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ONLINE May 10- 12 2021 International Symposium on SupraBiomolecular Systems 2021

Table of Contents

TBD 1 Prof. Andreas Herrmann

TBD 2 Prof. Rachel O’Reilly

Tackling challenges in nanomedicine with responsive supramolecular polymers and advanced mi- croscopy 3 Dr. Silvia Pujals, Mr. Edgar Fuentes, Dr. Lorenzo Albertazzi

Water Soluble Nanotubular Architectures from Amphiphilic Dinucleobases 4 Dr. Fatima Aparicio, Ms. Paula Blue Chamorro, Dr. Raquel Chamorro, Prof. David Gonzalez-Rodriguez

Glyconucleolipids as new drug delivery systems for Parkinson’s disease treatment 5 Mr. Anthony Cunha, Dr. Alexandra Gaubert, Prof. Philippe Barthélémy, Dr. Benjamin Dehay, Dr. Laurent Latxague

Membraneless compartments based on intrinsically disordered proteins: from biology towards new pro- tein materials 6 Prof. Paolo Arosio

Low Molecular Weight Oleogel formation via unique keto-enol-type nucleolipid supramolecular assembly 8 Mr. Arthur KLUFTS-EDEL, Ms. Bérangère Dessane, Dr. Aladin Hamoud, Dr. Geoffrey Prévot, Dr. Antoine Lo- quet, Dr. Brice Kauffmann, Prof. Philippe Barthélémy, Prof. Sylvie Crauste-Manciet, Dr. Valérie Desvergnes

Multi-responsive supramolecular fibers from peptide-based amphiphiles 9 Mr. Edgar Fuentes, Dr. Marieke Gerth, Dr. Jose Augusto Berrocal, Dr. Carlo Matera, Prof. Pau Gorostiza, Prof. Ilja Voets, Dr. Silvia Pujals, Dr. Lorenzo Albertazzi

Electrostatic Protein Assemblies Towards Biohybrid Photoactive Materials 10 Dr. Eduardo Anaya-Plaza, Prof. Mauri Kostiainen

Exploring Polyoxometalates as Non-destructive Staining Agents for Contrast-Enhanced Microfocus Com- puted Tomography of Biological Tissues 11 Ms. Sarah Vangrunderbeeck, Mr. Sébastien De Bournonville, Mrs. Hong Giang T. Ly, Prof. Wim De Borggraeve, Prof. Tatjana Parac-Vogt, Prof. Greet Kerckhofs

Hydrogels with Photo-Switchable Stiffness: A Tool to Mimic Extra Cellular Matrix 13 Prof. Nancy Lauth de Viguerie, Dr. Anne-Françoise Mingotaud, Ms. Camille Courtine, Dr. Florence Benoit- Marquié, Dr. Corinne Lorenzo, Dr. Childerick Severac, Dr. Jorge Royes

Sequence optimized peptide nanostructures as versatile biomaterials 14 Dr. Christopher Synatschke, Mrs. Kübra Kaygisiz, Prof. Tanja Weil

Supramolecular crosslinking: Advanced hydrogels for tissue engineering 16 Ms. Jasmina Gacanin, Dr. Christopher Synatschke, Prof. Tanja Weil

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Supramolecular entities as tools for methodological shift : from 2D to 3D cell culture 17 Mr. Omar El Hamoui, Mr. Tarek Sayde, Dr. Philippe Le Coustumer, Prof. Philippe Barthélémy, Prof. Serge Battu, Prof. Karen Gaudin, Prof. Gaëtane Lespes, Dr. Bruno Alies

Memantine improves memory and hippocampal proliferation in adult male rats 19 Dr. Maha Elbeltagy, Ms. Duha Atieh, Mr. Basil Abdin, Mr. Kenan Sheikh-Yasin, Mr. Ahmad Abdulraheem, Mrs. Doaa Qattan, Dr. Ahmed Salman

TRIfunctional NAnoparticle DESign to rescue neurosystem failure caused by protein aggregation (TRI- NADES) 21 Ms. Vanesa Redondo, Prof. Thomas Schrader

Supramolecular Halogen Bonding Anion Transporters 23 Ms. Laura Bickerton, Mr. Alistair Sterling, Prof. Paul Beer, Prof. Fernanda Duarte, Prof. Matthew Langton

Membrane Permeability and its Activation Energies in Dependence on Analyte, Lipid, and Phase Type Obtained by Fluorescent Artificial Receptor Membrane Assay 24 Dr. Mohamed Nilam, Ms. Solène Collin, Ms. Shreya Karmacharya, Prof. Andreas Hennig, Prof. Werner M. Nau

Sequence-selective DNA recognition with artificial ligands 25 Mr. Álvaro Serrano, Dr. Nahid Sadeghi, Mr. Max Peters, Dr. Shubhankar Gadre, Prof. Thomas Schrader

Innovative electrical interface for stamping out bacterial biofilm infections 26 Ms. Sanjana Auditto, Dr. Serena Carrara, Mr. Florent Rouvier, Dr. Frédéric Brunel, Dr. Charlotte Jeanneau, Dr. Michel Camplo, Prof. Michelle Sergent, Prof. Imad About, Dr. Jean-Michel Bolla, Prof. Jean-Manuel Raimundo

Formation of a G-quadruplex structure by a sequence found in pre-miRNA-let-7e 28 Mr. Tiago Santos, Mr. André Miranda, Mr. Lionel Imbert, Dr. Gilmar Salgado, Prof. Eurico Cabrita, Dr. Carla Cruz

Characterization of G-quadruplex structure in pre-MIR150: potential target for anti-cancer approaches in non-small cell lung cancer 29 Ms. Joana Figueiredo, Dr. Jean-Louis Mergny, Dr. Carla Cruz

Data mining approach to unveil property-activity correlation and sequence optimization of viral trans- duction enhancing peptides 30 Mrs. Kübra Kaygisiz, Dr. Arghya Dutta, Ms. Lena Rauch, Dr. Tristan Bereau, Dr. Christopher Synatschke, Prof. Jan Münch, Prof. Tanja Weil

Could a dual elongation of the AS1411 aptamer sequence induce a lock on structural polymorphism and maintenance of properties? 31 Mr. André Miranda, Mr. Tiago Santos, Dr. Eric Largy, Dr. Carla Cruz

Nucleolipids self-assemblies enhance metal ions binding 32 Dr. Bruno Alies, Prof. Philippe Barthélémy

Mirror-image β-sheets organize supramolecular water assemblies with opposite chirality 33 Mr. Ethan Perets, Mr. Daniel Konstantinovsky, Prof. Sharon Hammes-Schiffer, Prof. Elsa Yan

Protein Nanopores Reveal Single-Molecule Behavior of DNA & RNA 35 Prof. Cynthia Burrows

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CLICK-17, a DNA enzyme that harnesses ultra-low concentrations of either Cu+ or Cu2+ to catalyze the azide-alkyne ‘click’ reaction in water. 36 Prof. Dipankar Sen, Mr. Kun Liu, Prof. Hua-Zhong (Hogan) Yu

Self-assembled nucleic acid nanostructures for drug targeting, bioimaging and biosensing 37 Prof. Jorgen Kjems

TBD 38 Prof. Stefan Matile

Reducing Gram negative enterobacteria antibiotic resistance by conjugating synthetic oligonucleotides to a nucleolipid 39 Dr. Tina Kauss, Prof. Corinne Arpin, Dr. Clémentine Aubry, Ms. Patricia Korczak, Ms. Brune Vialet, Prof. Philippe Barthélémy

Cell-selective siRNA delivery using glycosylated dynamic covalent polymers self-assembled in situ by RNA-templating 40 Ms. Maëva Coste, Mr. Sebastien Ulrich

Emergent Assembly through DNA and Chemoenzymatically Synthesized Polypeptides 41 Dr. Prashant Gudeangadi, Prof. Michael Reithofer, Dr. Christopher Serpell

Self-assembly of tetraphenylethylene-DNA conjugates into DNA-addressable vesicular architectures 42 Mr. Simon Rothenbuehler, Dr. Ioan Iacovache, Dr. Simon M. Langenegger, Prof. Benoît Zuber, Prof. Robert Haener

Capturing the dynamic association of lanthanide complexes with biomolecules : a combined NMR and molecular simulations exploration. 43 Dr. Alessio Bartocci, Dr. Natacha Gillet, Prof. Elise Dumont

Single particle combinatorial multiplexed liposome fusion mediated by DNA 44 Ms. Mette Malle, Dr. Philipp Löffler, Mr. Søren S.-R. Bohr, Mr. Magnus Berg Sletfjerding, Mr. Nikolaj Alexander Risgaard, Dr. Simon Bo Jensen, Dr. Min Zhang, Prof. Per Hedegård, Prof. Stefan Vogel, Prof. Nikos S. Hatzakis

Bacteriophage T7 single-stranded DNA-binding protein displays template-catalyzed recycling 46 Dr. Jordi Cabanas-Danes, Mr. Matthew Halma, Mr. Longfu Xu, Prof. Gijs Wuite, Dr. Iddo Heller, Dr. A.M. van Oijen, Prof. Erwin Peterman

Cyclic poly(N-alkylated-glycine) as promising antimicrobial agents in infectology. 47 Mr. Pedro Salas-Ambrosio, Mr. Antoine Tronnet, Dr. Marc Since, Dr. Sandra Bourgeade-Delmas, Dr. Jean-Luc Stigliani, Dr. Amelie Vax, Prof. Sebastien Lecommandoux, Prof. Bruno Dupuy, Prof. Pierre Verhaghe, Dr. Colin Bonduelle

Hybrid Peptide-Agarose Hydrogels for High-Sensitivity 3D Immunoassays 49 Dr. Alessandro Gori, Dr. Greta Bergamaschi, Dr. Angelo Musicò, Dr. Roberto Frigerio, Dr. Alessandro Strada, Dr. Marina Cretich

SAP-NC hydrogels: a novel hybrid platform for bioanalytic applications 50 Dr. Greta Bergamaschi, Dr. Alessandro Gori, Dr. Alessandro Strada, Dr. Angelo Musicò, Prof. Paolo Bettotti

Switching Peptide Supramolecular Assemblies by Selective Mono-Substitution 51 Dr. Alessandro Strada, Dr. Greta Bergamaschi, Dr. Alessandro Gori, Dr. Angelo Musicò, Dr. Andrea Pizzi

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β-galactosidase instructed self-assembly of supramolecular bolaamphiphiles hydrogelators 52 Dr. Alexandra Gaubert, Dr. Julie Baillet, Mr. Julien Verget, Dr. Laurent Latxague, Prof. Philippe Barthélémy

The enhancement of the efficiency of the intraocular pressure decrease by ACE inhibitor within chitosan- covered calcium phosphate particles 54 Mrs. Ekaterina Popova, Dr. Victoria Tikhomirova, Mrs. Olga Beznos, Mrs. Natalia Chesnokova, Dr. Olga Kost

Influence of a low frequency magnetic field on the release of dextran from magnetic polyelectrolyte mi- crocapsules 55 Mr. Ivan Burmistrov, Ms. Daria Trushina, Mr. Alexander Mikheev, Mr. Maxim Veselov, Mrs. Tatiana Borodina, Prof. Natalia Klyachko, Mrs. Tatiana Bukreeva

Functionalization of Core-Shell Nanoparticles (ɣ- Fe2O3/silica) for Oriented Enzyme Immobilization 56 Mr. Hongtao JI, Dr. Thierry TRON, Dr. Karine HEUZE

Controlled deposition of cyclic peptides for the synthesis of monodisperse short peptide nanotubes for biomedical applications 57 Mrs. Kim Gaudin

A Noncovalent Model of Adaptive Shapeshifting Ligands for Biomolecules 58 Mr. Robert A. Ives, Dr. Paul R. McGonigal

Utilisation of molecular tweezer’s multivalency for targeting p97 protein pore 59 Mrs. Abbna Kirupakaran, Dr. Johannes van den Boom, Mr. Matthias Hayduk, Mr. Mike Blüggel, Dr. Christine Beuck, Prof. Peter Bayer, Prof. Jens Voskuhl, Prof. Hemmo Meyer, Prof. Thomas Schrader

Preparing for self-evolving synthetic cells: The importance of translation in evolving mixed-lipid com- partments 60 Prof. Peter Strazewski, Dr. Michele Fiore, Dr. Dimitri Fayolle, Mr. Augustin Lopez, Ms. Carolina Chieffo, Mrs. Anastasiia Shvetsova

Vitamin B12 – an intriguing bioheterocycle 61 Prof. Dorota Gryko

TBD 63 Dr. Dan Pantos

Self-assembling supramolecular dendrimers for biomedical applications 64 Dr. Ling Peng

Biohybrid materials in the context of systems chemistry 65 Dr. Andres de la Escosura, Ms. Noemí Nogal, Mr. Marcos Marcos Sanz, Mr. Alonso Puente, Mr. Martin Aleksiev, Mr. Santiago Guisán, Ms. Sonia Vela

Modulating Protein-Protein Interactions Using Dynamic Combinatorial Selection Methods 67 Prof. Andy Wilson

PROBING THE FUNCTION OF CELL BIOMIMETICS AND PROTOCELLS 68 Dr. Dietmar Appelhans, Dr. Silvia Moreno, Dr. Susanne Boye, Prof. Albena Lederer, Prof. Brigitte Voit

Programming Supramolecular Self-Assembly and Gelation with Chemical Clocks 69 Dr. Guido Panzarasa

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Regulating transmembrane ion transport using stimuli-responsive supramolecular carriers 70 Prof. Matthew Langton

Foldamer bundles binding amphiphiles in water: toward artificial glycolipid binding proteins 71 Dr. Sung Hyun Yoo, Dr. Gavin W. Collie, Dr. Jérémie Burrato, Dr. Estelle Morvan, Dr. Frédéric Rosu, Dr. Arup Roy, Dr. Morgane Pasco, Dr. Valérie Gabelica, Dr. Cameron D. Mackereth, Dr. Gilles Guichard

Suprabiomolecular sensor systems 72 Prof. Andreas Hennig

Flavin-based molecular rotors 74 Mrs. Dora Rasadean, Dr. Christopher Pudney, Dr. Dan Pantos

Optimization of functionalized H-Ferritin nanocages loaded with Navitoclax as targeted drug delivery agents against Cancer Associated Fibroblasts in vitro and in vivo 75 Dr. Leopoldo Sitia, Dr. Arianna Bonizzi, Dr. Serena Mazzucchelli, Dr. Sara Negri, Dr. Cristina Sottani, Dr. Elena Grignani, Dr. Maria Antonietta Rizzuto, Prof. Davide Prosperi, Dr. Luca Sorrentino, Dr. Carlo Morasso, Dr. Raffaele Allevi, Dr. Marta Sevieri, Dr. Filippo Silva, Dr. Marta Truffi, Prof. Fabio Corsi

ɑ-Galactosidase Enzyme Nanoformulated in Multifunctional Nanoliposomes for Fabry Disease Treatment 77 Ms. Judit Tomsen-Melero, Dr. Elisabet González-Mira, Mr. Josep Merlo-Mas, Dr. Edgar Cristóbal-Lecina, Dr. Vanessa Díaz-Riascos, Dr. Albert Font, Mr. Marc Moltó-Abad, Ms. Natalia García-Aranda, Dr. José Luis Corchero, Ms. Aida Carreño, Dr. Jannik Nedergaard Pedersen, Dr. Jeppe Lyngsø, Dr. Ionita Inbal, Dr. Daniel Pulido, Dr. Santi Sala, Prof. Jaume Veciana, Dr. Simó Schwartz, Jr., Prof. Dganit Danino, Prof. Jan Skov Pedersen, Dr. Andreu Soldevila, Dr. Miriam Royo, Dr. Alba Córdoba, Dr. Ibane Abasolo, Dr. Nora Ventosa

A plug-and-play RNA detection platform for sensing cell physiology and phenotype 79 Dr. James Chappell

Pushing the limits of the inhibitory multivalent effect in glycoscience 80 Mr. Philippe Compain

Metal-Modified DNA: Merging Bioinorganic Chemistry with Supramolecular Chemistry 81 Prof. Jens Müller

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TBD

Monday, 10th May - 09:00: Plenary 1 - Oral - Abstract ID: 105

Prof. Andreas Herrmann 1 1. Rwth Aachen University

TBD

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TBD

Monday, 10th May - 09:45: Plenary 1 - Oral - Abstract ID: 106

Prof. Rachel O’Reilly 1 1. University of Birmingham

TBD

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Tackling challenges in nanomedicine with responsive supramolecular polymers and advanced microscopy

Monday, 10th May - 10:50: Oral Session 1 - Oral - Abstract ID: 73

Dr. Silvia Pujals 1, Mr. Edgar Fuentes 1, Dr. Lorenzo Albertazzi 1 1. Institute for Bioengineering of Catalonia (IBEC)

Nanomedicine arise with the promise of selectively delivering therapeutic drugs to target sites, thus increasing its their effectivity while minimizing undesired side effects. However, tumour complexity and heterogeneity pose a great challenge to the design of effective therapeutic nanomaterials, and new approaches, from material design to screening in relevant models, are necessary. Many biological structures are made of multiple components that self-organize into complex architectures. Supramolecular polymers mimic this phenomenon, allowing a modular and tunable approach that eases sample preparation.[1] One of the most appealing features of supramolecular assemblies is their ability to respond to ex- ternal stimuli due to their noncovalent nature. With a combination of microscopy and spectroscopy techniques, self-assembly in water and responsiveness to temperature, light, pH, and ionic strength was demonstrated.[2] To study the behaviour of such complex nanomaterials in action a variety of optical microscopy techniques, in particular super resolution microscopy (SRM), are being used.[3,4] SRM can achieve a resolution down to 20 nm and represents an ideal tool to visualize nanosized objects in the biological environment. In particular, SMLM (Single Molecule Localization Microscopy) can be used to image a wide range of nanomaterials beyond the diffraction limit: nanoparticles, BTA fibers, peptidic nanostructures, etc. References [1] Casellas, N.M., Pujals, S., Bochicchio, D., Pavan, G.M., Torres, T., Albertazzi, L., García-Iglesias, M. Chem.Comm., 54(33) (2018) 4112-4115. [2] Fuentes E, Gerth M, Berrocal JA, Matera C, Gorostiza P, Voets IK, Pujals S, Albertazzi L. An Azobenzene-Based Single-Component Supramolecular Polymer Responsive to Multiple Stimuli in Water. J Am Chem Soc. (2020) 142(22):10069-10078. [3] Pujals S, Albertazzi L. Super-resolution Microscopy for Nanomedicine Research. ACS Nano. (2019) 13(9):9707- 9712. [4] Pujals, S., Feiner-Gracia, N., Delcanale, P., Voets, I., Albertazzi, L., Super-resolution microscopy: a powerful tool to unveil complex synthetic molecular systems, Nat. Rev. Chem. (2019) 3, 68–84.

