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(A) Structural elements and sequence of the natural T4Ff, and proposed structure of the (βAlaBpy)2-T4Ff helicate at the N-terminus of the T4Ff scaffold. The three chains of the T4Ff are shown with different colors (orange, blue, light gray) for clarity. The ΛΛ- chirality is induced by the natural twisting of the T4Ff N-terminal polyproline helices. (B) Synthetic procedure for obtaining the T4Ff helicates, and structure of the chelating Fmoc-βAlaBpy-OH amino acid.
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![(Left) Anisotropy titration of [(βAlaBpy)2-T4Ff]3Fe2 in 1 mM phosphate...](publication/328607832/figure/fig4/AS:11431281243103218@1715642444265/Left-Anisotropy-titration-of-bAlaBpy2-T4Ff3Fe2-in-1-mM-phosphate-buffer-10-mM-NaCl_Q320.jpg)
![EMSA DNA binding studies results for [(βAlaBpy)2-T4Ff]3Fe2 helicate....](publication/328607832/figure/fig5/AS:11431281243019760@1715642444483/EMSA-DNA-binding-studies-results-for-bAlaBpy2-T4Ff3Fe2-helicate-Lanes-1-6-200-nM_Q320.jpg)
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 stereoselect...
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Citations
... Others explored the potential of the linker to influence the overall structure. For example, Vázquez et al. designed peptide-linked Fe(II) and Co(III) [M 2 L 3 ] x+ complexes [35,40,41], and Sun et al. made use of a homochiral BINOL-containing (BINOL = 1,1'-bi-2-naphthol) scaffold in tandem with chiral amines to obtain enantiopure fac- (ΔΔ) and fac-(ΛΛ) helical species [42,43]. Formation of helical complexes over the related mesocate structures is often a factor of the ligand, for example, Lisboa et al. synthesised a series of [Fe 2 L 3 ] 4+ metallocylindrical arrays and found that while shorter linkers such as 1,3-substituted benzenes or pyridines between the 2,2'-bipyridine-coordinating motif favoured formation of helical structures, a longer 4,4'-diphenylmethylene linker gave a mixture of structurally related helicates and mesocates [8]. ...
... The discovery of the cytotoxic properties of cisplatin by Barnett Rosenberg in 1965 1 gave rise to the new eld of canonical dsDNA-targeted metallodrugs for cancer therapy. 2 More recently, as our knowledge of the structural polymorphism and biology of DNA expanded, the bioinorganic eld has shied towards targeting noncanonical DNA structures, including single-stranded DNA, i-motifs, G-quadruplexes (G4s), DNA triplexes and DNA junctions, such as three-way (3WJs) and fourway (Holliday) junctions, among others. [3][4][5][6][7][8][9][10][11][12][13] Here, we present a Cu II peptide helicate that selective recognises 3WJs both in vitro and at DNA replication foci in mammalian cells. This is the rst time such specicity has been achieved. ...
... 15 Metallosupramolecular helicates are the most promising agents for the selective recognition of these noncanonical DNA structures. [6][7][8][9][10][11][12] One of the key factors for their selectivity is the high complementarity between their shape and the trigonal symmetry of the branch point of the 3WJs. Indeed, this is a key structural feature of other 3WJ binders, such as triptycene derivatives, 16 C 3 -symmetric cationic azacryptands, 17 selfassembled supramolecular Fe II tetrahedral metallocages 18 and three-fold symmetric tripeptides. ...
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.
... have also been used to generate [M 2 L 3 ] 4+ triple-stranded helicates and recently Vázquez and co-workers have examined the DNA binding and cytotoxicity of some peptide linked [M 2 L 3 ] 4+ helicates [where M Fe(II) or Co(III)](Gamba et al., 2014;Gómez-González et al., 2018; Gomez-Gonzalez et al., 2021). ...
... The biological properties of the Fe(II) and Ru(II) systems were poor but the more robust Co(III) helicates were shown to bind to and condense DNA and in addition displayed good anticancer activity (Crlikova et al., 2020). Di(2,2′-bipyridine) ligands (Glasson et al., 2008;Glasson et al., 2011a;Glasson et al., 2011b) have also been used to generate [M 2 L 3 ] 4+ triple-stranded helicates and recently Vázquez and co-workers have examined the DNA binding and cytotoxicity of some peptide linked [M 2 L 3 ] 4+ helicates [where M Fe(II) or Co(III)] (Gamba et al., 2014;Gómez-González et al., 2018;Gomez-Gonzalez et al., 2021). ...
