides a rapid access to structurally-diverse iminosugar clusters for establishing structure–activity relationships in the context of multivalent glycosidase inhibition.

One-Pot Self-Assembly of Peptide-Based Cage-Type Nanostructures using Orthogonal Ligations

The designed arrangement of biomolecular entities within monodisperse nanostructures is an important challenge toward functional biomaterials. We report herein a method for the formation of water-soluble peptide-based cages using orthogonal ligation reactions-acylhydrazone condensation and thiol-maleimide addition. The results show that using preorganized cyclic peptides and heterobifunctional spacers as building blocks and a set of orthogonal and chemoselective ligation reactions enable cage formation in one pot from six components and through eight reactions. Molecular modelling simulations reveal the structural dynamics of these structures. Finally, we exploited the reactional dynamics of the acylhydrazone by demonstrating the controlled dissociation of the cage through directed component exchange.

A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.Artificial enzymes can be used to elicit reactions in cells. Here, the authors developed such an artificial catalyst combined with a genetic switch, and showed that it was readily taken up by human cells and able to kick off a reaction cascade resulting in the biosynthesis of the desired product.