Chelating effect in short polymers for the design of bidentate binders of increased affinity and selectivity

The design of new strong and selective binders is a key step towards the development of new sensing devices and effective drugs. Both affinity and selectivity can be increased through chelation and here we theoretically explore the possibility of coupling two binders through a flexible linker. We prove the enhanced ability of double binders of keeping their target with a simple model where a polymer composed by hard spheres interacts with a spherical macromolecule, such as a protein, through two sticky spots. By Monte Carlo simulations and thermodynamic integration we show the chelating effect to hold for coupling polymers whose radius of gyration is comparable to size of the chelated particle. We show the binding free energy of flexible double binders to be higher than that of two single binders and to be maximized when the binding sites are at distances comparable to the mean free polymer end-to-end distance. The affinity of two coupled binders is therefore predicted to increase non linearly and in turn, by targeting two non-equivalent binding sites, this will lead to higher selectivity

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy

Two-dimensional enzyme diffusion in laterally confined DNA monolayers

Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DNA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDNAs from the top-matrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top- and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon

Computational design of cyclic peptides for the customized oriented immobilization of globular proteins

The oriented immobilization of proteins, key for the development of novel responsive biomaterials, relies on the availability of effective probes. These are generally provided by standard approaches based on in vivo maturation and in vitro selection of antibodies and/or aptamers. These techniques can suffer technical problems when a non-immunogenic epitope needs to be targeted. Here we propose a strategy to circumvent this issue by in silico design. In our method molecular binders, in the form of cyclic peptides, are computationally evolved by stochastically exploring their sequence and structure space to identify high-affinity peptides for a chosen epitope of a target globular protein: here a solvent-exposed site of β2-microglobulin (β2m). Designed sequences were screened by explicit solvent molecular dynamics simulations (MD) followed by experimental validation. Five candidates gave dose-response surface plasmon resonance signals with dissociation constants in the micromolar range. One of them was further analyzed by means of isothermal titration calorimetry, nuclear magnetic resonance, and 250 ns of MD. Atomic-force microscopy imaging showed that this peptide is able to immobilize β2m on a gold surface. In short, we have shown by a variety of experimental techniques that it is possible to capture a protein through an epitope of choice by computational design

Insights on peptide topology in the computational design of protein ligands: the example of lysozyme binding peptides

Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for

Helium nanodroplet isolation ro-vibrational spectroscopy: methods and recent results

In this article, recent developments in HElium NanoDroplet Isolation (HENDI) spectroscopy are reviewed, with an emphasis on the infrared region of the spectrum. Topics discussed include experimental details, comparison of radiation sources, symmetry issues of the helium solvation structure, sources of line broadening, changes in spectroscopic constants upon solvation, and applications including formation of novel chemical structures.Comment: 24 pages, 8 figures, 3 tables; to be published in the Journal of Chemical Physic

Mismatch detection in DNA monolayers by atomic force microscopy and electrochemical impedance spectroscopy

6noThe availability of devices for cancer biomarker detection at early stages of the disease is one of the most critical issues in biomedicine. Towards this goal, to increase the assay sensitivity, device miniaturization strategies empowered by the employment of high affinity protein binders constitute a valuable approach. In this work we propose two different surface-based miniaturized platforms for biomarker detection in body fluids: the first platform is an atomic force microscopy (AFM)-based nanoarray, where AFM is used to generate functional nanoscale areas and to detect biorecognition through careful topographic measurements; the second platform consists of a miniaturized electrochemical cell to detect biomarkers through electrochemical impedance spectroscopy (EIS) analysis. Both devices rely on robust and highly-specific protein binders as aptamers, and were tested for thrombin detection. An active layer of DNA-aptamer conjugates was immobilized via DNA directed immobilization on complementary single-stranded DNA self-assembled monolayers confined on a nano/micro area of a gold surface. Results obtained with these devices were compared with the output of surface plasmon resonance (SPR) assays used as reference. We succeeded in capturing antigens in concentrations as low as a few nM. We put forward ideas to push the sensitivity further to the pM range, assuring low biosample volume (μL range) assay conditions.openopenNkoua Ngavouka, Maryse D.; Capaldo, Pietro; Ambrosetti, Elena; Scoles, Giacinto; Casalis, Loredana; Parisse, PietroNKOUA NGAVOUKA, MARYSE DADINA; Capaldo, Pietro; Ambrosetti, Elena; Scoles, Giacinto; Casalis, Loredana; Parisse, Pietr