Glycoligands and Co(II) glycocomplexes. Investigation of the variation of the sugar-scaffold on the structure and chirality measured by circular dichroism

Glycoligands using various monosaccharide platforms functionalised by three 2-picolyl groups and coordinated to Co(II) through the bidentate 2-picolyl ether moieties are interesting ligands as they efficiently induce chirality at the cobalt with a fine control of the structure through the central sugar scaffold.     

Synthesis and molecular modelling of double-functionalised nucleosides with aromatic moieties in the 5'-(S)-position and minor groove interactions in DNA zipper structures

A series of six double-functionalised nucleosides, in which aromatic moieties were inserted into the 5'-(S)-C-position, were synthesised and incorporated into DNA duplexes. The aromatic moieties were thymine-1-yl, phenyl, 1,2,3-triazol-1-yl, 1,2,3-triazol-4-yl, 4-(uracil-5-yl)-1,2,3-triazol-1-yl and 4-phenyl-1,2,3-triazol-1-yl. The DNA duplexes were studied with UV melting curves, CD spectroscopy and molecular modelling. The results showed that the aromatic moieties in some cases interact in the minor groove forming DNA zipper structures. The strongest specific interaction was found between two thymines or between a thymine and a phenyl group in a crossed (-3)-zipper motif (i.e., with two base pairs interspacing the modifications). Modelling revealed that the interaction is aromatic stacking across the minor groove. Also, the extended uracil-triazole moiety demonstrated zipper contacts in the minor groove as well as binding to the floor of the groove.     

Design of new bidentate ligands constructed of two Hoechst 33258 units for discrimination of the length of two A3T3 binding motifs

[structure: see text] The aim of this study is to develop bidentate minor-groove binders that bind the double binding motifs cooperatively. The new bidentate ligands (1) have been designed by connecting two Hoechst 33258 units with a polyether linker for cooperative binding with two remote A3T3 sites of DNA. The linker is introduced to the benzimidazole ring so that it is located at the convex side of the Hoechst unit. DNA binding affinity of the ligands was evaluated by measuring surface plasmon resonance (SPR), circular dichroism, and fluorescence spectra. Interestingly, the bidentate ligands (1) did not show affinity to DNA1 with a single A3T3 motif but showed selective affinity to DNA2 with two A3T3 motifs. The Long Bis-H (1L) having a long polyether linker showed specific binding to DNA2(6) with two A3T3 motifs separated by six nonbinding base pairs. The Long Bis-H (1L) has also shown specific binding to the three-way junction DNA4 with two A3T3 motifs. This study has demonstrated that DNA with double binding motifs can be selectively recognized by the newly designed bidentate ligands.     

Binding Survey of Ionizable Calix[4]‐1,2‐crown‐3 Nano‐baskets by Differential Pulse Voltammetry

Three nano‐baskets of p‐tert‐butylcalix[4]arene‐1,2‐crown‐3 were synthesized and their binding abilities towards alkali and alkaline earth metals as well as some lanthanides were studied using differential pulse voltammetry. The novelty of this study was the investigation of macrocyclic complexes by voltammetric behaviors of two acidic moieties in each scaffold. Their voltammetric behaviors were closely related to the complex formation by entrapment of cation into crown ether cavity and ion‐dipole interaction between cation and acidic moieties in calixcrowns. The results revealed that the sulfur atom in the crown ring as well as the position of crown‐3 ether in 1,2‐alternate instead of cone conformer changed the binding ability of the scaffold to more cations.     

Determination of Allosteric Effects and Interstrand Bidentate Interactions in DNA‐Peptide Molecular Recognition

To study DNA allostery, quantitative DNase I footprinting studies were carried out on a newly designed peptide His‐Hyp‐Lys‐Lys‐(Py)₄‐Lys‐Lys‐NH₂ (HypKK‐10) containing the XHypKK (Hyp = hydroxyproline) and polyamide motifs. The interconnection of DNA footprints of peptides HypKK‐10 and the parent peptide PyPro‐12 supports the proposal that interaction network cooperativity is preferred in DNA‐peptide interactions between multiple recognition sites. A simple method of determining interstrand bidentate interactions between the peptide moieties and DNA bases is introduced. It is envisaged that interstrand bidentate interactions also participate in the relay of conformational changes to recognition sites on the complementary strands. Circular dichroism studies of the titration of peptide HypKK‐10 with an oligonucleotide duplex indicate that this peptide binds in a dimeric fashion to DNA in the minor groove. This work may prompt the design of new DNA binding ligands for the study of DNA‐peptide allosteric interactions and DNA interaction network.     