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Water Soluble Nanotubular Architectures from Amphiphilic Dinucleobases

Monday, 10th May - 11:05: Oral Session 1 - Oral - Abstract ID: 17

Dr. Fatima Aparicio 1, Ms. Paula Blue Chamorro 2, Dr. Raquel Chamorro 1, Prof. David Gonzalez-Rodriguez 1 1. Universidad Autonoma de Madrid, 2. Universidad

Nature has demonstrated its capacity to form self-assembled tubular systems with specific functions; some ex- amples being the mosaic tobacco virus or tube-forming proteins, such as tubulin or aquaporin. Our biologically inspired project aims at establishing a strategy to prepare tubular nanostructures1 based on complementary dinucleobase monomers featuring an amphiphilic central block, which can self-assemble in aqueous media by diverse noncovalent interactions. Watson-Crick H-bonding1 produces macrocyclic tetramers2 with a hy- drophobic core that can stack through hydrophobic forces to yield the desired nanotubes.3a On the one hand, the hydrophilic chains oriented to the periphery would help to improve water solubility, while the lipophilic pore allows hosting molecules that are complementary in size and chemical affinity.3b The aqueous tubular self- assembly of dinucleobase amphiphilic monomers equipped with hydrophilic groups at the π-conjugated central block of different nature, anionic (carboxylate), neutral (glycol) or cationic (ammonium) was thoroughly stud- ied. When subjected to diverse (drastic) changes in the experimental conditions,3c these tubular nanostructures underwent disassembly into monomers, chiral reorganization, or a morphological restructuration into globular objects due to dehydration of the peripheral hydrophilic groups. References 1. M. J. Mayoral, C. Montoro-García, D. González-Rodríguez, inComprehensive Supramolecular Chemistry II (El- sevier), Oxford, 2017, pp. 191-257. 2. (a) C. Montoro-García, J. Camacho-García, A. M. López-Pérez, M. J. Mayoral, N. Bilbao, D. González-Rodríguez, Angew. Chem., Int. Ed., 2016, 55, 223. (b) C. Montoro-García, M. J. Mayoral, R. Chamorro, D. González-Rodríguez, Angew. Chem., Int. Ed., 2017,56, 15649. (c) C. Montoro-García, N. Bilbao, I. M. Tsagri, F. Zaccaria, M. J. Mayoral, C. Fonseca Guerra, D. González-Rodríguez, Chem. Eur. J., 2018, 24, 11983. 3. (a) V. Vázquez-González, M. J. Mayoral, R. Chamorro, M. M. R. M. Hendrix, I. K. Voets, and D. González- Rodríguez, J. Am. Chem. Soc., 2019, 141, 16432. (b) F. Aparicio, P. B. Chamorro, R. Chamorro, S. Casado, D. González-Rodríguez, Angew. Chem. Int. Ed., 2020, 59, 17091. (c) 5. P. B. Chamorro, F. Aparicio, R. Chamorro, N. Bilbao, S. Casado, D. González-Rodríguez, Org. Chem. Front.,2021. https://doi.org/10.1039/D0QO01110J

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Glyconucleolipids as new drug delivery systems for Parkinson’s disease treatment

Monday, 10th May - 11:20: Oral Session 1 - Oral - Abstract ID: 20

Mr. Anthony Cunha 1, Dr. Alexandra Gaubert 2, Prof. Philippe Barthélémy 2, Dr. Benjamin Dehay 3, Dr. Laurent Latxague 2 1. Université de Bordeaux, INSERM, U1212, CNRS UMR 5320, ARNA, ARN : Régulations Naturelle et Artificielle, ChemBioPharm and CNRS, IMN, UMR 5293, 33076 Bordeaux, France, 2. Université de Bordeaux, INSERM, U1212, CNRS UMR 5320, ARNA, ARN : Régulations Naturelle et Artificielle, ChemBioPharm, 33076 Bordeaux, France, 3. Université de Bordeaux, CNRS UMR 5293, IMN, 33076 Bordeaux, France

Treatment of neurodegenerative diseases have become one of the most significant challenges of the century due to their prevalence and increasing societal cost. Even if all the mechanisms linked to the physiopathology of Parkinson’s disease (PD) are not fully understood yet, increasing genetic and neuropathological evidence in- dicate that lysosomal impairment may be a common factor. A new interesting approach in PD treatment may thus rely on the restoration of the lysosomal function using therapeutic drugs such as trehalose, a non-reducing disaccharide acting as a chemical chaperone, whose neuroprotective effect has been shown to reduce proteins aggregates. However, potential in vivo applications require its vectorization thanks to the design of formula- tion strategies, especially to cross the blood-brain barrier (BBB). A promising formulation approach leverages the unique properties of trehalose based-nucleolipids (NLs) as bioinspired therapeutic amphiphilic carriers co-formulated with biocompatible and non-toxic PLGA nanoparticles (NPs) to improve the drug solubility and protection, the targeting efficiency, and the cellular internalization. Based on recent results, a new trehalose-based NL series, named GlycoNucleoLipids (GNLs), was designed us- ing click chemistry (CuAAC) and esterification reactions. The GNL skeleton, composed of a sugar (trehalose), a nucleoside (thymidine or adenine), and a lipidic chain, was then chemically modified to afford three different GNL derivatives displaying increasing polarities. Thymidine derivatives were co-formulation with PLGA and led to spherical NPs of 167.20 ± 2.36 nm in diameter which were colloidally stable at room temperature for at least one month and at 37°C for one week. The encapsulation efficiency and the average drug loading are cur- rently evaluated by UV-Visible spectroscopy. The NPs were tested in vitroon human neuronal cells (BE(2)-M17). After being incubated for 24h and 48h, no cytotoxicity was observed, highlighting the biocompatibility of the PLGA/GNLs NPs. Further biological assays were carried out and demonstrated a cellular uptake, internalization, and colocalization of the nano-objects with lysosomal markers. The combination of PLGA and trehalose-based GNLs in NPs has two advantages: i) a potential synergistic action of the trehalose and PLGA and ii) vectorization of the nano-objects. In vitro assays will be investigated to evalu- ate the trehalose delivery and activity to restore the lysosomal function into appropriate neuronal (BE(2)-M17) and dopaminergic (LUHMES) cells. Further investigations will also be performed to evaluate the BBB passage and the targeting of neuronal cells and intracellular organelles. These promising nonviral chemical nanovectors showed their potential and open a new path of discovery in PD treatment.

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Membraneless compartments based on intrinsically disordered proteins: from biology towards new protein materials

Monday, 10th May - 11:35: Oral Session 1 - Oral - Abstract ID: 82

Prof. Paolo Arosio 1 1. ETH Zurich

Introduction In the last few years, discoveries in biology are revealing that a class of intrinsically disordered protein se- quences plays an important role in the formation of cellular membraneless compartments by spontaneous liquid-liquid phase separation of proteins and nucleic acids. These compartments underlie several important functions and can act as microreactors, in which environment and composition are carefully regulated in space and time. These observations inspired us to exploit these intrinsically disordered protein sequences to mimic these membraneless compartments on the bench, towards the development of high-performance open microre- actors, with applications for instance in biocatalysis. Methods with biologically inspired intrinsically disordered protein domains we can regulate not only the dynamic pro- cess of phase separation but also the properties of the resulting compartments, including the uptake of client molecules, polarity and material properties. With this control we can, in turn, regulate biochemical reactions occurring within the compartments. Moreover, we develop microfluidic platforms to characterize and control multiple properties of the open compartments, including both thermodynamics and dynamics of phase separa- tion. Results and Discussion By using our multi-domain proteins consisting of globular domains and biologically-inspired intrinsically dis- ordered sequences we achieve a conscious coupling of function and phase separation: while the intrinsically disordered domain modulates the phase separation process and the properties of the resulting compartments, the globular domain of the chimeric protein can perform the desired activity. Moreover, we applied our flex- ible strategy not only to different proteins but also to inorganic nanoparticles, expanding the functionalities which can be included inside the networks, towards the generation of hybrid enzymatic and chemical catalytic reactions. References 1) Faltova L. et al, ACS Nano, 2018, 12, 9991-9999 2) Küffner A.M. et al., ChemSystemsChem, 2, 2020 3) Linsenmeier M.et al., Angewandte Chemie Int. Ed., 2019, 58, 1-7 4) Capasso Palmiero U. et al., Angewandte Chemie Int. Ed., 2020, 59, 8138-8142

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Low Molecular Weight Oleogel formation via unique keto-enol-type nucleolipid supramolecular assembly

Monday, 10th May - 11:50: Oral Session 1 - Oral - Abstract ID: 38

Mr. Arthur KLUFTS-EDEL 1, Ms. Bérangère Dessane 1, Dr. Aladin Hamoud 1, Dr. Geoffrey Prévot 1, Dr. Antoine Loquet 2, Dr. Brice Kauffmann 2, Prof. Philippe Barthélémy 1, Prof. Sylvie Crauste-Manciet 1, Dr. Valérie Desvergnes 1 1. Equipe ChemBioPharm, INSERM U1212, UMR 5320 CNRS-Université de Bordeaux, 2. Chimie et Biologie des Membranes et des Nano-objets (CBMN) UMR5248 CNRS-Université de Bordeaux

Over the years, gels covered a wide range of applications and interested increasingly scientists because of their incredible properties. They draw growing attention in drug delivery since they can control the release and rise formulation’s stability.[i] Among this family, oleogels (oily continuous phase) represent a major route of interest in medicine to spread and/or vectorize lipophilic active principle. Indeed, pharmacopoeia hold a large part of oily-like compounds, potential impact could be huge. Based on our team’s expertise in the formation of hydrogels, nucleolipids aspired to being a promising strategy.[ii] This fascinating family of amphiphilic bio- conjugates could have astounding self-assembly abilities depending on their molecular structures. It leads to an extensive panel of physical, chemical and/or biological properties. These bioinspired molecules contained one nucleoside part (or nucleotide) and one long carbon chain linked by a covalent bond. This work reports the design and synthesis of the first nucleolipidic Low Molecular Weight oleogelator (LMWG). Key step rests on bioinspired Stetter reaction which coupled nucleoside and lipid by 1,4-diketone link.[iii] This NL happened to be able to form a gel in FDA-approved oils like Miglyol 812 N. Nucleation networks are progressively formed until the gel is build. Following this result, a primary structure study demonstrated that each part of the molecule was necessary to make a gel in oil. To ensure a gelation process, weak interactions must be able to take place and particularly hydrogen bonds between the oleogelator molecules. NMR and XRD analyses revealed the pres- ence of an enol function resulting from keto-enol equilibrium, thus explaining the unique gelation capacity of this compound. Starting from this oleogelator, a more accurate structure/activity study is in the pipeline with the aim of making a rational design. Ultimately, identifying structural parameters that will affect properties of gels could be huge for the future design of drug delivery system. [i] Weiss, R., G. J. Am. Chem. Soc., 2014, 136, 7519-7530. [ii] (a) Rosemeyer, H. Chem. Biodivers. 2005, 2, 977-1062 (b) Gissot, A.; Camplo, M.; Grinstaff, M. W.; Barthélémy, P. Org. Biomol. Chem. 2008, 6, 1324-1333. (c) Baillet, J.; Desvergnes, V.; Hamoud, A.; Latxague, L.; Barthélémy, P. Adv. Mater. 2018 doi.org/10.1002/adma.201705078. [iii] (a) Breslow, R. J. Am. Chem. Soc. 1958, 80, 3719-3726 (b) Hamoud, A.; Barthélémy, P.; Desvergnes, V., Org. Biomol. Chem. 2018, 16, 1760-1769.

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Multi-responsive supramolecular fibers from peptide-based amphiphiles

Monday, 10th May - 12:05: Oral Session 1 - Oral - Abstract ID: 97

Mr. Edgar Fuentes 1, Dr. Marieke Gerth 2, Dr. Jose Augusto Berrocal 3, Dr. Carlo Matera 4, Prof. Pau Gorostiza 5, Prof. Ilja Voets 6, Dr. Silvia Pujals 1, Dr. Lorenzo Albertazzi 1 1. Institute for Bioengineering of Catalonia (IBEC), 2. Van’t Hoff Laboratory for Physical and Colloid Chemistry, Debye Institute for Nanomaterials Science, Utrecht University, 3. Adolphe Merkle Institute, Polymer Chemistry and Materials, University of Fribourg, 4. Department of Pharmaceutical Sciences - University of Milan, 5. Institute for Bioengineering of Catalonia, 6. Laboratory of Self-Organizing Soft Matter, Department of Chemical Engineering and Chemistry & Institute of Complex Molecular Systems (ICMS) Eindhoven University of Technology (TUE)

A strategy that is being intensively explored to efficiently deliver pharmaceuticals involves drug delivery sys- tems. These carriers should greatly improve the pharmacokinetics of drugs, while providing a selective delivery at the target tissue. Even though great advances are being done in the field, the lack of an effective targeting keeps this strategy away from real application. Supramolecular polymers could introduce some advantages compared with conventional drug delivery sys- tems. Supramolecular polymers are structures build from self-assembled monomeric units, held together by non-covalent interactions. Thus, it is a modular system in which each monomer can introduce multiple func- tionalities to the whole structure. Furthermore, their dynamic nature makes them ideal to respond to a stimulus and promote a controlled therapeutic release. Finally, their characteristic fiber-like shape makes them ideal for seeking and recognition of their target, due to the higher surface-volume ratio and the multivalency effect. In our group, we aim to generate a new family of peptide-based molecules able to self-assemble into highly stable and responsive supramolecular fibers. Our approach makes use of natural and synthetic amino acids in the molecular structure to fine tune the properties of the assemblies and imprint the advantages of synthetic peptides: high stability, high design modularity and straightforward synthesis by using Solid Phase Peptide Synthesis. Here we present a multi-responsive supramolecular system designed in our lab, in which assembly/disassembly of nanofibers can be achieved with light, pH, salt concentration and temperature. The molecular structure of the monomer (figure 1) is formed by a benzene tricarboxamide core (C3 symmetry and self-assembly driver), followed by three peptide like wedges, containing an azobenzene amino acid (responsiveness to light), and ended in lysine (gives responsiveness to pH and salt). This system represents a step forward in the study of responsive fiber-like structures as drug delivery systems.

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9 International Symposium on SupraBiomolecular Systems 2021

Electrostatic Protein Assemblies Towards Biohybrid Photoactive Materials

Monday, 10th May - 13:30: Oral Session 2 - Oral - Abstract ID: 36

Dr. Eduardo Anaya-Plaza 1, Prof. Mauri Kostiainen 1 1. Aalto University

Proteins represent one of the most versatile biomacromolecules employed by Nature to address structural, recognition, transport, and catalytic functions. These macromolecules present a pH-dependent surface charge, that makes them suitable candidates for self-assembled materials bound by electrostatic interactions. Re- cently, a very exciting field has arisen, developing highly ordered materials by combination of proteins and a crystallization agent.[1,2] Among them, a plethora of nanomaterials such as nanoparticles, micelles, oly- gomers/polymers or small molecules have been explored. The resulting materials are obtained by control on the pH, temperature, ionic strength of the media, as well as by the concentration, size, and charge of the co- crystallizing agent. Additionally, the selection of the protein shape plays a key role in the overall morphology of the materials. In this contribution, spherical (apoferritin, aFt),[3] toroidal (peroxiredoxin, Prdx),[4] and elongated (tobacco mosaic virus, TMV)[5] proteins will be overviewed. These proteins share a negative overall charge that, in combination with cationic photosensitizers, yields photoactive biohybrids. Their optical properties range from red-light emitters, to photoinduced singlet oxygen generators with potential application in water remediation.

[1] M. A. Kostiainen, P. Hiekkataipale, A. Laiho, V. Lemieux, J. Seitsonen, J. Ruokolainen, P. Ceci, Nature Nan- otechnology 2013, 8, 52–56. [2] V. Liljeström, A. Ora, J. Hassinen, H. T. Rekola, Nonappa, M. Heilala, V. Hynninen, J. J. Joensuu, R. H. A. Ras, P. Törmä, O. Ikkala, M. A. Kostiainen, Nature Communications 2017, 8, 671. [3] J. Mikkilä, E. Anaya-Plaza, V. Liljeström, J. R. Caston, T. Torres, A. de la Escosura, M. A. Kostiainen, ACS Nano 2016, 10, 1565–1571. [4] E. Anaya-Plaza, manuscript in preparation [5] E. Anaya￿Plaza, A. Aljarilla, G. Beaune, Nonappa, J. V. I. Timonen, A. Escosura, T. Torres, M. A. Kostiainen, Adv. Mater. 2019, 31, 1902582.

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10 International Symposium on SupraBiomolecular Systems 2021

Exploring Polyoxometalates as Non-destructive Staining Agents for Contrast-Enhanced Microfocus Computed Tomography of Biological Tissues

Monday, 10th May - 13:45: Oral Session 2 - Oral - Abstract ID: 25

Ms. Sarah Vangrunderbeeck 1, Mr. Sébastien De Bournonville 2, Mrs. Hong Giang T. Ly 1, Prof. Wim De Borggraeve 1, Prof. Tatjana Parac-Vogt 1, Prof. Greet Kerckhofs 3 1. Department of Chemistry, KU Leuven, 2. Skeletal Biology and Engineering Research Center, KU Leuven, 3. Biomechanics Lab – ContrasT Team, UCLouvain

The field of tissue engineering requires detailed ex-vivo tissue assessment. For that purpose, conventional histol- ogy is the gold standard in the field. However, it shows limited potential for assessing 3D biological tissues and quantifying their spatial distribution and interactions, as it is destructive and costly in terms of resources. By using contrast-enhancing staining agents (CESAs), contrast-enhanced X-ray microfocus computed tomography (CE-CT) could tackle these shortcomings and provide a solution for 3D soft tissue visualization. While many reported CESAs are destructive for the samples, our group validated a Hafnium-substituted Wells- Dawson polyoxometalate (Hf-WD POM) as CESA for skeletal tissue visualization. Thanks to the combination of the hydrophobic behavior of adipocytes and the non-covalent interactions of Hf-WD POM with the bone marrow and the vasculature, these tissues could be visualized at the single cell level using high resolution microCT scanning. In this study, different POM species, including the less expensive precursors of the metal substituted Hf-WD POM, were screened for their potential as CESAs. PBS solutions of four different Well-Dawson polyoxometa- lates were tested on murine tibiae and kidneys, and the resulting CE-CT images were analyzed for contrast and diffusion. Based on their staining capacity and speed of diffusion, as well as on their fate after tissue stain- ing experiments, we concluded that monolacunary Wells-Dawson polyoxometalate (Mono-WD POM) is a highly suitable CESA for non-destructive CE-CT imaging of soft tissues (Figure 1). Mono-WD POM is stable under physio- logical pH and temperature. Tissues that were stained with Mono-WD POM showed a similar contrast enhance- ment as those that were stained with Hf-WD POM and phosphotungstic acid, which have been typically used in the past. Therefore, the staining solution containing Mono-WD POM was further improved by the addition of lithium chloride (LiCl) in order to improve the solubility, and hence the contrast enhancement of soft tissues. We concluded that Mono-WD POM (35 mg/mL) dissolved in PBS, in the presence of 3.0 mg/mL LiCl resulted in the highest contrast enhancement of soft tissue without inducing tissue shrinkage. The design and synthesis of new CESAs contributes to a better visualization of biological tissue, as well as to a better understanding of the effect of pathologies on tissue architecture and hence function. [1] S. de Bournonville, S. Vangrunderbeeck, H. G. T. Ly, C. Geeroms, W. M. De Borggraeve, T. N. Parac-Vogt, G. Kerckhofs, Acta Biomater.2020, 105, 253-262.