Two new di(2,2′-bipyridine) ligands, 2,6-bis([2,2′-bipyridin]-5-ylethynyl)pyridine (L1) and bis(4-([2,2′-bipyridin]-5-ylethynyl)phenyl)methane (L2) were synthesized and used to generate two metallosupramolecular [Fe2(L)3](BF4)4 cylinders. The ligands and cylinders were characterized using elemental analysis, electrospray ionization mass spectrometry, UV-vis, ¹H-, ¹³C and DOSY nuclear magnetic resonance (NMR) spectroscopies. The molecular structures of the [Fe2(L)3](BF4)4 cylinders were confirmed using X-ray crystallography. Both the [Fe2(L1)3](BF4)4 and [Fe2(L2)3](BF4)4 complexes crystallized as racemic (rac) mixtures of the ΔΔ (P) and ΛΛ (M) helicates. However, ¹H NMR spectra showed that in solution the larger [Fe2(L2)3](BF4)4 was a mixture of the rac-ΔΔ/ΛΛ and meso-ΔΛ isomers. The host-guest chemistry of the helicates, which both feature a central cavity, was examined with several small drug molecules. However, none of the potential guests were found to bind within the helicates. In vitro cytotoxicity assays demonstrated that both helicates were active against four cancer cell lines. The smaller [Fe2(L1)3](BF4)4 system displayed low μM activity against the HCT116 (IC50 = 7.1 ± 0.5 μM) and NCI-H460 (IC50 = 4.9 ± 0.4 μM) cancer cells. While the antiproliferative effects against all the cell lines examined were less than the well-known anticancer drug cisplatin, their modes of action would be expected to be very different.
... For example, naphthalene diimides (NDIs) have a remarkable potential as anticancer drugs because of their wellproven ability to strongly interact with G-quadruplex (Salvati et al., 2016;Pirota et al., 2019;Platella et al., 2020a;Platella et al., 2020b). Encouraged by these results, many scientists use various optical methods (Gómez-González et al., 2018;Zhang and Wang, 2019;Lim and Hohng, 2020;Chen et al., 2021) and DFT theoretical calculations (Wu et al., 2012;Yao et al., 2013;Shi et al., 2015;Wang et al., 2019) to undertake an in-depth research on the interaction of intercalators and G-quadruplex, to reveal the structural details of this strong and specific binding. As we know, the visualization of these "host-guest" interactions based on X-ray crystallography will help understand the details of interactions between functional intercalations and targets, as well as the nucleotide conformational polymorphism changes. ...
A detailed understanding of the mismatched base-pairing interactions in DNA will help reveal genetic diseases and provide a theoretical basis for the development of targeted drugs. Here, we utilized mononucleotide fragment to simulate mismatch DNA interactions in a local hydrophobic microenvironment. The bipyridyl-type bridging ligands were employed as a mild stabilizer to stabilize the GG mismatch containing complexes, allowing mismatch to be visualized based on X-ray crystallography. Five single crystals of 2′-deoxyguanosine–5′–monophosphate (dGMP) metal complexes were designed and obtained via the process of self-assembly. Crystallographic studies clearly reveal the details of the supramolecular interaction between mononucleotides and guest intercalators. A novel guanine–guanine base mismatch pattern with unusual (high anti)–(high anti) type of arrangement around the glycosidic angle conformations was successfully constructed. The solution state ¹H–NMR, ESI–MS spectrum studies, and UV titration experiments emphasize the robustness of this g–motif in solution. Additionally, we combined the methods of single-crystal and solution-, solid-state CD spectrum together to discuss the chirality of the complexes. The complexes containing the g–motif structure, which reduces the energy of the system, following the solid-state CD signals, generally move in the long-wave direction. These results provided a new mismatched base pairing, that is g–motif. The interaction mode and full characterizations of g–motif will contribute to the study of the mismatched DNA interaction.
... Intermolecular interactions between the helicate and the 3WJ, electrostatic interactions and π-stacking, synergistically contributed to the binding. Vázquez and Vázquez López synthesized a chiral peptide helicate having two tris(bipyridine)-Fe II complexes, which was conjugated to a foldon protein afterward (Gamba et al., 2014;Gómez-González et al., 2018). The -isomer of the helical metallopeptide was found to bind to a DNA 3WJ more strongly than the enantiomeric -isomer. ...