Preferential binding specificity of silver cation to a single nucleobase over base pairs evaluated by abasic site-containing DNA

The binding specificity of silver cations to abasic (AP) site-containing DNA was electrochemically investigated by comparison with the fully matched DNA without the AP site. AP site-containing DNA is designed in a way that only the nucleotide opposite the AP site is variable to allow for coexistence of an unpaired nucleotide and a number of DNA base pairs. The surface of a gold electrode was modified by AP site-containing DNA duplex on which Ag⁺ binding specificity was evaluated. Electrochemical investigations on the AP-DNA-modified electrodes reveal that Ag⁺ preferentially associates to the unpaired nucleotides instead of the coexisted base pairs and shows sequence-dependant binding, especially stronger for purines than for pyrimidines. Additionally, the hydrogen bond pattern moieties of the unpaired nucleotides should be involved in Ag⁺ binding evidenced by a decrease of the redox signal when introducing a ligand with its hydrogen bond moiety complementary to the nucleotide deoxycytidine. This is the first attempt to make a comparison in one DNA molecule for metal ion binding to coexisted unpaired nucleotide and DNA base pairs. The present method demonstrates an easy way for investigating binding specificity of heavy metal ions to AP site in the presence of coexisted DNA base pairs.     

Natural and Synthetic Oligoarylamides: Privileged Structures for Medical Applications

The term “privileged structure” refers to a single molecular substructure or scaffold that can serve as a starting point for high-affinity ligands for more than one receptor type. In this report, a hitherto overlooked group of privileged substructures is addressed, namely aromatic oligoamides, for which there are natural models in the form of cystobactamids, albicidin, distamycin A, netropsin, and others. The aromatic and heteroaromatic core, together with a flexible selection of substituents, form conformationally well-defined scaffolds capable of specifically binding to conformationally well-defined regions of biomacromolecules such as helices in proteins or DNA often by acting as helices mimics themselves. As such, these aromatic oligoamides have already been employed to inhibit protein–protein and nucleic acid–protein interactions. This article is the first to bring together the scattered knowledge about aromatic oligoamides in connection with biomedical applications.     

Screening of threading bis-intercalators binding to duplex DNA by electrospray ionization tandem mass spectrometry

The DNA binding of novel threading bis-intercalators V1, trans-D1, and cis-C1, which contain two naphthalene diimide (NDI) intercalation units connected by a scaffold, was evaluated using electrospray ionization mass spectrometry (ESI-MS) and DNAse footprinting techniques. ESI-MS experiments confirmed that V1, the ligand containing the -Gly3-Lys- peptide scaffold, binds to a DNA duplex containing the 5'-GGTACC-3' specific binding site identified in previous NMR-based studies. The ligand formed complexes with a ligand/DNA binding stoichiometry of 1:1, even when there was excess ligand in solution. Trans-D1 and cis-C1 are new ligands containing a rigid spiro-tricyclic scaffold in the trans- and cis- orientations, respectively. Preliminary DNAse footprinting experiments identified possible specific binding sites of 5'-CAGTGA-5' for trans-D1 and 5'-GGTACC-3' for cis-C1. ESI-MS experiments revealed that both ligands bound to DNA duplexes containing the respective specific binding sequences, with cis-C1 exhibiting the most extensive binding based on a higher fraction of bound DNA value. Cis-C1 formed complexes with a dominant 1:1 binding stoichiometry, whereas trans-D1 was able to form 2:1 complexes at ligand/DNA molar ratios >or=1 which is suggestive of nonspecific binding. Collisional activated dissociation (CAD) experiments indicate that DNA complexes containing V1, trans-D1, and cis-C1 have a unique fragmentation pathway, which was also observed for complexes containing the commercially available bis-intercalator echinomycin, as a result of similar binding interactions, marked by intercalation in addition to hydrogen bonding by the scaffold with the DNA major or minor groove.     