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Hydrogels with Photo-Switchable Stiffness: A Tool to Mimic Extra Cellular Matrix

Monday, 10th May - 14:00: Oral Session 2 - Oral - Abstract ID: 84

Prof. Nancy Lauth de Viguerie 1, Dr. Anne-Françoise Mingotaud 1, Ms. Camille Courtine 1, Dr. Florence Benoit-Marquié 1, Dr. Corinne Lorenzo 2, Dr. Childerick Severac 2, Dr. Jorge Royes 3 1. Laboratoire des IMRCP, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, 118 Rte de Narbonne, F-31062 Toulouse cedex, France, 2. ITAV, Université de Toulouse, CNRS, UPS, 1 place Pierre Potier 31106 Toulouse Cedex 1, France, 3. IMDEA Nanociencia, C/ Faraday 9, 28049 Madrid, Spain

Cells in tissues interact continuously with their microenvironment influencing their behaviors, such as pro- liferation or cell differentiation. Mechanical stimuli can also lead to pathologies. It is thus to understand the relationship between cell response and mechanical stress. For this, smart materials controllable by external stimuli and capable of inducing a mechanical stress into the cell are needed. Azobenzene@cyclodextrin are well-known light-responsive host-guest complexes which have been used to cre- ate smart hydrogels [1]. Depending on its isomerization state, azobenzene (azo) and its derivatives can form complexes with β-cyclodextrin (β-CD). Light can thus trigger the formation or the breakage of the supramolec- ular complexes (Figure). In this work, visible-light responsive azo derivatives and their complexation in β-CD were studied. From azo@β- CD complexes, we designed hydrogels composed of a supramolecular photo-controllable polymer network [2]. The gels were furthermore crosslinked by thiol-maleimide reaction. In the end, controlling the composition of these hydrogels makes it possible to tune their stiffness and to change it in a controllable, light-assisted way. These hydrogels are promising tools to study the influence of matrix stiffness on the cellular and tissue behav- iors. [1] A. Harada, Y. Takashima, M. Nakahata, Acc. Chem. Res., 47, 2128, 2014 [2] J. Royes Mir, C. Coudret, C. Roux, F. Benoit￿Marquié, M. Cazalès, C. Séverac, C. Lorenzo, A.-F. Mingotaud, ChemPhotoChem, 1, 311-316, 2017

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13 International Symposium on SupraBiomolecular Systems 2021

Sequence optimized peptide nanostructures as versatile biomaterials

Monday, 10th May - 14:15: Oral Session 2 - Oral - Abstract ID: 37

Dr. Christopher Synatschke 1, Mrs. Kübra Kaygisiz 2, Prof. Tanja Weil 2 1. Max Planck Institute for Polymer Research, Mainz, Germany, 2. Max Planck Institute for Polymer Research, Mainz

Introduction: Self-assembling peptides (SAPs) are versatile building blocks for preparing bioactive materials and have been used as therapeutics, drug delivery agents, and cell scaffolds to name a few. Natural and un- natural amino acids provide the monomers for the design of sequences with desired chemical functionalities, which assemble into a diverse set of nanostructures (micelles, fibers, ribbons). While a near-infinite number of sequences can be generated, it is impossible to exhaustively prepare and test them. Therefore, sequence optimization is needed to identify high-performing materials from a small subset of sequences. Methods: Here, we performed sequence optimization of nanofiber-forming SAPs for two different applications, namely as viral transduction enhancers1 and as scaffolds for peripheral nerve regeneration.2 Spectroscopic and microscopic techniques were used to identify the molecular and nanoscopic structure of the SAPs. Subsequently, the physico-chemical information was correlated with biological behavior and design rules for highly efficient nanostructures could be identified. Results: Using a small peptide library of appr. 30 sequences, we identified a number of key parameters of the SAPs that are required for high bioactivity. These include the ability to form nanofibers, a positive zeta poten- tial and a high content of intramolecular beta-sheets. An alternating sequence of hydrophobic and positively charged amino acids was found as a general pattern which results in highly active materials. Discussion: By identifying underlying design rules for SAPs, a short peptide sequence was found that shows com- parable infectivity enhancement to a previously published sequence, enhancing factor-C, but at much shorter sequence length (7-mer vs. 12-mer). For peripheral nerve regeneration, SAPs were found that support neuronal growth in vitro and led to enhanced functional recovery after local injection in vivo. Interestingly, despite tar- geting different applications (virus delivery and tissue regeneration) the design rules for active SAPs were very similar. Further investigation is required to elucidate if a more general connection between bioactivity and SAP structure can be made. References: 1. Sieste S, Mack T, Lump E, Hayn M, Schütz D, Röcker A, et al. Supramolecular Peptide Nanofibrils with Op- timized Sequences and Molecular Structures for Efficient Retroviral Transduction. Adv Funct Mater 2021: ac- cepted. 2. Schilling C, Mack T, Lickfett S, Sieste S, Ruggeri FS, Sneideris T, et al. Sequence-Optimized Peptide Nanofibers as Growth Stimulators for Regeneration of Peripheral Neurons. Adv Funct Mater 2019, 29(24): 1809112.

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15 International Symposium on SupraBiomolecular Systems 2021

Supramolecular crosslinking: Advanced hydrogels for tissue engineering

Monday, 10th May - 14:30: Oral Session 2 - Oral - Abstract ID: 52

Ms. Jasmina Gacanin 1, Dr. Christopher Synatschke 1, Prof. Tanja Weil 1 1. Max Planck Institute for Polymer Research, Mainz

Introduction: In regenerative medicine there is a high demand for new biomaterials supporting cellular ad- hesion, growth, and differentiation to recover damaged tissue while offering responsiveness, biodegradability, stability, efficient recovery, controlled delivery of bioactive molecules, broad biocompatibility and minimally invasive application. Conventional hydrogels proposed for this purpose often lack the ability to meet all of these requirements. To overcome these limitations, we developed biohybrid materials for tissue engineering consisting of a biopolymer backbone that offers appealing properties like precise chain length and sequence and is grafted with two types of supramolecular gelators, namely DNA or nanofiber-forming peptides, to ex- plore the combination of covalent and supramolecular bonds in one versatile material. While the biopolymer provides enhanced stability, supramolecular crosslinking grafts from endogenous building blocks introduce a more dynamic behavior generating impressive material properties and good compatibility with various cell types. Methods: We developed hydrogels as attractive pharmacological systems for controlled delivery of bioactive proteins and selective cell population control in osteoporosis,[1] and as responsive, injectable cell matrices open- ing many attractive opportunities for 3D tissue engineering.[2] The synthesized biohybrids and their DNA or self-assembling peptide grafts as supramolecular gelators were characterized by chromatography, microscopy, spectroscopy and rheological techniques to identify chemical responsiveness and nanostructure formation, and examine material features including stability, degradation, mechanical properties, and regeneration ef- ficiencies. Cell tests were performed with different cell types (e.g., osteoclast, osteoblast, endothelial, neuronal, macrophage) to evaluate cytotoxicity, cell viability, migration, differentiation, and selective modulation of cell activities. Results: The DNA-grafted hydrogels offer programmability based on DNA-hybridization and achieve specific loading and spatiotemporally controlled release of active proteins reducing osteoclast formation and activity, making them an advanced system for local treatment of bone diseases. Peptides featuring stimulus-mediated nanofiber-formation based on molecular rearrangement and subsequent structural transition by supramolec- ular interactions were developed and grafted to a biopolymer to induce gelation with self-repair of defects by instantaneous reassembly, shear-thinning with exceptional in situ regeneration and ability of gel injection. Discussion: The supramolecular biohybrid hydrogels offer a broad range of programmable chemical, mechani- cal and biological properties, making them an appealing material for biomedical applications. Biocompatibility, controlled delivery or the ability to cultivate multiple cell types in injectable scaffolds demonstrates the mate- rials promise for regenerative medicine. [1] Gačanin, J., Kovtun, A., Fischer, S., et al., Adv. Healthcare Mater. 2017, 6, 1700392. [2] Gačanin, J., Hedrich, J., et al., Adv. Mater. 2019, 31, 1805044.

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Supramolecular entities as tools for methodological shift : from 2D to 3D cell culture

Monday, 10th May - 14:45: Oral Session 2 - Oral - Abstract ID: 70

Mr. Omar El Hamoui 1, Mr. Tarek Sayde 2, Dr. Philippe Le Coustumer 3, Prof. Philippe Barthélémy 4, Prof. Serge Battu 5, Prof. Karen Gaudin 6, Prof. Gaëtane Lespes 7, Dr. Bruno Alies 6 1. Institut des Sciences Analytiques et de Physico Chimie pour l’Environnement et les Matériaux, UMR CNRS 5254, UPPA / ARN : Régulations Naturelles et Artificielles, Inserm U1212, UMR 5320 CNRS, Université de Bordeaux, 2. ARN : Régulations Naturelles et Artificielles, Inserm U1212, UMR 5320 CNRS, Université de Bordeaux / Contrôle de l’Activation cellulaire, Progression Tumorale et Résistance thérapeutique, EA 3842, Université de Limoges, 3. Bordeaux Imaging Center, UMS 3420 CNRS-INSERM, Université de Bordeaux, 4. Equipe ChemBioPharm, INSERM U1212, UMR 5320 CNRS-Université de Bordeaux, 5. Contrôle de l’Activation cellulaire, Progression Tumorale et Résistance thérapeutique, EA 3842, Université de Limoges, 6. ARN : Régulations Naturelles et Artificielles, Inserm U1212, UMR 5320 CNRS, Université de Bordeaux, 7. Institut des Sciences Analytiques et de Physico Chimie pour l’Environnement et les Matériaux, UMR CNRS 5254, UPPA

Introduction: Research has made drastic progresses with cell culture, leading to some improvement in the understanding of cellular and molecular mechanisms. Nevertheless, two-dimensional (2D) in vitro studies may reflect results that differ from those in vivo because of the flattened way cells grow on plate. Such differences in cell morphology can account for some variabilities between in vitro and in vivo results. Furthermore, in vivo studies are strictly regulated, hard and long to lead, and ethically questionable. New methods based on three-dimensional (3D) cell culture using supramolecular gel matrix, where cells could grow in a proper spatial organization, can be implemented to overcome these limitations, forming a bridge between in vitro and in vivo. Here, we investigate a class of biomaterials consisting of supramolecular assembly of amphiphilic glyco-nucleolipid-based gelators (GNBA) that appears to exhibit close properties of interest for our purpose. Although in order to be used as a 3D cell culture scaffold, the latter has to present similar biophysical properties to the extracellular matrix (ECM) and be easily manipulated during cell culturing. Thus, characterizations under different gel conditions have been carried out in order to discriminate among the targeted properties.

Methods: Electron microscopy and shear rheology were done in order to characterize the architecture and the mechani- cal properties of these scaffolds. Kinetics and thermodynamics features were studied by UV-vis spectroscopy owing to the optical properties of these hydrogels. UV-vis spectroscopy and fluorescence spectrophotometry were conducted to follow the diffusion of different biomolecules through the gels and their external medium. 3D cell cultures were done using GNBA-based matrix by seeding cancer stem cells (U87 glioblastoma cell line) under sol-gel transition. Biological characterizations were performed during the cultures by measuring cell viability, metabolic activity and morphology.

Discussion: Our results show fibrillar architecture and viscoelastic properties in cell culture media mimicking the ECM. Rheological properties are easily tunable by adapting the GNBA concentration, allowing to simply reach an aimed viable stiffness. Their kinetic and thermal features (i.e. thermal reversibility of the gelation and high gel- sol transition temperature) permit easy handling and stability for culture incubation. Cells incorporated into these hydrogels show small-sized spheroids growing for at least 1 month and are still alive and proliferating until the end of the cell cultures, supporting a proof-of-concept of nucleolipid-based hydrogel matrix. Next steps consist of refining a 3D cell culture protocole in order to grow multicellular tumor spheroids for toxicity

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assays.

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18 International Symposium on SupraBiomolecular Systems 2021

Memantine improves memory and hippocampal proliferation in adult male rats

Monday, 10th May - 15:20: Flash Session 1 - Poster - Abstract ID: 19

Dr. Maha Elbeltagy 1, Ms. Duha Atieh 2, Mr. Basil Abdin 2, Mr. Kenan Sheikh-Yasin 2, Mr. Ahmad Abdulraheem 2, Mrs. Doaa Qattan 2, Dr. Ahmed Salman 1 1. Department of Anatomy and Histology, Faculty of Medicine, The University of Jordan, Amman, Jordan, 2. Faculty of Medicine, The University of Jordan, Amman, Jordan

Introduction Neurogenesis occurs during the embryological development of the brain. Post-developmental neural prolifer- ation occurs only in the hippocampus, the subventricular zone of the lateral ventricles, and the striatum. Doxorubicin (DOX) is an anthracycline agent that results in cognitive deterioration and memory impairment, whereas memantine (MEM) is an NMDA receptor antagonist that is approved for the treatment of Alzheimer dementia. Many studies have revealed MEM’s positive impact on memory and demonstrated that it stimulates neuronal division in the hippocampus. This study aimed to assess the effect of MEM on spatial memory and hippocampal proliferation in adult male rats treated with DOX. Methods Forty male Sprague-Dawley rats were divided into four groups of ten rats each according to agent: control, MEM (2.5 mg/kg), DOX (2 mg/kg), and DOX with MEM. The rats were given seven intraperitoneal injections every other day. We tracked the rat’s weights to assess the weight-reducing effects of the drugs. To test spatial memory, the rats were subjected to the novel location recognition (NLR) task 30 minutes after the last injection. Additionally, Ki67 immunohistochemistry was performed to examine hippocampal proliferation. Results The results showed no performance difference in the NLR task for the MEM-injected group compared to the control group. The DOX-injected group failed to differentiate between the two locations in the NLR task (p = 0.2). However, the co-administration of MEM with DOX improved the spatial memory of the rats (p = 0.04). There was a significant increase in Ki67-positive cells in the MEM-treated group compared to the saline-treated group. Treatment with DOX impaired hippocampal proliferation compared to treatment with MEM or saline. The co-administration of MEM with DOX improved hippocampal proliferation compared to treatment with DOX alone. There was significant weight reduction in the DOX group in comparison to the control group, but MEM attenuated DOX-induced weight loss. Conclusions Rats treated with DOX displayed a drop in memory, hippocampal proliferation, and weight, whereas the co- administration of MEM with DOX improved memory, hippocampal proliferation, and doxorubicin-induced weight loss.

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20 International Symposium on SupraBiomolecular Systems 2021

TRIfunctional NAnoparticle DESign to rescue neurosystem failure caused by protein aggregation (TRINADES)

Monday, 10th May - 15:20: Flash Session 1 - Poster - Abstract ID: 51

Ms. Vanesa Redondo 1, Prof. Thomas Schrader 2 1. University of Duis, 2. university of dui

Alzheimer’s disease (AD) is the most prevalent form of dementia, with 50 million people affected around the world. Aβ is one of the proteins involved in AD, which can misfold and form plaques that collect between neurons and disrupt cell function. To this day, an effective treatment against Aβ deposition has yet to be found. With this challenge in mind, a trifunctional nanoparticle was designed to prevent the toxic effect of Aβ aggre- gation. Each nanoparticle is equipped with three elements: a selector, which binds to small neurotoxic Aβ oligomers; a breaker, which disrupts the existing β-sheet by dissociating its hydrogen bonds; an artificial pro- tease, which cuts the absorbed protein molecules into smaller fragments, regenerating the original trifunctional state and therefore making the cycle catalytic. With this approach, the trifunctional nanoparticle is turned into a disaggregation nanomachine, which may completely abolish the aggregation propensity of Aβ and rescue cell viability. A total organic synthesis was carried out to obtain β-sheet selectors, β-sheet breakers, and artificial proteases. β- sheet selectors and breakers exhibited a significant reduction of Aβ fibrils in vitro. Preliminary fluorescence assays were carried out, finding that the artificial proteases can inhibit and disag- gregateAβ protein. The effectivity of the artificial proteases to cleave different peptides will be evaluated by proteomic analysis using mass spectrometry. Finally, the synthesized ligands will be supported on gold nanoparticles obtained by Pulsed Laser Ablation in Liquids (PLAL). The effectivity of the nanomachine will be evaluated by several assays (CD, Fluorescence spec- troscopy, AFM, TEM…)

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21 International Symposium on SupraBiomolecular Systems 2021

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22 International Symposium on SupraBiomolecular Systems 2021

Supramolecular Halogen Bonding Anion Transporters

Monday, 10th May - 15:20: Flash Session 1 - Poster - Abstract ID: 64

Ms. Laura Bickerton 1, Mr. Alistair Sterling 1, Prof. Paul Beer 1, Prof. Fernanda Duarte 1, Prof. Matthew Langton 1 1. University of Oxford

There are many cases within nature where the transport of anions across lipid bilayer membranes is crucial to maintain homeostasis. Ions have very low membrane permeability and thus require the action of either a mem- brane channel, pumps or mobile carriers to facilitate their transmembrane transport. There are over 400 genes which encode for ion channels in the human genome1 and mutations within these genes leads to life-altering channelopathies including Bartter syndrome, Best disease, and Cystic Fibrosis. There is therefore a pressing need to develop synthetic carriers to facilitate ion transport across membranes. In comparison to hydrogen bonding (HB), halogen bonding (XB) – the attractive intermolecular interaction between a polarized halogen atom and a Lewis base – has strict linear geometry making them highly directional, and more hydrophobic.2 Despite these favorable properties, their utilisation in synthetic anion transporters is underdeveloped.3 Here we show that novel iodotriazole halogen bonding anion carriers and their C-H hydrogen bonding analogues can be synthesized via high yielding and versatile click chemistry, to afford highly active and readily accessible anion transporters. The ability of the carriers to transport anions across lipid bilayers was determined using fluorescence assays in multiple vesicle-based experiments, to determine the effect of the XB and HB interactions on anion transport selectivity.4 References 1P. Imbrici, A. Liantonio, G. M. Camerino, A. Mele, A. Giustino, S. Pierno, A. De Luca, D. Tricarico, J. F. Desaphy and D. Conte, Front. Pharmacol., 2016, 7, 121. 2L. C. Gilday, S. W. Robinson, T. A. Barendt, M. J. Langton, B. R. Mullaney and P. D. Beer, Chem. Rev, 2015, 115, 7118-7195. 3A. V. Jentzsch, D. Emery, J. Mareda, S. K. Nayak, P. Metrangolo, G. Resnati, N. Sakai and S. Matile, Nat. Commun, 2012, 3, 905. 4L. E. Bickerton, A. J. Sterling, P. D. Beer, F. Duarte and M. J. Langton, Chem. Sci, 2020, 11, 4722-4729.