Various nano-sized supramolecular architectures have been constructed from DNA molecules via sequence-dependent self-assembly. A DNA three-way junction (3WJ), consisting of three oligonucleotides that are partially complementary to each other, is one of the simplest DNA supramolecular structures. This minireview covers studies on DNA 3WJ motifs bridged by an interstrand metal complex with some related works. The incorporation of interstrand metal complexes into DNA has attracted increasing attention because it potentially allows for metal-dependent regulation of the thermal stability and the structure of DNA supramolecules. Metal-bridged DNA 3WJs were synthesized from three DNA strands containing a bipyridine (bpy)-modified nucleotide in the presence of appropriate metal ions. The bpy-modified DNA strands were crosslinked by an interstrand 3:1 metal complex [NiII(bpy)3 etc.] at the junction core. As a result, the thermal stability of the 3WJs was significantly enhanced upon metal complexation. Furthermore, metal-mediated structural transformation between DNA duplexes and 3WJs was demonstrated by using the same bpy-modified DNA strands. A mixture of bpy-modified strands and their natural complementary strands were self-assembled exclusively into duplexes in the absence of any transition metal ions. In contrast, addition of NiII ions induced the formation of 3WJs through the formation of an interstrand NiII(bpy)3 complex, which served as a template for the 3WJ assembly. Because DNA 3WJ structures are essential structural motifs for DNA-based nanoarchitectures, the metal-mediated stabilization and structural induction of metal-locked 3WJs would lead to many potential applications to artificial DNA architectures.
T help (Th), stimulation of toll-like receptors (pathogen-associated molecular patterns, PAMPs), and antigen organization and repetitiveness (pathogen-associated structural patterns, PASPs) were shown numerous times to be important in driving B-cell and antibody responses. In this study, we dissected the individual contributions of these parameters using newly developed “Immune-tag” technology. As model antigens, we used eGFP and the third domain of the dengue virus 1 envelope protein (DV1 EDIII), the major target of virus-neutralizing antibodies. The respective proteins were expressed alone or genetically fused to the N-terminal fragment of the cucumber mosaic virus (CMV) capsid protein—nCMV, rendering the antigens oligomeric. In a step-by-step manner, RNA was attached as a PAMP, and/or a universal Th-cell epitope was genetically added for additional Th. Finally, a PASP was added to the constructs by displaying the antigens highly organized and repetitively on the surface of CMV-derived virus-like particles (CuMV VLPs). Sera from immunized mice demonstrated that each component contributed stepwise to the immunogenicity of both proteins. All components combined in the CuMV VLP platform induced by far the highest antibody responses. In addition, the DV1 EDIII induced high levels of DENV-1-neutralizing antibodies only if displayed on VLPs. Thus, combining multiple cues typically associated with viruses results in optimal antibody responses.
Investigations on binding strength differences of non-covalent protein complex components were performed by mass spectrometry. T4 fibritin foldon (T4Ff) is a well-studied miniprotein, which together with its biotinylated version served as model system to represent a compactly folded protein to which an Intrinsically Disordered Region (IDR) was attached. The apparent enthalpies of the gas phase dissociation reactions of the homo-trimeric foldon F-F-F and of the homo-trimeric triply biotinylated foldon bF-bF-bF have been determined to be rather similar (3.32 kJ/mol and 3.85 kJ/mol) but quite distinct from those of the singly and doubly biotinylated hetero-trimers F-F-bF and F-bF-bF (1.86 kJ/mol and 1.08 kJ/mol). Molecular dynamics simulations suggest that the ground states of the (biotinylated) T4Ff trimers are highly symmetric and well comparable to each other, indicating that the energy levels of all four (biotinylated) T4Ff trimer ground states are nearly indistinguishable. The experimentally determined differences and/or similarities in enthalpies of the complex dissociation reactions are explained by entropic spring effects, which are noticeable in the T4Ff hetero-trimers but not in the T4Ff homo-trimers. A lowering of the transition state energy levels of the T4Ff hetero-trimers seems likely because the biotin moieties, mimicking intrinsically disordered regions (IDRs), induced asymmetries in the transition states of the biotinylated T4Ff hetero-trimers. This transition state energy level lowering effect is absent in the T4Ff homo-trimer, as well as in the triply biotinylated T4Ff homo-trimer. In the latter, the IDR-associated entropic spring effects on complex stability cancel each other out. ITEM-FIVE enabled semi-quantitative determination of energy differences of complex dissociation reactions, whose differences were modulated by IDRs attached to compactly folded proteins.
The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it combines four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.
DNA has been a key target for cancer therapy, with a range of compounds able to bind and either impair its processing or induce damage. Targeting DNA with small molecules in a truly sequence specific way, to impair gene specific processes, remains out of reach. The ability of DNA to assume different structures from the classical double helix allows access to more specific ligand binding modes and, potentially, to new avenues of treatment. In this review, we illustrate the small molecules that have been reported to bind to three- and four-way junctions.
