A copper(ii) peptide helicate selectively cleaves DNA replication foci in mammalian cells.

The use of copper-based artificial nucleases as potential anticancer agents has been hampered by their poor selectivity in the oxidative DNA cleavage process. An alternative strategy to solve this problem is to design systems capable of selectively damaging noncanonical DNA structures that play crucial roles in the cell cycle. We designed an oligocationic CuII peptide helicate that selectively binds and cleaves DNA three-way junctions (3WJs) and induces oxidative DNA damage via a ROS-mediated pathway both in vitro and in cellulo, specifically at DNA replication foci of the cell nucleus, where this DNA structure is transiently generated. To our knowledge, this is the first example of a targeted chemical nuclease that can discriminate with high selectivity 3WJs from other forms of DNA both in vitro and in mammalian cells. Since the DNA replication process is deregulated in cancer cells, this approach may pave the way for the development of a new class of anticancer agents based on copper-based artificial nucleases.

Cell-Surface-Retained Peptide Additives for the Cytosolic Delivery of Functional Proteins.

The delivery of functional proteins remains a major challenge in advancing biological and pharmaceutical sciences. Herein, we describe a powerful, simple, and highly effective strategy for the intracellular delivery of functional cargoes. Previously, we demonstrated that cell-penetrating peptide (CPP) additives equipped with electrophilic thiol-reactive moieties temporarily attach to the cellular membrane, thereby facilitating the cellular uptake of protein- and antibody-CPP cargoes through direct membrane transduction at low concentrations. Now, we hypothesize that CPP-additives with an increased retention on the cellular membrane will further enhance intracellular uptake. We discovered that adding a small hydrophobic peptide sequence to an arginine-rich electrophilic CPP-additive further improved the uptake of protein-CPP conjugates, whereas larger hydrophobic anchors showed increased cytotoxicity. Cell viability and membrane integrity measurements, structure-activity relationship studies, and quantitative evaluation of protein-CPP uptake revealed important design principles for cell-surface-retained CPP-additives. These investigations allowed us to identify a nontoxic, thiol-reactive CPP-additive containing the hydrophobic ILFF sequence, which can deliver fluorescent model proteins at low micromolar concentrations. This hydrophobic CPP-additive allowed the addition of protein cargoes for intracellular delivery after initial additive incubation. Time-lapse fluorescence microscopy and membrane tension analysis of cells treated with fluorescent ILFF-CPP-additives supported the claim of increased cell surface retention and suggested that the protein-CPP cargoes enter the cell through a mechanism involving lowered cell membrane tension. Finally, we demonstrated that our newly engineered hydrophobic CPP-additive enabled the uptake of a functional macrocyclic peptidic MDM2-inhibitor and a recombinant genome editing protein. This indicates that the developed hydrophobic CPP-additive holds promise as a tool to enhance the intracellular delivery of peptide and protein cargoes.

Dimeric Metal-Salphen Complexes Which Target Multimeric G-Quadruplex DNA.

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization.

Structure-Uptake Relationship Study of DABCYL Derivatives Linked to Cyclic Cell-Penetrating Peptides for Live-Cell Delivery of Synthetic Proteins.

Modifying cyclic cell-penetrating deca-arginine (cR10) peptides with 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time-lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL-R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure-uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell-uptake efficiency when conjugated to mono-Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.

Selective recognition of A/T-rich DNA 3-way junctions with a three-fold symmetric tripeptide.

Non-canonical DNA structures, particularly 3-Way Junctions (3WJs) that are transiently formed during DNA replication, have recently emerged as promising chemotherapeutic targets. Here, we describe a new approach to target 3WJs that relies on the cooperative and sequence-selective recognition of A/T-rich duplex DNA branches by three AT-Hook peptides attached to a three-fold symmetric and fluorogenic 1,3,5-tristyrylbenzene core.

Stereoselective Self-Assembly of DNA Binding Helicates Directed by the Viral ß-Annulus Trimeric Peptide Motif.

Combining coordination chemistry and peptide engineering offers extraordinary opportunities for developing novel molecular (supra)structures. Here, we demonstrate that the ß-annulus motif is capable of directing the stereoselective assembly of designed peptides containing 2,2'-bipyridine ligands into parallel three-stranded chiral peptide helicates, and that these helicates selectively bind with high affinity to three-way DNA junctions.

Dynamic Stereoselection of Peptide Helicates and Their Selective Labeling of DNA Replication Foci in Cells*.

Although largely overlooked in peptide engineering, coordination chemistry offers a new set of interactions that opens unexplored design opportunities for developing complex molecular structures. In this context, we report new artificial peptide ligands that fold into chiral helicates in the presence of labile metal ions such as FeII and CoII . Heterochiral ß-turn-promoting sequences encode the stereoselective folding of the peptide ligands and define the physicochemical properties of their corresponding metal complexes. Circular dichroism and NMR spectroscopy in combination with computational methods allowed us to identify and determine the structure of two isochiral ΛΛ-helicates, folded as topological isomers. Finally, in addition to the in-vitro characterization of their selective binding to DNA three-way junctions, cell-microscopy experiments demonstrated that a rhodamine-labeled FeII helicate was internalized and selectively stains DNA replication factories in functional cells.

Directed Self-Assembly of Trimeric DNA-Bindingchiral Miniprotein Helicates.

We propose that peptides are highly versatile platforms for the precise design of supramolecular metal architectures, and particularly, for the controlled assembly of helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff) can been engineered on its N-terminus with metal-chelating 2,2'-bipyridine units that stereoselectively assemble in the presence of Fe(II) into parallel, three-stranded peptide helicates with preferred helical orientation. Modeling studies support the proposed self-assembly and the stability of the final helicate. Furthermore, we show that these designed mini-metalloproteins selectively recognize three-way DNA junctions over double-stranded DNA.