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23 International Symposium on SupraBiomolecular Systems 2021

Membrane Permeability and its Activation Energies in Dependence on Analyte, Lipid, and Phase Type Obtained by Fluorescent Artificial Receptor Membrane Assay

Monday, 10th May - 15:20: Flash Session 1 - Poster - Abstract ID: 66

Dr. Mohamed Nilam 1, Ms. Solène Collin 2, Ms. Shreya Karmacharya 2, Prof. Andreas Hennig 1, Prof. Werner M. Nau 2 1. Institute for Chemistry of New Materials, University of Osnabrück, Barbarastraße 7, 49069 Osnabrück, 2. Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1, 28759 Bremen

Time-resolved monitoring of the permeability of analytes is of utmost importance in membrane research. Exist- ing methods are restricted to single-point determinations or flat synthetic membranes, limiting access to biologi- cally relevant kinetic parameters (permeation rate constant, permeation coefficients). We now use the recently introduced fluorescent artificial receptor membrane assay (FARMA) as a method to monitor in real-time the permeation of indole derivatives through liposomal membranes of different lipid compositions. The method is based on the liposomal encapsulation of a chemosensing ensemble or “fluorescent artificial receptor”, con- sisting of 2,7-dimethyldiazapyrenium (MDAP) as a fluorescent dye and cucurbit[8]uril (CB8) as a macrocyclic receptor, that responds to the complexation of a permeating aromatic analyte by a fluorescence quenching.[1] FARMA does not require fluorescent labeling of the analytes and allows access to permeability coefficients in the range from 10-8 cm s-1 to 10-4 cm s-1. The effect of temperature on the permeation rate of a series of indole derivatives across the phospholipid membranes was studied. The activation energies for permeation through POPC/POPS phospholipid membranes were in the range of 28-96 kJ mol–1. To study the effect of different lipid phases on the membrane permeability, we performed experiments with DPPC/DOPS vesicles, which showed a phase transition from a gel phase to a liquid-crystalline phase, where the activation energies for the permeation process were expected to show a dramatic change. Accordingly, for the permeation of the indole derivatives into the DPPC/DOPS liposomes, discontinuities were observed in the Arrhenius plots, from which the perme- ation activation energies for the distinct phases could be determined, for example, for tryptamine 245 kJ mol–1 in the gel phase and 47 kJ mol–1 in the liquid-crystalline phase.[2] References [1] F. Biedermann, G. Ghale, A. Hennig, W. M. Nau, Commun. Biol2020, 3, 383. [2] M. Nilam, S. Collin, S. Karmacharya, A. Hennig, W. M. Nau, ACS Sens.2021, 6, 175-182.

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24 International Symposium on SupraBiomolecular Systems 2021

Sequence-selective DNA recognition with artificial ligands

Monday, 10th May - 15:20: Flash Session 1 - Poster - Abstract ID: 93

Mr. Álvaro Serrano 1, Dr. Nahid Sadeghi 1, Mr. Max Peters 1, Dr. Shubhankar Gadre 1, Prof. Thomas Schrader 1 1. Institute of Organic Chemistry, University of Duisburg-Essen, Universitätsstr. 7, 45141 Essen, Germany

Nucleic acid recognition is a fundamental biological process and a major prerequisite for gene expression and other important biological events. For sequence-selective binding to be achieved, DNA ligands must target either the major or the minor groove, where the base sequence becomes freely accessible. Deliberate interference with specific DNA fragments by synthetic binding agents bears great potential for applications in molecular biology, such as site-specific modulation of gene expression, modulation of protein binding or targeting of DNA damage, thus providing a powerful tool for gene-specific manipulation of DNA. Apart from Dervan’s seminal work in the minor groove,1 there is, until today, a frustrating lack of entirely ar- tificial non-invasive sequence-selective major groove binders for double stranded DNA that operate efficiently without sequence constraints. TFOs (Triplex Forming Oligonucleotides), the classical antigeneagents, suffer from weaker interactions at physiological pH and charge repulsion between the phosphodiester backbones and, despite many chemical modifications, the recognition of mixed purine/pyrimidine sequences by TFOs remains a challenge. In this context, we have designed a modular system for the sequence selective molecular recognition of dou- ble stranded DNA by triplex formation in the major groove. It consists of four artificial base pair binders (AT/TA/GC/CG) linked to a backbone, which are specifically arranged to be complementary to any given DNA sequence. 1. D. M. Chenoweth, D. A. Harki, J. W. Phillips, C. Dose, P. B. Dervan: Cyclic Pyrrole-Imidazole Polyamides Tar- geted to the Androgen Response Element, J. Am. Chem. Soc.2009, 131, 7182-7188; K. A. Muzikar, N. G. Nickols, P. B. Dervan: Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression, Proc.Natl. Acad. Sci. USA2009, 106, 16598-16603.

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25 International Symposium on SupraBiomolecular Systems 2021

Innovative electrical interface for stamping out bacterial biofilm infections

Monday, 10th May - 15:20: Flash Session 1 - Poster - Abstract ID: 44

Ms. Sanjana Auditto 1, Dr. Serena Carrara 2, Mr. Florent Rouvier 3, Dr. Frédéric Brunel 2, Dr. Charlotte Jeanneau 4, Dr. Michel Camplo 2, Prof. Michelle Sergent 5, Prof. Imad About 4, Dr. Jean-Michel Bolla 3, Prof. Jean-Manuel Raimundo 2 1. Aix-Marseille Univ, CNRS, CINAM, Marseille, France., 2. Aix-Marseille Univ, CNRS, CINAM, Marseille, 3. Aix-Marseille Univ, INSERM, SSA, IRBA, MCT, Marseille, 4. Aix-Marseille Univ, CNRS, ISM, Inst Movement Sci, Marseille, 5. Aix-Marseille Université, Avignon Université, CNRS, IRD, IMBE, Marseille

Bacterial infections are one of the major threats to public health and food safety which makes it urgent to com- bat by developing materials or strategies limiting or preventing these bacterial proliferations and biofilm infec- tions.[1] Although medical implants have led to dramatic improvement in patient’s health and well-being, there are drawbacks that include surgical risks during placement or removal, implant failure and more specifically microbial infections. These implant-associated infections are mainly caused by the bacterial biofilm formation in which bacteria are more recalcitrant towards treatments. Implant surfaces are non-vascularized abiotic ma- terials rendering the common strategies inappropriate and ineffective.[2] In this context we have designed and developed innovative and smart interfaces based on phosphonium SAMs[3] that can be electrically activated on-demand for eradicating bacterial infections on solid surfaces. Upon electroactivation, using a low potential of 0.2V for 1 hour, a successful stamping out of Gram-positive and Gram-negative bacteria strains has been clearly highlighted on SAM-modified titanium surfaces. Using these conditions, Staphylococcus aureus and Klebsiellapneumoniae were killed up to 95% and 90% respectively with full eradication if time is prolongated (Fig. 1). Moreover, no harmful activity has been observed towards eukaryotic cells which clearly demonstrates the biocompatible character of these novel surfaces for further implementation. Figure 1: (a) and (b)

([1]) Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S.; Mendelson, M.; Monnet, D. L.; Pulcini, C.; Kahlmeter, G.; Kluytmans, J.; Carmeli, Y.; Ouellette, M.; Outterson, K.; Patel, J.; Cavaleri, M.; Cox, E. M.; Houchens, C. R.; Grayson, M. L.; Hansen, P.; Singh, N.; Theuretzbacher, U.; Magrini, N. Lancet Infect.Dis. 2018, 18 (3), 318-327. ([2]) Bjarnsholt, T.; Jensen, P. Ø.; Moser, C.; Høiby, N. Editors, Biofilm infections, Springer-Verlag New York, NY, 2011, 314pp. ([3]) a) Brunel, F.; Lautard, C.; Garzino, F.; Giorgio, S.; Raimundo, J. M.; Bolla, J. M.; Camplo, M. Bioorg. Med. Chem. Lett. 2016, 26 (15), 3770–3772; b) Brunel, F.; Lautard, C.; Giorgio, S.; Garzino, F.; Raimundo, J. M.; Bolla, J. M.; Camplo, M. Bioorg. Med. Chem. Lett. 2018, 28 (5), 926-929; c) Brunel, F.; Lautard, C.; Garzino, F.; Raimundo, J. M.; Bolla, J. M.; Camplo, M. Bioorg. Med. Chem. Lett. 2020, 30(18), 127389; d) Raimundo, J.-M.; Camplo, M.; Bolla, J.-M.; Brunel, F.; Lautard, C.; PCT Int. Appl. 2020, WO 2020008000 A1 20200109; e) Carrara, S.; Rouvier, F.; Auditto, S.; Brunel, F.; Janneau, C.; Camplo, M.; Sergent, M.; About, I.; Bolla, J.-M.; Raimundo, J.-M. ACS Appl. Mater. Interfaces, 2020, submitted.

26 International Symposium on SupraBiomolecular Systems 2021

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27 International Symposium on SupraBiomolecular Systems 2021

Formation of a G-quadruplex structure by a sequence found in pre-miRNA-let-7e

Monday, 10th May - 15:50: Flash Session 2 - Poster - Abstract ID: 35

Mr. Tiago Santos 1, Mr. André Miranda 2, Mr. Lionel Imbert 3, Dr. Gilmar Salgado 4, Prof. Eurico Cabrita 5, Dr. Carla Cruz 1 1. CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal, 2. CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique,6200-506 Covilhã, Portugal, 3. Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), F-38044, Grenoble, France, 4. Univ. Bordeaux, ARNA Laboratory, INSERM, U1212, CNRS UMR 5320, IECB, F-33600, Pessac, France, 5. UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

Introduction: RNA G-quadruplexes (rG4) have been shown to play an important role in some biological pro- cesses, including the regulation of microRNA (miRNA) biogenesis and function. Also, it was recently shown that rG4 oligonucleotides found in pre-miRNA-149 and pre-miRNA-92b bind to ligands and proteins. The protein nucleolin (NCL) was shown to bind rG4s and to modulate their folding and/or stability. Among several pre- miRNA with potential ability to form G4s, pre-miRNA-let-7e have shown to be deregulated in several cancer types. Therefore, it is important to unveil and characterize its capability to form a G4 structure, and determine the role that some ligands may have in its folding and binding capacity to NCL. Methods:Herein, the putative rG4 sequence (5’-GGGCUGAGGUAGGAGG-3’) found in pre-miRNA-let-7e was used to check its ability to form an rG4 structure and bind ligands and proteins. In order to evaluate the formation and stabilization of the putative rG4 found in pre-miRNA-let-7e G-quadruplex sequence, we employed circu- lar dichroism (CD), nuclear magnetic resonance (NMR) and UV spectroscopy. The kinetic parameters were determined by using time-dependent CD and isothermal difference spectra (IDS). Thereafter, in order to in- vestigate the ability of ligands to stabilize or destabilize the rG4 structure, we employed CD- and FRET- melt- ing experiments and have used well-known G4 ligands. The formation of the supramolecular complexes pre- miRNA/ligand, pre-miRNA/NCL and pre-miRNA/ligand/NCL complexes were checked by polyacrylamide gel elec- trophoresis (PAGE). Results/Discussion:Our results suggested the formation of an rG4 structure by a sequence found in pre-miRNA- let-7e. The NMR spectra of KCl titration experiments highlighted a set of eight resonances in the imino region, which arise upon addition of increasing amounts of KCl. Additionally, CD and time-dependent experiments proved that the formation of rG4 is K+-dependent. The stability of the rG4 sequence was achieved by adding G4 ligands as demonstrated by CD- and FRET-melting experiments. Overall, the results suggest that the rG4 sequence of pre-miRNA-let-7e can form a stable rG4 in the presence of K+, G4 ligands and NCL. Acknowledgements: Tiago Santos acknowledges FCT for the doctoral fellowship PD/BD/142851/2018, inte- grated in the PTNMR PhD Programme (PD/00065/2013). André Miranda acknowledges the fellowship grant from PTNMR project (PINFRA/22161/2016-B4). This work was supported by PESSOA programme ref. 5079, project ref. IF/00959/2015 and PTNMR Network (ROTEIRO/0031/2013-PINFRA/22161/2016). The authors acknowledge Jérôme Boisbouvier. This work benefited from access to the IBS - Institut de Biologie Structurale - Grenoble / France, an Instruct-ERIC centre. Financial support was provided by Instruct-ERIC (PID: 10168).

28 International Symposium on SupraBiomolecular Systems 2021

Characterization of G-quadruplex structure in pre-MIR150: potential target for anti-cancer approaches in non-small cell lung cancer

Monday, 10th May - 15:50: Flash Session 2 - Poster - Abstract ID: 50

Ms. Joana Figueiredo 1, Dr. Jean-Louis Mergny 2, Dr. Carla Cruz 1 1. CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Tel: +351 275 329 076; Fax: +351 275 329 099., 2. Laboratoire d’Optique et Biosciences, Institut Polytechnique de Paris, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France. Institute of Biophysics of the Czech Academy of Sciences, Brno 612 65, Czech Republic.

RNA molecules can fold into noncanonical structures such as the four-stranded structures known as G- quadruplexes (G4). RNA G4s are present under physiologically conditions and are involved in the regulation of several key cellular process, including the regulation of miRNA biogenesis and function. Dysregulation of miR- NAs is associated with Non-Small Cell Lung Cancer (NSCLC) development. The human MIR-150 is significantly upregulated in NSCLC compared to normal cells and have been associated to NSCLC progression, acting as an oncogene. The formation of these G4 structures in stem-loop region of pre-miRNAs interferes with the enzyme Dicer activity decreasing mature miRNA production inside the cell. In this study we used several biophysics techniques to show that pre-MIR150 forms a G4 structure that coexist in equilibrium with the canonical hairpin structure, potentially controlling the production of the mature MIR-150. We have used the full-length pre-MIR150 and one short sequence comprise the G-rich motif responsible to G4 formation (rG4). Nuclear magnetic resonance (NMR) spectra showed that G4 formation in rG4 sequence depends on the relative amounts of mono- and divalent metal ions, such K+ and Mg2+present in solution and it is influenced by increase of temperature, folding into G4 conformation at 57 °C. In addition, we showed that increasing concentrations of PhenDC3, a well-known G4 ligand, favors the G4 conformer. Circular dichroism (CD) spectra indicated the formation of a parallel G4 structure. The thermal stability of rG4 is very high (Tm= 69.1 °C) and was further increased in the presence of PhenDC3 at 10 molar equivalents (Tm= 87.5 °C). The binding affinities between rG4 and PhenDC3 were determined by fluorescence titrations and have KD value in the micromolar range. The

fluorescent intercalator displacement (FID) assay demonstrated that PhenDC3 furnishes low DC50value (0.94

µM). PhenDC3 also showing a cytotoxicity effect (IC50 = 0.83 µM) on KRAS-mutant NSCLC cell line (A549). Thus, we identified a G4 structure within pre-MIR150 that could be a therapeutic target in NSCLC. Acknowledgment: J. Figueiredo acknowledges a doctoral fellowship grant from the FCT – Foundation for Sci- ence and Technology ref. SFRH/BD/145106/2019.

29 International Symposium on SupraBiomolecular Systems 2021

Data mining approach to unveil property-activity correlation and sequence optimization of viral transduction enhancing peptides

Monday, 10th May - 15:50: Flash Session 2 - Poster - Abstract ID: 4

Mrs. Kübra Kaygisiz 1, Dr. Arghya Dutta 1, Ms. Lena Rauch 2, Dr. Tristan Bereau 3, Dr. Christopher Synatschke 1, Prof. Jan Münch 2, Prof. Tanja Weil 1 1. Max Planck Institute for Polymer Research, Mainz, 2. Institute of Molecular Virology, Ulm University Medical Center, 3. Van ’t Hoff Institute for Molecular Sciences and Informatics Institute, University of Amsterdam

Introduction: Amyloid-like nanofibers from self-assembled peptides provide a versatile platform for biomateri- als applications.1 We previously reported on the 12-residue peptide EF-C that forms amyloid nanofibers and ef- ficiently enhances retroviral gene transfer.2 Peptides derived from this sequence were highly potent enhancers of viral transduction3 and furthermore useful scaffolds for cellular growth.4 Methods: Here, we present our work on a data mining approach with a library consisting of more than 160 peptides by systematically varying the amino acid sequence and length of EF-C derivatives. To optimize the sequence of transduction enhancing peptides, the amino acids were coarse grained in terms of hydrophilicity and charge. Using a data mining algorithm, these coarse-grained sequences were correlated with transduction efficiencies (low, medium, high). Furthermore, the peptide library was characterized by various physicochemical methods and a property-activity correlation was established via regression analysis. Results: The most promising peptide patterns from the coarse-grained pattern analysis were synthetically re- translated into novel peptides and their improved activity was experimentally verified. The property-activity correlation unveiled amyloid character and positive surface charge of the self-assembled peptide fibrils as key material properties for efficient viral transduction. Discussion: Therapeutic applications of peptides as enhancers for viral gene transfer are still limited by mod- erate transduction efficiencies. Data mining was applied as a powerful tool to study and optimize peptide se- quences from a relatively small initial data set. Important parameters leading to high activity were found by a systematical property-activity correlation study. We envision that the identified key material properties can be generalized for self-assembling peptides from different origins to identify highly efficient transduction en- hancers. References: 1 K. Kaygisiz, C. V. Synatschke, Biomater. Sci. 2020,8, 6113-6156. 2 M. Yolamanova, C. Meier, T. P. J. Knowles, T. Weil, F. Kirchhoff, J. Münch et al., Nat. Nanotechnol. 2013, 8, 130–136. 3 S. Sieste, T. Mack, E. Lump, M. Hayn, D. Schütz, C. Meier, K. Kaygisiz, F. Kirchhoff, F. S. Ruggeri, C. V. Synatschke, J. Münch, T. Weil, submitted. 4 C. Schilling, T. Mack, S. Lickfett, S. Sieste, F. S. Ruggeri, A. Dutta, T. Bereau, T. P. J. Knowles, C. V. Synatschke, T. Weil, B. Knöll et al., Adv. Funct. Mater. 2019, 29, 1–15.

Data mining approach to unveil property activity correlation and sequence optimization of viral transduction enhancing peptides kaygisiz2021.jpg

30 International Symposium on SupraBiomolecular Systems 2021

Could a dual elongation of the AS1411 aptamer sequence induce a lock on structural polymorphism and maintenance of properties?

Monday, 10th May - 15:50: Flash Session 2 - Poster - Abstract ID: 58

Mr. André Miranda 1, Mr. Tiago Santos 1, Dr. Eric Largy 2, Dr. Carla Cruz 1 1. CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal, 2. Laboratoire Acides Nucléiques: Régulations Naturelle et Artificielle, Université de Bordeaux, INSERM & CNRS, (ARNA, U1212, UMR5320), IECB, 2 rue Robert Escarpit, 33607 Pessac, France

Aptamers are a class of oligonucleotides (RNA or DNA) that can recognize and bind their targets (proteins, cells, bacteria, and viruses) with affinities range from KD of pM to µM. The aptamers formed from G-rich sequences, besides recognizing the target, are capable of folding into G-quadruplexes (G4) structures and take out advan- tages (chemical and thermodynamic stability, nucleases resistance, low immunogenicity, and enhanced cellular uptake). The AS1411 has been reported as an aptamer of nucleolin (NCL), which is a protein overexpressed at the surface of several cancer cells, and studied for cancer therapy and diagnosis. Despite the broad applicability of AS1411, it suffers from a large conformational polymorphism that limits the characterization of its structural features, namely the resolution of 3D structures. Based on these considerations, we hypothesized that it would be possible to decrease the conformational polymorphism by retaining the original sequence but adding short 5′ and 3′ flanking sequences capable of hybridizing with one another. Thus, we have designed AS1411-N6 and evaluated it using several biophysical techniques (Circular dichroism, UV, Nuclear Magnetic Resonance spectroscopies) in terms of the effects of KCl on the structure and stability, and to check whether it can adopt one or more conformations. We found that AS1411-N6 is less polymorphic than AS1411, and its folding into a G4 in presence of K+ promotes the hybridization of the flanking sequences into a duplex. Also, we studied the binding properties of ligands TMPyP4, PhenDC3, PDS, 360A, and BRACO-19 in terms of stability, binding, topology maintenance of AS1411-N6, and NCL recognition. To achieve this, and additionally to previously enounced biophysical methods, FRET-melting assay, Surface Plasmon Resonance, and fluorimet- ric experiments were performed. The melting experiments revealed promising stabilizer effects of PhenDC3, 360A, and TMPyP4, and the affinity calculations showed that the stabilization of AS1411-N6 with 360A led to a remarkable enhancement of the target affinity towards NCL. The affinity determined between AS1411-N6 and NCL denoting a strong interaction and complex formation was assessed by PAGE in which the electrophoretic profile of AS1411-N6 showed bands of the dimeric form in the presence of the ligands and NCL. Overall, our results and strategy open up a framework for the design and development of new engineered G4-forming ap- tamer beacons, by locking the structure with 5′ and 3′ end complementary strands for the NCL targeting.