Synthesis of Multivalent Galactosides as Targeting Ligand for Gene Delivery

A series of novel bifunctional glycolipid ligands designed to bind with high affinity and specificity to the asialoglycoprotein receptor (ASGP-R) has been synthesized and assayed in vitro on human hepatoma cells, HepG2, derived from parenchymal liver cells. The compounds bear five β-linked Gal moieties linked to the core scaffold, hexa-antennary alcohol, for interaction with the binding site of the ASGP-R. The liposome/DNA complexes containing the glycolipid ligands are efficiently recognized by ASGP-R and exhibited high affinity and transfection activity.     

Miniature homeodomains: high specificity without an N-terminal arm

Recently, we described a strategy for the design of miniature proteins that bind DNA and protein surfaces with high affinity and selectivity. This strategy involves identifying the functional epitope required for macromolecular recognition by a natural protein and presenting it on a small, stable protein scaffold. In previous work, high-affinity DNA recognition was achieved only when the miniature protein contained the complete functional epitope. Here we report a miniature homeodomain that recognizes its 6-bp target site in the nanomolar concentration range at 25 degrees C, despite the absence of DNA contact residues located along the homeodomain N-terminal arm. We conclude that miniature proteins can achieve high affinity and selectivity for DNA by design even when the functional epitope is incomplete by using pre-organization to effectively compensate for lost protein-DNA contacts. In this case it has been possible to miniaturize both the recognition surface and the structural framework of a globular protein fold.     

Synthesis of 2-aminobenzophenone-based Schiff base Pd(II) complexes: Investigation on crystal structure,biological behavior of DNA/protein-binding,molecular docking,and in vitro anticancer activities

Two new monobasic bidentate ligands and their Pd(II) complexes have been synthesized and characterized by analytical and spectroscopic methods. The structures of the complexes were confirmed by single-crystal X-ray diffraction. The bimolecular binding of the ligands and complexes has been carried out and described. Interestingly, both the bidentate chelating ligands replaced all the triphenyl arsine and chloride ions from the metal precursor in the formation of new complexes and were found to be approximately square planar. The interaction of the ligands and the complexes with calf thymus DNA and bovine serum albumin was studied by electronic and emission spectroscopy techniques, which suggested an intercalation mode of binding. It is well-known that the viscosity of a DNA solution increases if any compound added binds to it through intercalation because this process lengthens the DNA helix due to the increased separation of the DNA base pairs when the compound slides in between, whereas a partial, nonclassical intercalation could bend (or kink) the DNA helix, which leads to a reduction in length and thereby reducing its viscosity. By contrast, there will be no change in the viscosity when the compounds bind with DNA grooves or by partial intercalation, which was further confirmed by viscosity measurements and molecular docking studies. It has been found that the compounds cleaved supercoiled DNA into nicked DNA without any external agent. The in vitro cytotoxicity studies of the ligands and complexes against human lung (A549) and breast (MCF7) cancer cell lines showed significant activity for both species.     

The recognition of a noncanonical RNA base pair by a zinc finger protein.

BACKGROUND: The zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence. RESULTS: We constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex. CONCLUSIONS: It is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures.     

Binding of D- and L-captopril inhibitors to metallo-beta-lactamase studied by polarizable molecular mechanics and quantum mechanics