31 International Symposium on SupraBiomolecular Systems 2021

Nucleolipids self-assemblies enhance metal ions binding

Monday, 10th May - 15:50: Flash Session 2 - Poster - Abstract ID: 76

Dr. Bruno Alies 1, Prof. Philippe Barthélémy 2 1. University of Bordeaux, 2. Equipe ChemBioPharm, INSERM U1212, UMR 5320 CNRS-Université de Bordeaux

Nucleolipids are biomimetic synthetic molecules. Like their natural analogues (Acetyl-CoA, Tunicamycin…), these hybrid compounds consist of a hydrophilic part (nucleoside and/or nucleotide) and a hydrophobic part (aliphatic chain). Due their amphiphilic properties, they can self-assemble into various structures (liposomes, micelles, fibers…). Thanks to their low toxicity, they have been used in many fields, ranging from cell culture to vectorization. In order to reach further applications, we explored the interactions between nucleolipids and metal ions. Using a specific nucleolipids, we recently demonstrated their ability to coordinate and detect silver ions in water even at very low concentrations (3 ppb). In this work, we show that nucleolipids supramolecu- lar assemblies can be used to detect other metal ions using fluorescence. In order to gain insight into metal coordination to these structures, we focused on how supramolecular assemblies of nucleolipids impact metal ions coordination with special emphasis for toxic metal ions. Using UV-visible spectroscopy, we determined the stoichiometry and affinity of nucleolipids supramolecular assemblies for metal ions. We demonstrated how re- duction of entropy, due spontaneous self-organization of nucleolipids, enhance metal ions affinities compared to nucleosides. We also explore the ability of nucleolipids-based hydro- and oleogel to decontaminate water soiled with mercury ions.

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32 International Symposium on SupraBiomolecular Systems 2021

Mirror-image β-sheets organize supramolecular water assemblies with opposite chirality

Monday, 10th May - 15:50: Flash Session 2 - Poster - Abstract ID: 67

Mr. Ethan Perets 1, Mr. Daniel Konstantinovsky 1, Prof. Sharon Hammes-Schiffer 1, Prof. Elsa Yan 1 1. Yale University

Introduction Hydration modulates the structures and functions of biomacromolecules. However, probing water superstruc- tures in hydration shells remains difficult, due to the large background signal from bulk water and the dy- namic nature of the solvation layer. To overcome these challenges, we combined chiral vibrational sum fre- quency generation (SFG) spectroscopy with computational modeling to study supramolecular assemblies of water molecules surrounding an antiparallel β-sheet protein (Figure 1). Methods We employ chiral SFG spectroscopy to reveal the presence of a chiral supramolecular structure of water molecules around an antiparallel β-sheet protein. Molecular dynamics data is employed to model the spec- troscopic response of the chiral water superstructures around the protein. A direct comparison between the experimental and predicted chiral SFG spectra of supramolecular water assemblies around the protein is un- dertaken (Figure 2). Results We combine chiral SFG spectroscopy with computational modeling to reveal the intricate structure of the sol- vation shell surrounding an antiparallel β-sheet protein. We demonstrate experimentally that the supramolecular water assemblies have a chiral architecture, and that this chirality is induced by the chirality of the protein. Enantiomeric (L-) or (D-) proteins give rise to enan- tiomeric water superstructures (Figure 3). Molecular dynamics simulations reveal the supramolecular architecture of a chiral water superstructure around the protein. Computational modeling shows that the spectroscopic response of the chiral water as- semblies can be understood in terms of water molecules that form stronger or weaker hydrogen bonds with the protein. Computational results suggest that β-sheet protein secondary structures can induce the formation of chiral water superstructures, but that α-helix structures cannot due to the limited contact between water and the peptide backbone. Vibrational couplings of water molecules are demonstrated to be necessary for generat- ing the chiral SFG response. Discussion Our work establishes the existence of a chiral supramolecular assembly of water molecules around a protein secondary structure. We demonstrate how chiral SFG spectroscopy can be used as a structural biology technique to examine the solvation shells of biomacromolecules. We speculate that the chiral water superstructures around biomacromolecules could be involved in biomolec- ular recognition and may have played a role in the origins of biological homochirality. Future synergistic collaborations between theory and experiment should allow for better understanding of the role of the chiral solvation shell in protein folding and denaturation.

33 International Symposium on SupraBiomolecular Systems 2021

Figure2 direct comparison of experimental and Figure1 chiral supramolecular water assembly computational spectra of the chiral water around beta-sheet protein.jpg superstructure around beta-sheet protein.jpg

Figure3 mirror image proteins give rise to water superstructures with opposite chirality.jpg

34 International Symposium on SupraBiomolecular Systems 2021

Protein Nanopores Reveal Single-Molecule Behavior of DNA & RNA

Monday, 10th May - 16:40: Plenary 2 - Oral - Abstract ID: 9

Prof. Cynthia Burrows 1 1. University of Utah

Self-assembled protein nanopores such as α-hemolysin (α-HL) create a transmembrane channel when inserted into lipid bilayers, and the resulting pore has appropriate dimensions for capture of duplex DNA, DNA hairpins, and G-quadruplexes on the cis side of the pore, while only single-stranded DNA or RNA can translocate through the channel to the trans side under an applied electrical potential. We used both WT and site-directed mutants to study the capture of various DNA and RNA secondary structures, with or without base modifications, in a single-molecule analysis. In contrast, Oxford Nanopore Technology has been used to optimize single-molecule sequencing of DNA and RNA strands wherein we have adapted the data analysis to also provide information about the presence of base modifications in, for example, SARS-CoV-2 mRNA.

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35 International Symposium on SupraBiomolecular Systems 2021

CLICK-17, a DNA enzyme that harnesses ultra-low concentrations of either Cu+ or Cu2+ to catalyze the azide-alkyne ‘click’ reaction in water.

Monday, 10th May - 17:25: Plenary 2 - Oral - Abstract ID: 116

Prof. Dipankar Sen 1, Mr. Kun Liu 1, Prof. Hua-Zhong (Hogan) Yu 1 1. Simon Fraser University

To enable the optimal, biocompatible and non-destructive application of the highly useful copper (Cu+)- mediated alkyne-azide ‘click’ cycloaddition in water, we have isolated and characterized a 79- nucleotide DNA enzyme or DNAzyme, ‘CLICK-17’, that harnesses as low as sub-micromolar Cu+; or, surprisingly, Cu2+(without added reductants such as ascorbate) to catalyze conjugation between a variety of alkyne and azide substrates, including small molecules, proteins and nucleic acids. CLICK-17’s Cu+ catalysis is orders of magnitude faster than that of either Cu+ alone or of Cu+ complexed to PERMUT- 17, a sequence-permuted DNA isomer of CLICK- 17. With the less toxic Cu2+, CLICK-17 attains rates comparable to Cu+, under conditions where both Cu2+ alone and Cu2+ complexed with a classic accelerating ligand, THPTA, are wholly inactive. Cyclic voltammetry shows that CLICK-17, unlike PERMUT-17, powerfully perturbs the Cu(II)/Cu(I) redox potential. CLICK- 17 thus pro- vides a unique, DNA-derived ligand environment for catalytic copper within its active site. To date, CLICK-17 has been successfully used for the efficient generation of self-assembled DNA monolayers, and in the accurate quantitation of copper in industrial sources. Furthermore, as a bona fide Cu2+-driven enzyme, with potential for being evolved to accept only designated substrates, CLICK-17 and future variants promise the fast, safe, and substrate-specific catalysis of ‘click’ bioconjugations, potentially on the surfaces of living cells.

36 International Symposium on SupraBiomolecular Systems 2021

Self-assembled nucleic acid nanostructures for drug targeting, bioimaging and biosensing

Tuesday, 11th May - 09:00: Plenary 1 - Oral - Abstract ID: 108

Prof. Jorgen Kjems 1 1. iNANO, Aarhus university

TBD

37 International Symposium on SupraBiomolecular Systems 2021

TBD

Tuesday, 11th May - 09:45: Plenary 1 - Oral - Abstract ID: 109

Prof. Stefan Matile 1 1. University of Geneva

TBD

38 International Symposium on SupraBiomolecular Systems 2021

Reducing Gram negative enterobacteria antibiotic resistance by conjugating synthetic oligonucleotides to a nucleolipid

Tuesday, 11th May - 10:50: Oral Session 1 - Oral - Abstract ID: 31

Dr. Tina Kauss 1, Prof. Corinne Arpin 2, Dr. Clémentine Aubry 1, Ms. Patricia Korczak 1, Ms. Brune Vialet 1, Prof. Philippe Barthélémy 1 1. ARNA Laboratory, ChemBioPharm team, INSERM U1212, CNRS UMR 5320, University of Bordeaux, F-33076 Bordeaux, France, 2. University of Bordeaux, CNRS UMR5234

Antibiotic resistance has become a major issue in public health. According to World Health Organization classi- fication, the resistance is particularly critical for one of the most used antibiotic sub-family; the third-generation cephalosporins. One of the main resistance mechanisms in Enterobacteriaceae, is the production of Extended- Spectrum β-lactamases (ESBL, and more specifically Escherichia coli ESBL), responsible for antibiotic degra- dation and appearance of resistance. Alternatively to existing strategies of antibiotic association or research for new antibiotics, our strategy consists in using chemically modified oligonucleotides to reduce β-lactamase expression. The antibiotic co-administered becomes efficient again. Our results demonstrated that the oligonucleotide therapy is an efficient approach to reduce the resistance of bacteria to antibiotic treatment. Lipid oligonucleotides (LONs) were proved to be an efficient strategy in both, (i) intracellular delivering the oligonucleotide sequences in the prokaryotic cells and (ii) decreasing the Mini- mum Inhibitory Concentrations (MIC) of resistant bacteria to a third-generation cephalosporin, the ceftriaxone. These findings demonstrated the strong antimicrobial potential of this LON strategy targeting the ß-lactamase activity on both clinical and laboratory strains. Chemical modifications were explored I view of their impact on resistance decrease. Our results support the concept that the self-delivery of oligonucleotide sequences via lipid conjugation may be extended to other antimicrobial drugs, which opens novel ways to fight against the antibiotic resistance.

39 International Symposium on SupraBiomolecular Systems 2021

Cell-selective siRNA delivery using glycosylated dynamic covalent polymers self-assembled in situ by RNA-templating

Tuesday, 11th May - 11:05: Oral Session 1 - Oral - Abstract ID: 101

Ms. Maëva Coste 1, Mr. Sebastien Ulrich 1 1. Université de Montpellier

Oligonucleotides like siRNA have a high potential as therapeutic agents which is illustrated by the recent approvals of 3 drugs: Onpattro™ (2018), Givlaari™ (2019) and Oxlumo™ (2020). However, vectors are required to promote their transfection in cells and, unfortunately, cheap and versatile synthetic vectors have not yet delivered their promises.[1] An effective gene delivery vector should be able to complex effectively nucleic acids in biological fluids, pass biological barriers, and actively release nucleic acids inside the targeted cells. Thus, it is clear that smart dynamic vectors have to be developed in order to achieve such active delivery process which could rival viral vectors.[2] Our group has developed a strong interest in dynamic and adaptive vectors,[3] and in this context, we have been exploring the potential of dynamic covalent polymers.[4] More specifically, we recently studied a dynamic covalent library made of complementary peptide derivatives which are both water-soluble and cationic. Interestingly, we designed one of them to bear a clickable residue onto which targeting ligands can be grafted. While DCPs are traditionally formed only at high concentration – macrocycles of low valency unfit for nucleic acid recognition being preferentially formed at low concentrations – we found here that the presence of siRNA allows the in situ formation of DCPs in aqueous media at unusually low concentrations. More interestingly, using a glycosylated building block, we showed that siRNA-templated DCPs are capable of cell-selective siRNA delivery mediated through receptor-assisted endocytosis. This work shows the first example of a self-adapted siRNA vector which further promotes cellular uptake and cell-selective delivery through multivalent ligand display.[5] Overall, these results represent a stepping stone towards the self-fabrication of targeted nucleic acid vectors.

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40 International Symposium on SupraBiomolecular Systems 2021

Emergent Assembly through DNA and Chemoenzymatically Synthesized Polypeptides

Tuesday, 11th May - 11:20: Oral Session 1 - Oral - Abstract ID: 59

Dr. Prashant Gudeangadi 1, Prof. Michael Reithofer 2, Dr. Christopher Serpell 1 1. University of Kent, 2. University of Vienna

Peptide-oligonucleotide conjugates are currently explored for applications in the applied biomedical field,1 but their nanoscale assembly has not yet been investigated to a significant extent. Our work focuses on introduc- ing new multiscale diversity to DNA and peptide nanotechnology through orthogonal hierarchical and emer- gent assembly. Previously, we produced nucleobase-peptide-amphiphile hybrids which showed hierarchical assembly to give DNA-sensitive hydrogels.2 We are now studyingin situ polymerisation of peptide chain on DNA strand. Although conventional solid phase peptide synthesis can be used, the tedious multistep reactions with several protection and de-protection steps restrict their large-scale production.3 Ring-opening polymerisation of amino acid N-carboxyanhydrides (NCAs) can also be widely employed for the synthesis of polypeptides with precise control of molecular weight. This method is beneficial but has a poor sequence control.4 On the other hand, chemoenzymatic reactions have emerged as an advantageous method over other solution-based synthe- ses because of their environmental benignity. These reactions are also an atom-economical and facile approach that uses mild aqueous media and is scalable to industrial production.5 Recently, elastin like polypeptides were synthesised by this method, which showed temperature dependent structural transition very similar to native elastin.6 In this work, for the first time, peptide chain elongation was achieved from a DNA strand using enzymes. The initial N-terminal of phenylalanine unit was linked to the DNA-fragment through a succinic acid linker. En- zymatic polymerisation was then carried out using papain under mild aqueous conditions. L-glutamic acid diethyl ester was used for polymerisation reaction. The polymerisation reaction yielded mixture of peptides and DNA-peptide conjugates containing up to 8-mer peptide units. The formation of DNA-peptide conjugates was confirmed by mass spectrum, and the resulting emergent assembly studied by AFM, DLS, and SEM. References:

1. 1. N. Venkatesan and B. H. Kim, Chem. Rev., 2006, 106, 3712. 2. C. J. Serpell, M. Barłóg, K. Basu, J. F. Fakhoury, H. S. Bazzi and H. F. Sleiman, Mater. Horiz., 2013, 1, 348. 3. R. B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149–2154.

4. F. Guzman, S. Barberis and A. Illanes, Electron. J. Biotechnol., 2007, 10, 279. 5. K. Tsuchiya and K. Numata, Macromol. Biosci., 2017, 1700177. 6. P. G. Gudeangadi, K. Tsuchiya and K. Numata, Polym. Chem.,2018, 9,2336.

41 International Symposium on SupraBiomolecular Systems 2021

Self-assembly of tetraphenylethylene-DNA conjugates into DNA-addressable vesicular architectures

Tuesday, 11th May - 11:35: Oral Session 1 - Oral - Abstract ID: 23

Mr. Simon Rothenbuehler 1, Dr. Ioan Iacovache 2, Dr. Simon M. Langenegger 1, Prof. Benoît Zuber 2, Prof. Robert Haener 1 1. Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland, 2. Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3012 Bern, Switzerland

Aggregation-induced emission (AIE) type chromophores are non-emissive in the molecularly dissolved state but become highly fluorescent in their aggregated state. In this emerging field, implementations of the AIE phe- nomenon include biomolecular sensing or optoelectronic devices, to name a few. DNA conjugates describe the fusion of natural DNA single strands with artificial nucleotide surrogates and thus, expand the scope of DNA. Supramolecular assembly of such DNA conjugates lead to functional constructs, with applications in biomate- rials science and DNA nanotechnology. In this contribution, we present our recent findings of a combination of the two previously described concepts. DNA conjugates, terminally functionalized with AIE-active tetraphenylethylene (TPE) units, have been prepared. As visualized by cryo-electron microscopy (cryo-EM), these TPE-DNA conjugates self-assemble into two different vesicular constructs, varying in the alignment of the DNA hybrids (Figure). The divergent accessibility of the two DNA architectures is demonstrated by ethidium bromide intercalation experiments and the incorporation of a Cy3-labelled DNA via strand exchange, leading to DNA-addressable, light-harvesting constructs. This dynamic behavior of these DNA-constructed vesicles renders them versatile intermediates for nanotechnology applications, such as DNA-triggered delivery devices.

Simon Rothenbuehler, Ioan Iacovache, Simon M. Langenegger, Benoît Zuber, Robert Haener, Nanoscale, 2020, 12, 21118–21123. Caroline D. Boesch, Jovana Jevric, Nutcha Buerki, Markus Probst, Simon M. Langenegger, Robert Haener, Bio- conjugate Chem., 2018, 29, 1505–1509. Ju Mei, Nelson L. C. Leung, Ryan T. K. Kwok, Jacky W. Y. Lam, Ben Zhong Tang, Chem. Rev., 2015, 115, 11718– 11940.

Figure self-assembly of tetraphenylethylene-dna conjugates into dna-addressable vesicular architectures.png

42 International Symposium on SupraBiomolecular Systems 2021

Capturing the dynamic association of lanthanide complexes with biomolecules : a combined NMR and molecular simulations exploration.

Tuesday, 11th May - 11:50: Oral Session 1 - Oral - Abstract ID: 30

Dr. Alessio Bartocci 1, Dr. Natacha Gillet 1, Prof. Elise Dumont 1 1. Ecole Normale Supérieure de Lyon

Interaction of lanthanide ions or complexes with biomolecules (proteins, peptides, but also lipid and DNA) is versatile with timely applications such as (chiral-) sensing, paramagnetic tags, and even assisted nucleation. Yet, only ~100 structures of proteins in presence of lanthanide ions or complexes are currently reported in the Protein Data Bank. In order to predict the binding sites of two lanthanide complexes (tris-dipicolinate and a crystallophore shown below) with peptides and proteins, we have developed a simulation protocol based on classical then QM/MM molecular dynamics simulations and a machine-learning based post-processing that captures the versatile as- sociation of these lanthanide complexes either with peptides or proteins. First the tris-dipicolinate lanthanide complexe interaction is mostly electrostatically-driven, yet is competitive for a decapeptide [1] and ubiquitine, and I will present the synergistic view gained by our simulations and paramagnetic NMR measurements. The so-called crystallophore complex (Ln-Xo4) offers a much more versatile range of interactions, which can be duly quantified along our simulations. Our approach here affords a direct cross-talk with crystallization assays for proteins, where we can situate the interaction between Xo4 agents and surface residues occurring at the very beginning of the nucleation process. The same computational protocol can be invoked to investigate binding of sulfonated calixarenes as molecular glues at protein-protein interfaces [2,3]. [1] S. Denis-Quanquin, A. Bartocci, F. Szczepaniak, F. Riobé, O. Maury, E. Dumont, N. Giraud, chem- rxiv https://doi.org/10.26434/chemrxiv.13297694.v1 [2] S. Engilberge, M. L. Rennie, E. Dumont, P. B. Crowley, ACS Nano, 2019, 13:10343 [3] A. Bartocci, N. Gillet, T. Jiang, F. Szczepaniak, E. Dumont, J. Phys. Chem. B, 2020,

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43 International Symposium on SupraBiomolecular Systems 2021

Single particle combinatorial multiplexed liposome fusion mediated by DNA

Tuesday, 11th May - 12:05: Oral Session 1 - Oral - Abstract ID: 100

Ms. Mette Malle 1, Dr. Philipp Löffler 2, Mr. Søren S.-R. Bohr 1, Mr. Magnus Berg Sletfjerding 1, Mr. Nikolaj Alexander Risgaard 2, Dr. Simon Bo Jensen 1, Dr. Min Zhang 1, Prof. Per Hedegård 1, Prof. Stefan Vogel 2, Prof. Nikos S. Hatzakis 1 1. University of Copenhagen, 2. University of Southern Denmark

The supramolecular organization of nano compartments is key for the construction of bottom-up synthetic biosystems. Such systems could serve to miniaturize high-throughput (HTP) methods transforming screen- ing and discovery processes in the field of synthetic biochemistry and biomedical sciences. Conventional HTP methods operate at large material and time scales, which may be alleviated by operating at a much smaller scale, and radically higher parallelization. We thus present Single PARticle Combinatorial multiplexed Lipo- some fusion mediated by DNA (SPARCLD)1, offering a versatile platform for a parallelized, non-deterministic, multi-step library generation within arrays of zeptoliter nanocontainers (Fig 1a). These target nanocontainers were engrafted with up to 6 different lipidated nucleic acid2 (LiNAs, Fig. 1b) strands and immobilized within a microscopy channel slide at high density (100-300 per field-of-view, 4.2×104 containers per mm2) and allowed to fuse with up to 6 different populations of cargo nanocontainers carrying one of the complementary LiNA strands. For real-time TIRF imaging, each cargo population was barcoded by a distinct ratio of membrane fluo- rophores, allowing their identification based on the three-channel intensity data using a supervised machine- learning algorithm (Fig. 1c). When target and cargo populations were engrafted with complementary LiNAs, efficient (>93%), and leakage free fusion was observed, while almost no non-specific fusion was seen in con- trols with non-complementary LiNAs. When six cargo populations were brought into contact with arrays of target liposomes, a stochastic combinatorial fusion was observed. Algorithm based barcode classification, sig- nal convolution, and event detection revealed the distinct permutations of fusion sequences for each target nanocontainer. This way, the direct observations of >16000 fusion events were accurately classified, and 566 distinct fusion sequences were identified (Fig. 1d). We observed up to 7 successive docking events per target, suggesting that the method is capable of HTS with more than a quarter million combinatorial outcomes. SPARCLD utilizes the stochasticity in the order of nanocontainer fusion events as well as heterogeneous particle sizes, turning the otherwise limiting and inher- ent problem in conventional assays into an experimental advantage3,4. The method is both efficient and leakage free. With up to 10 barcodes available and classified and 11 LiNA strands, the method is highly adjustable and expandable for a specific assay needed. We envision that SPARCLD will be applied for multiplexed and combinatorial chemistry in nanoreactors for the discovery or screening of carbohydrates or nucleic acids, using minute material and time.

44 International Symposium on SupraBiomolecular Systems 2021

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45 International Symposium on SupraBiomolecular Systems 2021

Bacteriophage T7 single-stranded DNA-binding protein displays template-catalyzed recycling

Tuesday, 11th May - 14:00: Flash Session 1 - Poster - Abstract ID: 11

Dr. Jordi Cabanas-Danes 1, Mr. Matthew Halma 1, Mr. Longfu Xu 1, Prof. Gijs Wuite 1, Dr. Iddo Heller 1, Dr. A.M. van Oijen 2, Prof. Erwin Peterman 1 1. Vrije Universiteit Amsterdam, 2. University of Groningen

Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) is an essential component of the phage replica- tion machinery. During DNA replication, gp2.5 binds and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. Here, we use opti- cal tweezers combined with confocal fluorescence microscopy to investigate the real-time binding dynamics of gp2.5 to ssDNA. Our data show that bound gp2.5 reduces the contour length of ssDNA, in well agreement with structural data proposing a DNA binding cleft capable of sandwiching nucleotide triplets between aromatic residues. Also, we demonstrate that increasing the tension on the DNA template reduces gp2.5 binding signifi- cantly, thereby supporting a binding model that requires mechanical bending of the DNA, as nucleotide triplet stacking would imply. Next, we report two major trends that hint a binding model relying, at least partially, on the formation of hydrogen bonds between the protein and the ssDNA template. On the one hand, the protein exhibits a more stable binding on GC-rich than on AT-rich DNA sequences, in agreement with the intrinsic sta- bility of DNA base pairs. On the other hand, we observed that if gp2.5 binds to ssDNA in absence of secondary structures, which can arise from base pairing, their formation is prevented in comparison to bare ssDNA. Lastly, we found evidence that re-association after unbinding is enhanced with regards to direct protein binding from solution, which would lead to an efficient spatial redistribution of gp2.5 during synthesis of successive Okazaki fragments. Together, our findings provide a more defined molecular picture of the role of gp2.5 during DNA replication.

46 International Symposium on SupraBiomolecular Systems 2021

Cyclic poly(N-alkylated-glycine) as promising antimicrobial agents in infectology.

Tuesday, 11th May - 14:00: Flash Session 1 - Poster - Abstract ID: 89

Mr. Pedro Salas-Ambrosio 1, Mr. Antoine Tronnet 2, Dr. Marc Since 3, Dr. Sandra Bourgeade-Delmas 4, Dr. Jean-Luc Stigliani 2, Dr. Amelie Vax 1, Prof. Sebastien Lecommandoux 1, Prof. Bruno Dupuy 5, Prof. Pierre Verhaghe 2, Dr. Colin Bonduelle 1 1. Laboratoire de Chimie des Polymères Organiques (LCPO), 2. LCC-CNRS, 3. Normandie Univ, UNICAEN, 4. UMR 152 PHARMA-DEV, 5. Institut Pasteur

Protein-like polymers combine the structural advantages of proteins with improved stability and processabil- ity, which is beneficial for many applications.1,2 In this field, polypeptoids, or poly N-substituted-glycines, are a promising class of polymers that offer unique properties in biology.3 In this context, the development of antibac- terial structures is of major concern and the design of polypeptoid backbones is an approach that is scarcely explored in infectology (figure 1). Figure 1. Synthetic strategy for preparing antimicrobial polypeptoids by copolymerizing N-alkylated-N- carboxyanhydride (NNCA) monomers. In this work, we developed antibacterial polypeptoids by ring expansion polymerization (REP), a process me- diated by lithium bis(trimethylsilyl)amide (LiHMDS = B).4 Indeed, this original approach was first developed to access to cyclic polysarcosine, a topology that was well characterized by size-exclusion chromatography (SEC: lower elution time as compared to linear backbones, figure 2A) and by MALDI-ToF (set of ions corresponding to cyclic polysarcosine and bearing a Münchnone exocyclic moiety, figure 2B). Using this same methodology, we designed a library of copolymers 1-11 (table 1) using N-benzyl-NNCA (analogue of phenylalanine-NCA) and N-Cbz-aminobutyl-NNCA (analogue of Cbz-lysine-NCA). Figure 2. Macromolecular characterizations of cyclic polysarcosine at M/B=100 (A: SEC in DMF and B: MALDI TOF). All the copolymers were tested against Clostridioides difficile, a Gram-positive bacterium responsible for severe hospital-acquired intestinal infections (table 1). The minimum inhibitory concentrations (MIC) were evaluated in parallel with the in vitro cytotoxicity (CC50) over human intestinal epithelial cells (Caco-2) to calculate the selectivity indices (SI = CC50/MIC). By varying the cationic content of copolymers, we optimized the antibacterial activity and copolymer 6 displayed a good activity, comparable to LL37 (an antimicrobial peptide) or antibiotic controls. Interestingly, this statistical copolymer was significantly less toxic than the copolymer diblock counter- parts (10-11). We also observed that compound 6preserved its activity after trypsin digestion, in marked contrast to LL37. These results showed that cyclic polypeptoids are promising candidates as antibacterial agents. Table 1. In vitro antibacterial activity against C. difficile and cytotoxicity over Caco-2 cell line (r: random copoly- mers and b: block copolymers). References (1) Kricheldorf, H. R. Angew. Chem, Int. Ed. 2006, 45 (35), 5752–5784. (2) Salas-Ambrosio, P. et al. Biomacromolecules 2021, 22 (1), 57–75. (3) Chan, B. A.et al. Biopolymers 2017, 109 (1), 1–25. (4) Salas-Ambrosio, P. et al. J. Am. Soc. Chem. 2021. https://doi.org/10.1021/jacs.0c13231

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48 International Symposium on SupraBiomolecular Systems 2021

Hybrid Peptide-Agarose Hydrogels for High-Sensitivity 3D Immunoassays

Tuesday, 11th May - 14:00: Flash Session 1 - Poster - Abstract ID: 91

Dr. Alessandro Gori 1, Dr. Greta Bergamaschi 1, Dr. Angelo Musicò 1, Dr. Roberto Frigerio 1, Dr. Alessandro Strada 2, Dr. Marina Cretich 1 1. National Research Council of Italy, 2. National Research Council, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC-CNR), Milan, Italy

High-throughput analytical platforms are prominent tools in the rapid delivery of novel diagnostic assays. In this scenario, a growing niche is represented by 3D droplet microarrays, where hydrogels are used as matri- ces to stably entrap biomolecules onto analytical surfaces under solution-like conditions, providing relevant advantages such as increased loading capacity, smaller nonspecific binding and enhanced signal-to-noise ra- tio. Herein, we report on the use of a hybrid hydrogel for microarray applications obtained through the com- bination of a self-assembling peptide (namely Q3 peptide) with low-temperature gelling agarose. In this blend, agarose acts as the hydrogel core structural component, while Q3 addition is crucial to balance out its rheolog- ical properties and to easily introduce bio-functionalization of the hydrogel. The novel material was used to fabricate microdroplet arrays, and we demonstrate its real-case application in 3D immunoassays. Remarkably, 3D assays yield a 50-fold increase in sensitivity compared to canonical assays in 2D format, as here ultimately exemplified by profiling Covid-19 patients sera immunoreactivity.

49 International Symposium on SupraBiomolecular Systems 2021

SAP-NC hydrogels: a novel hybrid platform for bioanalytic applications

Tuesday, 11th May - 14:00: Flash Session 1 - Poster - Abstract ID: 92

Dr. Greta Bergamaschi 1, Dr. Alessandro Gori 2, Dr. Alessandro Strada 1, Dr. Angelo Musicò 2, Prof. Paolo Bettotti 3 1. National Research Council, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC-CNR), Milan, Italy, 2. National Research Council of Italy, 3. Nanoscience Laboratory, Department of Physics, University of Trento, Trento, Italy

Hybrid hydrogels have emerged as a class of nanomaterials with wide-ranging uses that can combine the ben- eficial properties of responsive supramolecular low-molecular-weight gelators with robust polymer gelators. In self-assembled multi-component hybrid hydrogels, each component maintains its own unique functionality, allowing to obtain appealing biomaterials with high-water content, microporous structure, and tunable me- chanical stability. Here we show our latest results on the development of a novel hybrid hydrogel that combine the use of nanocel- lulose (NC) and self-assembling peptides (SAP), allowing the fine modulation of the mechanical properties of the tridimensional network and enables the introduction of complementary chemical functionalization thanks to the use of ad-hoc designed peptides. Rheological and structural experiments have been performed to unravel the relationship between material properties and their application as cell culture matrix.

50 International Symposium on SupraBiomolecular Systems 2021

Switching Peptide Supramolecular Assemblies by Selective Mono-Substitution

Tuesday, 11th May - 14:00: Flash Session 1 - Poster - Abstract ID: 94

Dr. Alessandro Strada 1, Dr. Greta Bergamaschi 2, Dr. Alessandro Gori 2, Dr. Angelo Musicò 2, Dr. Andrea Pizzi 3 1. National Research Council, Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC-CNR), Milan, Italy, 2. National Research Council of Italy, 3. Politecnico di Milano - Department of Chemistry, Materials, and Chemical Engineering “Giulio Natta”

Supramolecular peptide assemblies are experiencing a growing interest as versatile nanomaterials.1 In partic- ular, peptide-hydrogels are being investigated in several biomedical applications, e.g. regenerative medicine, drug delivery, biosensing, 3D matrices for cell culture and bioassays.2 In the present work, we synthetized a small library of short peptides, capable of forming hydrogel under mi- cromolar concentrations. We investigated their self-assembly behaviors and the resulting hydrogels properties through a set of experimental techniques (e.g. fluorescence spectroscopy, circular dichroism, infrared spec- troscopy, and transmission electron microscopy, contact angle). These peptide derivatives exhibited remark- ably distinct supramolecular self-assembling and macroscopic hydrogel properties, that can be tuned by subtle modifications. These results represent a step forward in the development of new smart materials with tunable functional properties toward bioanalytical applications.

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51 International Symposium on SupraBiomolecular Systems 2021

β-galactosidase instructed self-assembly of supramolecular bolaamphiphiles hydrogelators

Tuesday, 11th May - 14:00: Flash Session 1 - Poster - Abstract ID: 33

Dr. Alexandra Gaubert 1, Dr. Julie Baillet 2, Mr. Julien Verget 1, Dr. Laurent Latxague 1, Prof. Philippe Barthélémy 1 1. Université de Bordeaux, INSERM, U1212, CNRS UMR 5320, ARNA, ARN : Régulations Naturelle et Artificielle, ChemBioPharm, 33076 Bordeaux, France, 2. Université de Bordeaux, INSERM, U1212, CNRS UMR 5320, ARNA, ARN : Régulations Naturelle et Artificielle, ChemBioPharm, 33076 Bordeaux, France _ currently Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305, USA

In the last decades, biomedical research has become a worldwide challenge to address health issues with the development of more effective and selective approaches. In this context, stimuli responsive biomaterials have emerged as powerful platforms thanks to their unique property to sense their surroundings and to change their macroscopic behavior in time and space. Several in vivo pathological states can be tied to an enzymatic path- way deregulation, such as senescent or certain cancer cells known to overexpress β-galactosidase (β-gal) [1],[2]. The β-gal catalysis is therefore a pertinent and selective trigger for the design of new responsive biomaterials, which can act as a therapeutic reservoir or dynamic self assembling system. Supramolecular hydrogels based on bioinspired Low Molecular Weight Gelators (LMWG) have been widely developed and used in biomedical applications thanks to their relevant mechanical properties, their inherent biocompatibility and biodegrad- ability. Among them, original substrates called glyconucleo-bolaamphiphiles (GNBAs) have shown promising applications as LMWGs and can be easily tailored to respond to biological triggers. In this work, β-gal instructed supramolecular GNBAs assemblies have been developed [3]. Two bolaamphiphile progelators containing the sensitive lactose moiety at both end of the structure were synthetized using CuAAC chemistry. The cleavage of the β(1®4) glycosidic bond was followed by 1H NMR spectroscopy and successfully led to the release of gelator structures in solution. The spontaneous formation of various morphologies ranging from nanoparticules to nanofibers were observed by TEM. Interestingly, the application of heat induced the formation of gels displaying entangled bundles of nanofibers and different kinetics depending on the gelator structure and gelation abilities. Rheological experiments demonstrated gel states, yet more fragile toward the applied constraint compared to the corresponding gelator based hydrogel. The intrinsic parameters such as thixotropic properties and gel-sol transition - mostly related to the supramolecular network - remained similar. Controlling in situ the self-assembly of supramolecular networks via the β-gal trigger open new perspectives to control the cellular functions. β-gal instructed hydrogels represent thus a promising new tool in the therapeutic arsenalfor the design of more selective approaches in biomedicine. [1] D. J. Kurz, S. Decary, Y. Hong and J. D. Erusalimsky, J. Cell. Sci., 2000, 113, 3613–3622 [2] T. Nishihara, S. Kuno, H. Nonaka, S. Tabata, N. Saito, S. Fukuda, M. Tomita, S. Sando and T. Soga, Chem. Commun. (Camb.), 2018, 54, 11745–11748 [3] J. Baillet, A. Gaubert, J. Verget, L. Latxague and P. Barthélémy, Soft Matter,2020, 16, 7648-7651

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53 International Symposium on SupraBiomolecular Systems 2021

The enhancement of the efficiency of the intraocular pressure decrease by ACE inhibitor within chitosan-covered calcium phosphate particles

Tuesday, 11th May - 14:30: Flash Session 2 - Poster - Abstract ID: 71

Mrs. Ekaterina Popova 1, Dr. Victoria Tikhomirova 1, Mrs. Olga Beznos 2, Mrs. Natalia Chesnokova 2, Dr. Olga Kost 1 1. Faculty of Chemistry, M. V. Lomonosov Moscow State University, 119234, Moscow, Russia, 2. Helmholtz National Medical Research Center of Eye Diseases

Ophthalmic therapy is preferably carried out by topical medication. Such treatment is most convenient for the patient but almost ineffective for diseases involving the internal structures of the eye due to the extremely low efficiency of drug penetration into intraocular tissues (less than 5 %). Since the drug concentration increase leads to the development of local and systemic side effects, the best way to increase the efficiency of drug de- livery into the eye is to prolong the contact time of the drug with the anterior surface of the eye. This could be achieved by the encapsulation of the drug into nano or microcarriers. However, the inclusion of low molecular weight drugs into carriers remains a challenge. We demonstrate an increase in the efficiency of the intraocular pressure (IOP) decrease when using a low molec- ular weight angiotensin-converting enzyme (ACE) inhibitor enalaprilat in the composition of inorganic biocom- patible, biodegradable, and non-toxic calcium phosphate particles. To increase the affinity of the carriers to the cornea of the eye, which has a negatively charged mucin layer on its surface, we coated our particles with a positively charged polysaccharide, chitosan. For coating, we used two different chitosans, low molecular (av- erage MW 5 kDa) chitosan and glycol chitosan (72 kDa). Particles containing enalaprilat and coated with 5 kDa chitosan were characterized by a hydrodynamic diameter 180 nm and ζ potential +7 mV, while particles coated with glycol chitosan were characterized by a hydrodynamic diameter 260 nm and ζ potential +16 mV. Chitosan- coated carriers demonstrated high stability at 4°С and very high capacity, enalaprilat incorporation into the particles being about 70%. The in vivo experiments on rabbits demonstrated that IOP decrease reduction in normotensive rabbits is more effective when enalaprilat is used in the composition of chitosan-coated parti- cles than in the free form. Enalaprilat included in the carriers was found to remain in the lacrimal fluid twice as long as free enalaprilat. Moreover, IOP decrease was observed for a remarkably long period in the case of enalaprilat within these particles. Thus, the inclusion of drugs into these carriers can be very effective for the treatment of eye diseases. Such particles can supply the drug to the inner regions of the eye, maintaining its concentration at a level sufficient to reduce IOP.