The bacterial Zn2+ metallo-beta-lactamase from B. fragilis is a zinc-enzyme with two potential metal ion binding sites. It cleaves the lactam ring of antibiotics, thus contributing to the acquired resistance of bacteria against antibiotics. The present study bears on the binuclear form of the enzyme. We compare several possible binding modes of captopril, a mercaptocarboxamide inhibitor of several zinc-metalloenzymes. Two diastereoisomers of captopril were considered, with either a D- or an L-proline residue. We have used the polarizable molecular mechanics procedure SIBFA (Sum of Interactions Between Fragments ab initio computed). Two beta-lactamase models were considered, encompassing 104 and 188 residues, respectively. The energy balances included the inter and intramolecular interaction energies as well as the contribution from solvation computed using a continuum reaction field procedure. The thiolate ion of the inhibitor is binding to both metal ions, expelling the bridging solvent molecule from the uncomplexed enzyme. Different competing binding modes of captopril were considered, either where the inhibitor binds in a monodentate mode to the zinc cations only with its thiolate ion, or in bidentate modes involving additional zinc binding by its carboxylate or ketone carbonyl groups. The additional coordination by the inhibitor's carboxylate or carbonyl group always occurs at the zinc ion, which is bound by a histidine, a cysteine, and an aspartate side chain. For both diastereomers, the energy balances favor monodentate binding of captopril via S-. The preference over bidentate binding is small. The interaction energies were recomputed in model sites restricted to captopril, the Zn2+ cations, and their coordinating end side chains from beta-lactamase (98 atoms). The interaction energies and their ranking among competing arrangements were consistent with those computed by ab initio HF and DFT procedures.     

Directed evolution of high-affinity antibody mimics using mRNA display

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.     

Synthesis and functional evaluation of DNA-assembled polyamidoamine dendrimer clusters for cancer cell-specific targeting

We sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5'-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics.     

Multivalent chelators for spatially and temporally controlled protein functionalization

Site-specific protein modification—e.g. for immobilization or labelling—is a key prerequisite for numerous bioanalytical applications. Although modification by use of short peptide tags is particularly attractive, efficient and bio-orthogonal systems are still lacking. Here, we review the application of multivalent chelators (MCH) for high-affinity yet reversible recognition of oligohistidine (His)-tagged proteins. MCH are based on multiple nitrilotriacetic acid (NTA) moieties grafted on to molecular scaffolds suitable for conjugation to surfaces, probes or other biomolecules. Reversible interaction with the His-tag is mediated via transition metal ions chelated by the NTA moieties. The small size and biochemical compatibility of these recognition units and the possibility of rapid dissociation of the interaction with His-tagged proteins despite sub-nanomolar binding affinity, enable distinct and versatile handling and modification of recombinant proteins. In this review, we briefly introduce the key principles and features of MCH–His-tag interactions and recapitulate the broad spectrum of bioanalytical applications with a focus on quantitative protein interaction analysis on micro or nano-patterned solid surfaces and specific protein labelling in living cells.

Cyclohexyl "base pairs" stabilize duplexes and intensify pyrene fluorescence by shielding it from natural base pairs

In this study, we investigated the stability and structure of artificial base pairs that contain cyclohexyl rings. The introduction of a single pair of isopropylcyclohexanes into the middle of DNA slightly destabilized the duplex. Interestingly, as the number of the "base pairs" increased, the duplex was remarkably stabilized. A duplex with six base pairs was even more stable than one containing six A-T pairs. Thermodynamic analysis revealed that changes in entropy and not enthalpy contributed to duplex stability, demonstrating that hydrophobic interactions between isopropyl groups facilitated the base pairing, and thus stabilized the duplex. NOESY of a duplex containing an isopropylcyclohexane-methylcyclohexane pair unambiguously demonstrated its "pairing" in the duplex because distinct NOEs between the protons of cyclohexyl moieties and imino protons of both of the neighboring natural base pairs were observed. CD spectra of duplexes tethering cyclohexyl moieties also showed a positive-negative couplet that is characteristic of the B-form DNA duplex. Taken together, these results showed that cyclohexyl moieties formed base pairs in the DNA duplex without severely disturbing the helical structure of natural DNA. Next, we introduced cyclohexyl base pairs between pyrene and nucleobases as an "insulator" that suppresses electron transfer between them. We found a massive increase in the quantum yield of pyrene due to the efficient shielding of pyrene from nucleobases. The cyclohexyl base pairs reported here have the potential to prepare highly fluorescent labeling agents by multiplying fluorophores and insulators alternately into DNA duplexes.     

Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules

We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.