54 International Symposium on SupraBiomolecular Systems 2021

Influence of a low frequency magnetic field on the release of dextran from magnetic polyelectrolyte microcapsules

Tuesday, 11th May - 14:30: Flash Session 2 - Poster - Abstract ID: 99

Mr. Ivan Burmistrov 1, Ms. Daria Trushina 1, Mr. Alexander Mikheev 2, Mr. Maxim Veselov 2, Mrs. Tatiana Borodina 1, Prof. Natalia Klyachko 2, Mrs. Tatiana Bukreeva 1 1. Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences, 119333, Lenin avenue 59, Moscow, Russia, 2. Lomonosov Moscow State University, 119991, Leninskie gory 1, Moscow, Russia

Magnetically controlled targeted drug delivery is one of the promising directions of targeted drug delivery tech- nologies. The permeability of capsules can be controlled using a magnetic field. Application of a low frequency non heating magnetic field for these purposes offers prospects of high penetration ability into tissues, high lo- cality and safety, which makes this method suitable for in vivo use. In this work, we study the influence of a low frequency magnetic field with different frequencies (lower than 300 Hz) on the release from the Poly(allylamine hydrochloride)/Poly(styrene sulfonate) multilayer capsules modified with magnetite nanoparticles. The capsules were obtained by the sequential adsorption of the poly- electrolytes on the surface of calcium carbonate core templates. Magnetic nanoparticles were obtained by co- precipitation method. The capsules and magnetic nanoparticles characterized using scanning electron micro- scope (SEM) Jeol JSM-7401F, transmission electron microscope (TEM) FEI Osiris. We suppose that under the irradiation by magnetic field, some defects in the shell of magnetic microcapsules may temporarily appear. These defects contribute to the release of the cargo from the microcapsules. To deter- mine the increase in release from microcapsules, we used TRITC (tetramethylrhodamine isothiocyanate) labeled dextran with high molecular weight (65-85 kDa). To monitor changes in the release of TRITC-dextran under the influence of the low frequency magnetic field we measured the time dependence of adsorption of a suspension of the polyelectrolyte microcapsules upon irradiation with a magnetic field. The size of the microcapsules was determined using SEM. Using TEM, we determined size of magnetic nanopar- ticles and that the nanoparticles are uniform distributed in the microcapsules shell. From the analysis of the kinetic release curves we found that for the most effective frequency, the release of dextran increased by 4 times compared to other frequencies. The authors of the work are grateful to Artemov V.V. for the study of the samples by scanning electron mi- croscopy and D.N. Khmelenin. for the study of samples by transmission electron microscopy. This work was performed using the equipment of the Shared Research Center FSRC “Crystallography and Photonics” RAS and was supported by the Ministry of Science and Higher Education of the Russian Federation (project RFMEFI62119X0035) and by the grant of the President of the Russian Federation (МК-1109.2021.1.3).

55 International Symposium on SupraBiomolecular Systems 2021

Functionalization of Core-Shell Nanoparticles (ɣ- Fe2O3/silica) for Oriented Enzyme Immobilization

Tuesday, 11th May - 14:30: Flash Session 2 - Poster - Abstract ID: 81

Mr. Hongtao JI 1, Dr. Thierry TRON 2, Dr. Karine HEUZE 1 1. University of Bordeaux - ISM UMR5255, 2. University Aix-Marseille - ISM2 UMR7313

Introduction : In the field of biocatalysis, immobilization of enzymes results in more efficient and cost-effective sustainable processes. We propose a surface functionalization of Magnetic Nanoparticles (MNPs) with linear and dendritic functional coupling agents, leading to a large chemical flexibility and allowing a larger number of potential sites of immobilization. These functional coupling agents introduced three functional terminal groups- NH2, CHO, N3, which are suitable for an oriented and selective grafting toward the two types of targeted enzymes. To achieve both an oriented, more accessible and more selective enzyme immobilization, a strategy was pro- posed to offer the perspective to orient the Laccase(EC 1.10.3.2, from plant, fungi or bacteria) at the surface of nanoparticles, as opposed to functionalization methods that lead to a random orientation of the catalyst at the surface of a given material. In principle, the ratio of efficient catalyst/particle should greatly benefit from a proper and homogenous orientation of the enzymes active sites at the surface of the particles. Methods : In this study, we describe the synthesis of silylated coupling agents and the characterization of func- tional nanoparticles (300nm, γ-Fe2O3@Silica) obtained after covalent grafting of the coupling agents through hydrolysis-condensation reactions. Further, the oriented immobilization of Lac3 (with catalytic site at the op- posite of reactive functional group) and Unik157 variant (with catalytic site close to the reactive functional group) enzymes was performed though selective covalent bonding between reactive functions of the enzymes and the corresponding coupling agents. Catalytic activity (UV detection and Elisa tests) of the new hybrid bio- catalysts was also investigated. Results : Functional MNPs@Silica nanoparticles (MNP@SiO2@NH2, MNP@SiO2@CHO, MNP@SiO2@N3) have been syn- thesized with function loadings values between 20 to 2000 µmol/g of particles. Specific activity results showed some enzyme orientation effects with Lac3 and Unik157 and with ratios (Lac3/Unik157) of 2.7 for MNP@SiO2@CHO@Enz, 1.25 for MNP@SiO2@N3@Enz and 0.14 for MNP@SiO2@2BN3@Enz. Discussion : Immobilizations of enzymes are depending of the covalent reactions involved. We observed various en- zyme loadings and also various specific activities. Generally, Lac3 exhibited higher activity than the unik157 showing the influence of the orientation of the enzyme on the surface of MNPs. Also, in the case of MNP@SiO2@2BN3@Enz this orientation effect is probably hidden by a high enzyme loading and then an inten- sive mass-transfer limitations. This provide an over crowed microenvironment close to the catalytic site, and led to molecular state changes compare to the native ones.

56 International Symposium on SupraBiomolecular Systems 2021

Controlled deposition of cyclic peptides for the synthesis of monodisperse short peptide nanotubes for biomedical applications

Tuesday, 11th May - 14:30: Flash Session 2 - Poster - Abstract ID: 87

Mrs. Kim Gaudin 1 1. University of Strasbourg

Organic nanotubes are promising nanomaterials displaying many potential applications in nanotechnology in the chemical, physical, biological, or medical field.1 Among all the existing organic nanotubes, peptide nan- otubes are self-assembled structures with significant advantages, such as biocompatibility, biodegradability, pH-dependent construction, and easy synthesis in large amounts.2 For the formation of peptide nanotubes, the supramolecular stacking of cyclic peptides is a valuable approach for the size apprehension of nanotubes. Cyclic peptides composed of an even number of alternating d- and l-amino acids adopt a rigid flat and disk-like confor- mation, in which the amide bonds protrude perpendicularly to the plane of the ring. This conformation allows the peptides to stack through β-sheet hydrogen bonding, resulting in the formation of tubular structures. The functional properties are designable, depending on the cyclic peptide sequence. The diameter of the nanotubes is accurately determined by the size of the cyclic peptides and can be tuned down to sub-nanometer scale.3 How- ever, their self-assembly construction makes their length very difficult to control: indeed, peptide nanotubes are generally long (several micrometers) with a high length polydispersity. The uncontrolled length distribu- tion can induce batch-to-batch fluctuation, resulting in potential variability in terms of organ biodistribution, cellular internalization, elimination, and fate. Therefore, there is a strong need of synthesizing monodisperse very short nanotubes for future clinical translation. In this poster presentation, I will explain the aim of my PhD project focusing on the synthesis of short peptide nanotubes with a high length monodispersity for biomedical applications. The strategy relies on the layer-by- layer construction of the nanotubes, alternating positively and negatively charged cyclic peptides, therefore allowing to control the self-assembly process through electrostatic repulsions. I will also present our prelimi- nary results on the synthesis and characterization of positively and negatively charged cyclic peptides. References: (1) Shimizu, T.; Ding, W.; Kameta, N. Soft-Matter Nanotubes: A Platform for Diverse Functions and Applications. Chem. Rev. 2020, 120 (4), 2347–2407. (2) Mandal, D.; Nasrolahi Shirazi, A.; Parang, K. Self-Assembly of Peptides to Nanostructures. Org. Biomol. Chem. 2014, 12 (22), 3544–3561. (3) Chapman, R.; Danial, M.; Koh, M. L.; Jolliffe, K. A.; Perrier, S. Design and Properties of Functional Nanotubes from the Self-Assembly of Cyclic Peptide Templates. Chem. Soc. Rev. 2012, 41 (18), 6023.

57 International Symposium on SupraBiomolecular Systems 2021

A Noncovalent Model of Adaptive Shapeshifting Ligands for Biomolecules

Tuesday, 11th May - 14:30: Flash Session 2 - Poster - Abstract ID: 55

Mr. Robert A. Ives 1, Dr. Paul R. McGonigal 1 1. Durham University

Despite the potentials of shape-adaptive chemistry in sensing applications, very little is known about how even the simplest shapeshifting molecules adapt their configurations in controlled environments. Previously, flux- ional carbon cages such as bullvalene have been successfully1 exploited in developing chemical sensors for biologically active compounds. Functionalisation of bullvalene allows for access to adaptive molecules that have high levels of structural complexity. Yet until recently2, low-yielding and time-consuming syntheses has hampered access to highly substituted bullvalenes. We have shown how shapeshifting barbaralanes adapt their conformation when transitioning from solution to the solid state3, however a fundamental leap is needed to take advantage of more exotic shapeshifting molecules. Towards this end, we are investigating a bullvalene func- tionalised with two redox-active viologen moieties. Upon reduction, intramolecular dimerisation of viologen4 units will allow us to exercise stimulus-responsive control over the shapeshifting equilibrium of the bullvalene (see Figure 1). This poster will present spectroscopic and electrochemical analyses of this responsive bullvalene. We hope that the insights gained into the influence of supramolecular interactions over shapeshifting bullva- lene equilibria will inform us about the potential for shapeshifting molecules to be used as a constitutionally adaptive ligand for complex biomolecules, such as proteins. References 1. K. K. Larson, M. He, J. F. Teichert, A. Naganawa and J. W. Bode, Chem. Sci., 2012, 3, 1825–1828. 2. O. Yahiaoui, L. F. Pašteka, B. Judeel and T. Fallon, Angew. Chemie - Int. Ed., 2018, 57, 2570–2574. 3. A. N. Bismillah, J. Sturala, B. M. Chapin, D. S. Yufit, P. Hodgkinson and P. R. McGonigal, Chem. Sci., 2018, 9, 8631–8636. 4. K. Wadhwa, S. Nuryyeva, A. C. Fahrenbach, M. Elhabiri, C. Platas-Iglesias and A. Trabolsi, J. Mater.Chem. C, 2013, 1, 2302–2307.

Figure 1 potential conformation adopted by viologen functionalised bullvalene.png

58 International Symposium on SupraBiomolecular Systems 2021

Utilisation of molecular tweezer’s multivalency for targeting p97 protein pore

Tuesday, 11th May - 14:30: Flash Session 2 - Poster - Abstract ID: 57

Mrs. Abbna Kirupakaran 1, Dr. Johannes van den Boom 2, Mr. Matthias Hayduk 1, Mr. Mike Blüggel 2, Dr. Christine Beuck 2, Prof. Peter Bayer 2, Prof. Jens Voskuhl 1, Prof. Hemmo Meyer 2, Prof. Thomas Schrader 1 1. Institute of Organic Chemistry, University of Duisburg-Essen, Universitätsstr. 7, 45141 Essen, Germany, 2. University of Duisburg-Essen

Nowadays molecular tweezers have been developed as supramolecular tools which selectively address accessi- ble lysines and arginines on protein surfaces.1 This unique interaction has been used to interfere with pathologic protein aggregation and also to inhibit protein-protein interactions. We asked ourselves if multiple tweezers could also be utilised to explore and block the function of protein pores due to the presence of multiple copies of basic amino acids. One such example is the segregase p97 which is relevant for protein quality control inside cells. It contains a central dynamic pore which runs through the whole self-assembled protein machinery and is discussed to take place in recognition and unfolding of lesioned protein termini. The precise function of the pore and its mechanism of action are still unclear.2 We now decided to synthesize a symmetrical multivalent tweezer array with defined distances and a rigid core containing aromatic systems or fluorophore, which can target the spherical arrangement of lysines or arginines lining the pore wall. This tailored hybrid tweezer oligomer should be able to bind cooperatively and tightly to the ring of basic amino acids and hence prevent unfolded peptide strands to penetrate the pore. To this end, we have attached an azide function to the parent tweezer and will employ “click” chemistry to couple multiple tweezers to different oligoalkyne. This can be the first example of an artificial plug for a protein pore. References 1M. Fokkens, T. Schrader, F.-G. Klärner, J. Am.Chem. Soc. 2005, 127, 14415-14421; 2V. E. Pye, I. Dreveny, L. C. Briggs, C. Sands, F. Beuron, X. Zhang, P.S. Freemont: Going through the motions: The ATPase cycle of p97. J. Struct. Biol. 2006, 156, 12-28.

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59 International Symposium on SupraBiomolecular Systems 2021

Preparing for self-evolving synthetic cells: The importance of translation in evolving mixed-lipid compartments

Tuesday, 11th May - 15:20: Plenary 2 - Oral - Abstract ID: 110

Prof. Peter Strazewski 1, Dr. Michele Fiore 1, Dr. Dimitri Fayolle 1, Mr. Augustin Lopez 1, Ms. Carolina Chieffo 1, Mrs. Anastasiia Shvetsova 1 1. Université de Lyon, Univ. Claude Bernard Lyon 1, Institut de Chimie et Biochimie Moléculaires et Supramoleculaires, équipe CO2Glyco

The experimental observation of life-like behaviour self-evolving from a complex chemical system needs many players on the same ground, in our case lipids, nucleic acids, peptides, dehydrating agents, all influencing one another in not always fully predictable ways. We are preparing for this stepwise by testing the most promising conditions for feeding giant lipid vesicles with more lipids, so as to make their membranes grow in size and the vesicles divide to produce more vesicles. We are exploiting various condensation conditions to reversibly join membranophilic mixed-sequence peptides with short nucleic acids and, at the same time, to phoshorylate alcohols to phosphate esters. We are developing analytical tools from fluorophores and rapid clickers to be able to detect minute amounts of biomacromolecules that are found encapsulated in the vesicles. In this talk we will focus on the general principles of information transfer carried over through translation, and the feeding of glass sphere-supported giant vesicles with membranogenic amphiphiles, as reported in refs. 72 and 76 at http://www.icbms.fr/co2glyco/effectif/strazewski .

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60 International Symposium on SupraBiomolecular Systems 2021

Vitamin B12 – an intriguing bioheterocycle

Tuesday, 11th May - 16:05: Plenary 2 - Oral - Abstract ID: 111

Prof. Dorota Gryko 1 1. Institute of Organic Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw

Vitamin B12is a mammoth and complex molecule of vast biochemical importance as its various forms play a crit- ical role in enzymatic processes.1Recently, it has also been studied as a potential drug or imaging agent carrier[2] due to its dietary uptake pathway assuring a proper intake, and because of its constant demand in rapidly di- viding cells. In order for B12 to act as a carrier, its structure must be modified to allow selective coupling of biologically active moieties and at the same time to maintain a high affinity to transport proteins. Selective and high-yielding functionalizations of B12 are highly desirable; however, the complexity of cobalamin’s structure makes this extremely challenging.

Our group has introduced new methods allowing us to achieve this goal. Now, B12 can be selectively and directly attached to alkynes (via CuAAC),3 acids (via amide bond formation)4 or thiols (via disulfide bond formation).5

Also reduction-free, direct alkynylation of vitamin B12 at cobalt center that leads to previously unknown heat and light stable acetylide cobalamins has been developed.6 The selective orthogonal conjugation at both the Co center and 5’-OH group can also be achieved.6 Recently, we have developed a method that involves modifications at previously unexplored meso position.

However, a complete understanding of vitamin B12 reactivity has not yet been achieved hindering the re- searcher’s ability to easily manipulate this molecule. Hence, our goal is to shine a light on vitamin B12 chemistry by fully elaborating on its elusive properties and take advantage of its biological activity. References:

1. Banerjee R. Chemistry and Biochemistry of B12, John Wiley & Sons, Inc, 1999. 2. Wierzba, A.; Hassan, S.; Gryko, D. Asian J.Org.Chem. 2019, https://doi.org/10.1002/ajoc.201800579 3. Chromiński M.; Gryko D. Chem. - Eur. J. 2013, 19, 5141. 4. Jackowska A.; Giedyk M.; Chromiński M.; Gryko D. Org. Biomol. Chem. 2018, 16, 936-943.. 5. Wierzba A. J.; Wojciechowska M.; Trylska J.; Gryko D. Bioconjugate Chem. 2016, 27, 189. 6. Chromiński M.; Lewalska A.; Gryko D. Chem. Commun. 2013, 49, 11406. 6. Chromiński M.; Lewalska, A.; Karczewski, M.; Gryko., D. J. Org.Chem. 2014, 79, 7532.

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62 International Symposium on SupraBiomolecular Systems 2021

TBD

Wednesday, 12th May - 09:00: Plenary 1 - Oral - Abstract ID: 112

Dr. Dan Pantos 1 1. Department of Chemistry, University of Bath

TBD

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Self-assembling supramolecular dendrimers for biomedical applications

Wednesday, 12th May - 09:45: Plenary 1 - Oral - Abstract ID: 113

Dr. Ling Peng 1 1. Aix-Marseille University

TBD

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Biohybrid materials in the context of systems chemistry

Wednesday, 12th May - 10:50: Oral Session 1 - Oral - Abstract ID: 6

Dr. Andres de la Escosura 1, Ms. Noemí Nogal 1, Mr. Marcos Marcos Sanz 1, Mr. Alonso Puente 2, Mr. Martin Aleksiev 2, Mr. Santiago Guisán 1, Ms. Sonia Vela 1 1. Universidad Autonoma de Madrid, 2. Universidad Autónoma de Madrid

The study of complex molecular networks and supramolecular assemblies is a clear objective in the field of sys- tems chemistry, which is expected to have a great impact in the area of origins-of-life research and as biohybrid materials for biomedicine [1,2]. With regards to the origins of life, a pertinent question is whether artificial cells could be constructed from non-natural components. To answer this question, we have research lines on nucleic acid hybrids, replicating nucleopeptide networks [3] (see scheme of our general strategy in Figure 1) and nu- cleolipid compartments [4]. Merging these components is an interesting approach because it allows exploring some properties of life without the restrictions of the historical pathway that Darwinian evolution took. Concerning biohybrid materials, we focus on supramolecular approaches toward biohybrids for biomedical light management [5], which combine different photoactive molecules with peptide, protein and nucleic acid nanostructures (Figure 2). For example, we have recently studied the electrostatic co-crystallization of a cationic porphyrinoid and negatively charged TMV virus, together with singlet oxygen generation by the resulting crys- tals [6]. Electrostatically assembled zinc Pc-DNA origami complexes have also been demonstrated [7]. In this presentation we will shortly discuss some of these research lines. References:

1. A. de la Escosura, “The Informational Substrate of Chemical Evolution: Implications for Abiogenesis”. Life 2019, 9, 66. 2. K. Ruiz-Mirazo, C. Briones, A. de la Escosura, “Chemical Roots of Biological Evolution: The Origins of Life as a Process of Development of Autonomous Functional Systems”. Open Biol.2017, 7, 170050. 3. A. de la Escosura, G. Ashkenasy, et al., “Primitive Selection of the Fittest Emerging Through Functional Synergy in Nucleopeptide Networks”. Proceed, Natl. Acad. Sci. USA, in press. 4. S. Morales-Reina, C. Giri, M. Leclercq, S. Vela-Gallego, I. de la Torre, J. R. Caston, M. Surin, A. de la Esco- sura, “Programmed Recognition between Complementary Dinucleolipids to Control the Self-Assembly of Lipidic Amphiphilles”. Chem. Eur J.2019, 26, 1082-1090. 5. V. Almeida-Marrero, E. van de Winckle, E. Anaya-Plaza, T. Torres, A. de la Escosura, “Porphyrinoid Bio- hybrid Materials as an Emergent Toolbox for Biomedical Light Management”. Chem. Soc.Rev.2018, 47, 7369. 6. E. Anaya-Plaza, A. Aljarilla, G. Beaune, Nonappa, J. V. I. Timonen, A. de la Escosura, T. Torres, M. Kosti- ainen, Adv. Mater.2019, 31, 1902582. 7. A. Shaukat, S. Julin, V. Linko, E. Anaya-Plaza, T. Torres, A. de la Escosura, M. Kostiainen, “Phthalocyanine- DNA Origami Complexes with Enhanced Stability and Optical Properties”. Chem. Commun.2020, 56, 7341- 7344.

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66 International Symposium on SupraBiomolecular Systems 2021

Modulating Protein-Protein Interactions Using Dynamic Combinatorial Selection Methods

Wednesday, 12th May - 11:05: Oral Session 1 - Oral - Abstract ID: 22

Prof. Andy Wilson 1 1. University of Leeds

Protein-protein interactions (PPIs) regulate virtually all cellular signalling processes thus developing methods to modulate PPIs represents a significant challenge of immense biochemical and medical importance.1 However, methods to target intracellular (PPIs) using synthetic molecules are not well established.2 PPI interfaces are often shallow and large compared with typical small molecule binding sites, making it difficult to identify molecules which bind with sufficient affinity and specificity to be effective modulators. This presentation will describe two complementary techniques that exploit dynamic combinatorial selection to identify ligands that modulate PPIs. The presentation will firstly illustrate an approach to manipulate protein self-assembly, in which disulfide tethering3 is used to rapidly explore chemical space and identify “post-translational chemical modifications” that stabilize specific PPIs, to inhibit amyloid assembly. We will show how a disulfide tethering, was used to iden- tify a series of covalent ligands targeted to a dynamic site on an amyloidogenic variant of β2-microglobulin (ΔN6). These ligands were capable of stabilising a non-amyloidogenic, tetrameric form of this protein, an oligomer which is normally only transiently populated and thus not amenable to high-resolution structural studies. In the presence of a covalent ligand, we successfully obtained a crystal structure of this oligomer, showing that these PPI interfaces can be stabilised by small molecules which act as “molecular glue” between adjacent protein subunits, and which stabilise conformations of the protein that promote the formation of specific interaction surfaces.4 Secondly, peptides can also be used as a scaffold/ligand to identify non-covalent small molecule binders at PPI interfaces. The presentation will illustrate how a dynamic hydrazone screening assay can be identify peptide- small molecule combinations/ligations capable of inhibiting a PPI of interest: in this case, the binding of GKAP to SHANK1-PDZ – a challenging βstrand mediated interaction.5 The resulting peptide-fragment hybrids have the potential to be developed further as protease-resistant peptide-small molecule inhibitors or to be used as leads for the development of high affinity small molecule binders and chemical probes. More excitingly, crystallo- graphic and molecular dynamics analyses will show this “dynamic ligation” screening approach represents a high-throughput, site-directed method for exploring ligandable binding pockets at and around PPI interfaces. 1. Cancer Cell2017, 32 (1), 9-25. 2. Chem. Biol.2014, 21 (9), 1102-1114. 3. Drug Discov.Today: Technol.2013, 10 (4), e501-e508. 4. J. Am. Chem. Soc.2020, 142 (49), 20845-20854. 5. Chem. Sci.2021 10.1039/D0SC05694D

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67 International Symposium on SupraBiomolecular Systems 2021

PROBING THE FUNCTION OF CELL BIOMIMETICS AND PROTOCELLS

Wednesday, 12th May - 11:20: Oral Session 1 - Oral - Abstract ID: 98

Dr. Dietmar Appelhans 1, Dr. Silvia Moreno 1, Dr. Susanne Boye 1, Prof. Albena Lederer 1, Prof. Brigitte Voit 1 1. Leibniz-Insitut für Polymerfoschung Dreden e.V., Hohe Straße 6, 01069 Dresden, Germany

Engineering of multifunctional vesicular (multi)compartments for mimicking specific cellular functions and protocells is one promising approach for overcoming protein lack in organ tissues and human diseases as well as for imitating prebiotic live.1,2 The vesicular compartments have to fulfil various key characteristics (e.g. tune- able by external stimuli (e.g. light and redox), controlling membrane functions for exchanging biomolecules, controlled release of biomolecules, retaining cargo inside of vesicular cavity),3-6 while multicompartments7,8 should also possess orthogonal-responsive membrane properties to control spatiotemporal and spatially sepa- rated biological pathways for establishing protocells. Overall, this would result in, for example, the establish- ment of next-generation therapeutics, life-like cells and bio-nanotechnology approaches. This contribution will show the fabrication of pH-responsive polymersome-based hollow capsules7 and pro- teinososomes. It will also highlight the common integration of biologically-active particles in microgel and polymersome compartments within proteinosomes, including the controlled action of enzymes, for the estab- lishment of protocells besides the use of enzyme-loaded polymersomes as artificial organelles. 1. Schwille P., Science 33: 1252, 2011. 2. Mann S., Acc. Chem. Res. 45: 2131, 2012. 3. Gaitzsch J. et al., Angew. Chem. Int. Ed. 51: 4448, 2012. 4. Gräfe D. et al., Nanoscale 6: 10752, 2014. 5. Liu X. et al., J. Am. Chem. Soc. 140: 16106, 2018. 6. Gumz H. et al., Adv. Sci. 9: 1801299, 2019. 7. Moreno S. et al., Biomacromolecules 21: 5172, 2020.

8. Liu X. et al., Angew. Chem. Int. Ed. 56: 16233, 2017 9. Wen P. et al., Small 17: 2005749, 2021. 10. Wang, X. et al., Adv. Sci. accepted, 2021.

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Programming Supramolecular Self-Assembly and Gelation with Chemical Clocks

Wednesday, 12th May - 11:35: Oral Session 1 - Oral - Abstract ID: 5

Dr. Guido Panzarasa 1 1. ETH Zurich

Living systems can grow a huge variety of materials with the highest degree of sophistication, and an overall efficiency that remains largely unparalleled by artificial fabrication techniques. Moreover, living materials are adaptive i.e. they exist and perform autonomously under dissipative conditions. These features are made possible by the ability to control complex reactions networks, carefully organized in spatiotemporal sequences. Developing autonomous chemical systems that could imitate the properties of living matter is a challenge at the meeting point of materials science and systems chemistry. Chemical clocks thus become versatile tools to program in time the autonomous and transient self-assembly of organic as well as inorganic building blocks. The design of such ad hoc reaction networks is at the core of my current research efforts. I will show how to “clock” molecules, polymers and metal cations into different structures, from nanoparticles to gels, without the need for external control, and demonstrate how this approach paves the way to the development of (almost) living artificial materials.

69 International Symposium on SupraBiomolecular Systems 2021

Regulating transmembrane ion transport using stimuli-responsive supramolecular carriers

Wednesday, 12th May - 11:50: Oral Session 1 - Oral - Abstract ID: 14

Prof. Matthew Langton 1 1. Department of Chemistry, University of Oxford

Transmembrane ion transport across lipid bilayer membranes in biology is controlled by membrane proteins, which in turn are regulated by external stimuli, including small molecules, membrane potential and light. Syn- thetic supramolecular analogues have emerged as a complementary method to facilitate transmembrane ion transport, with potential applications as therapies for diseases arising from mis-regulated ion channels, and as tools for physiological research.1 However, whilst the activity of natural ion channels and transporters can be precisely controlled by external stimuli, stimuli-responsive supramolecular ion channels and mobile ion carri- ers are rare, and achieving high levels of control (to switch between inactive “OFF” states and ion transporting “ON” states), with spatial and/or temporal precision, is particularly challenging.2 Photo-regulated transporters are particularly attractive targets, because of the possibility of achieving both spatial and temporal control over ion transport in a readily adaptable synthetic system. Such systems are rare, and employ almost exclusively UV light-triggered transport mechanisms, which is ultimately poorly suited to biological applications due to poor tissue penetration and collateral cell damage. Here recent work towards developing stimuli-responsive ion transporters is described. In particular, progress on developing two-colour responsive molecular photo-switches that act as supramolecular transmembrane an- ion carriers will be presented.3 In these systems, reversible switching of the photo-switches within the lipid bi- layer membrane is achieved using biocompatible visible wavelengths of light, such that temporal control over transmembrane anion transport is achieved through alternating irradiation with red and blue light (Figure).

1. J. T. Davis, P. A. Gale and R. Quesada, Chem. Soc. Rev. 2020, 49, 6056-6086 2. M. J. Langton, Nat. Rev. Chem. 2021, 5, 46-61 3. A. Kerckhoffs, M. J. Langton, Chem. Sci. 2020, 11, 6325–6331

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70 International Symposium on SupraBiomolecular Systems 2021

Foldamer bundles binding amphiphiles in water: toward artificial glycolipid binding proteins

Wednesday, 12th May - 12:05: Oral Session 1 - Oral - Abstract ID: 104

Dr. Sung Hyun Yoo 1, Dr. Gavin W. Collie 2, Dr. Jérémie Burrato 1, Dr. Estelle Morvan 1, Dr. Frédéric Rosu 1, Dr. Arup Roy 3, Dr. Morgane Pasco 1, Dr. Valérie Gabelica 1, Dr. Cameron D. Mackereth 1, Dr. Gilles Guichard 1 1. Institut Européen de Chimie et Biologie (IECB), Univ. Bordeaux, Pessac, 2. Discovery Sciences, IMED Biotech unit, AstraZeneca, Cambridge, 3. CSTD, CSIR-NEIST, Assam

Protein mimicry using artificial scaffolds has attracted considerable interest as it may provide molecules with improved or new activity profiles for various applications in life science. Synthetic folded oligomers (“Foldamers”) which display advantageous features such as chemical-structural variety, folding fidelity, se- quence programmability and more importantly, bioorthogonality have emerged as useful scaffolds to cre- ate original protein mimics. In particular, it has been shown that water-soluble amphiphilic foldamer se- quences may be engineered to create complex and atomically precise self-assembled nanostructures with the size of small proteins. However, very few studies have exploited specific features of these foldamer-based supramolecular architectures such as cavities to create receptors that would recognize specific guest molecules. Biomolecule encapsulation via molecular recognition can be a promising basic platform for catalysts and drug- delivery systems rivaling natural counterparts (i.e. proteins). Here, we report that foldamer bundles from aqueous self-assembly of N,N’-linked oligourea helices can bind diverse amphiphiles possessing long hydro- carbon chains. Solid, solution and gas phase analysis of the various receptor-substrate complexes using X-ray crystallography, NMR, CD and IMS-MS revealed atomic-resolution structures of the supramolecular complexes as well as receptor-substrate interaction in water. Structural features and the binding principle reminiscent of human glycolipid transfer proteins suggest foldamer supramolecular architectures to be prospective “artificial proteins”.

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71 International Symposium on SupraBiomolecular Systems 2021

Suprabiomolecular sensor systems

Wednesday, 12th May - 14:00: Oral Session 2 - Oral - Abstract ID: 8

Prof. Andreas Hennig 1 1. Universität Osnabrück

The combination of supramolecular chemistry with biomolecular processes affords new avenues in the de- sign of useful sensors and assays. We have previously explored the differential response of supramolecular chemosensors to the substrates and products of enzymatic reactions to afford new label-free fluorescence-based enzyme assays with potential applications in drug discovery and metabolite screening (Figure 1) and demon- strated the possibility to transfer this principle to magnetic resonance imaging (MRI).[1] We have further shown that supramolecular systems afford new possibilities to monitor membrane transport of ions, low molecular weight drugs, and peptides including otherwise spectroscopically silently translocating cell-penetrating pep- tides (Figure 2).[2] One of the main challenges in the development of supramolecular sensor systems is to enhance their sensi- tivity, which is commonly addressed by a refined receptor design to increase the affinity between analyte and sensor. We now show that a pH gradient across the lipid bilayer membrane of liposome-encapsulated supramolecular sensor systems causes an imbalance of the influx and efflux rates of basic analyte, which leads to a significantly increased concentration of the analyte in the liposome interior (Figure 3). To demonstrate the utility of our liposome-enhanced sensors, we have selected various biologically relevant amines and ob- served a significant increase in sensitivity of all analytes. For example, the sensitivity of the cucurbit[8]uril/2,7- dimethyldiazapyrenium (CB8/MDAP) host-dye pair towards the neurotransmitter serotonin was increased by a factor of ca. 75, and the sensitivity of the hydroxypropyl-β-cyclodextrin/berberine (HP-β-CD/BE) host-dye pair towards phenethylamine was increased by a factor of ca. 130 (Figure 3c). We believe that our liposome-enhanced sensing principle provides a widely applicable method to increase the sensitivity of suprabiomolecular sensor systems. It should be also transferable to acidic analytes as well as to other mechanisms than protonation and deprotonation, which cause an imbalance of the influx and efflux rates of the analytes. [1] (a) Nat. Methods 2007, 4, 629; (b) J. Am. Chem. Soc.2009, 131, 11558; (c) Chem. Eur. J.2012, 18, 3444; (d) Chem. Sci. 2015, 6, 6069; (e) Angew. Chem. Int. Ed. 2015, 54, 13444; (f)J. Am. Chem. Soc.2016, 138, 13022; (g) SLAS Discovery 2017, 22, 906; (h) Anal. Bioanal. Chem.2017, 409, 6485; (i) ChemistryOpen 2019, 8, 1350. [2] (a) Chirality 2008, 20, 932; (b) Org. Biomol. Chem. 2009, 7, 1784-1792; (c) Nat. Chem.2010, 2, 533; (d) Angew. Chem. Int. Ed.2017, 56, 15742; (e)J. Am. Chem. Soc.2019, 141, 20137; (f) ACS Sensors 2021, 6, 175.

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73 International Symposium on SupraBiomolecular Systems 2021

Flavin-based molecular rotors

Wednesday, 12th May - 14:15: Oral Session 2 - Oral - Abstract ID: 65

Mrs. Dora Rasadean 1, Dr. Christopher Pudney 2, Dr. Dan Pantos 1 1. Department of Chemistry, University of Bath, 2. Department of Biology and Biochemistry, University of Bath

The development of synthetic nanomachines that mimic the macroscopic and biological worlds is in full swing1,2 and its importance has been supported by the Nobel Prize award in 2016. Numerous molecular machines are reported and controlling their rotation/motion is the key towards successful construction.1–3 Inspired by the ubiquitous flavoenzymes, we report the first generation of synthetic flavin-based molecular rotors. The stator is an isoalloxazine core in which the benzene ring is replaced by pyridine. The rotor unit is attached on the N10 position of isoalloxazine following a protocol we have recently published.4 The rotors series includes three scaffolds with cyclic, alkyl chain and aromatic functionalities and chiral centre in the alpha position; the last rotor is an N10-isopropyl-isoalloxazine (Fig. 1A: the two stations of rotor 1 ). The driving force behind their motion is the ability of the proton in the alpha position to form intramolecular hydrogen bonding (Fig.1A) with nitrogen atoms in positions 1 (imide station) and 9 (pyridine station). We aim to control the rotation and di- rectionality of these molecules by disrupting and reforming the hydrogen bonding by acid-base equilibria and reduction of the isoalloxazine core. The electronic, fluorescent and chiroptical properties of rotors are explored by 1D and 2D NMR (Fig.1C: partial VT 1H NMR spectra of rotor 1 in DMSO-d6 showing the coalescence of the alpha protons of the two stations across the indicated temperature range), circular dichroism, absorption and fluorescent spectroscopies. TDDFT calculations (Fig.1B: calculated plot of total energy at different degrees of rotation of rotor 1) complement the experimental data. References: 1. N. Koumura, R. W. J. Zijlstra, R. A. van Delden, N. Harada and B. L. Feringa, Nature, 1999, 401, 152–155. 2. M. R. Wilson, J. Solà, A. Carlone, S. M. Goldup, N. Lebrasseur and D. A. Leigh, Nature, 2016, 534, 235–240. 3 . Y. Wu, G. Wang, Q. Li, J. Xiang, H. Jiang and Y. Wang, Nat. Commun., 2018, 9, 1953. 4 . D.-M. Răsădean, T. Machida, K. Sada, C. R. Pudney and G. D. Pantoș, Tetrahedron, 2021, 131925.

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Optimization of functionalized H-Ferritin nanocages loaded with Navitoclax as targeted drug delivery agents against Cancer Associated Fibroblasts in vitro and in vivo

Wednesday, 12th May - 14:30: Oral Session 2 - Oral - Abstract ID: 69

Dr. Leopoldo Sitia 1, Dr. Arianna Bonizzi 1, Dr. Serena Mazzucchelli 1, Dr. Sara Negri 2, Dr. Cristina Sottani 2, Dr. Elena Grignani 2, Dr. Maria Antonietta Rizzuto 3, Prof. Davide Prosperi 3, Dr. Luca Sorrentino 4, Dr. Carlo Morasso 2, Dr. Raffaele Allevi 1, Dr. Marta Sevieri 1, Dr. Filippo Silva 1, Dr. Marta Truffi 2, Prof. Fabio Corsi 2 1. Dipartimento di Scienze Biomediche e Cliniche “L. Sacco”, Università di Milano, 2. Istituti Clinici Scientifici Maugeri IRCCS, 27100 Pavia, 3. Department of Biotechnology and Biosciences, University of Milan-Bicocca, 20126 Milan, 4. Colorectal Surgery Unit, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, 20133 Milan

In the fight against cancer, new players are gaining attention and new therapies are being developed to target not only the cancer cells but also relevant components of the tumor microenvironment. In this context, Cancer- associated fibroblasts (CAFs) play a pivotal role in cancer progression, metastasis formation, and in inducing drug resistance. To enable specific targeting and delivery of cytotoxic agents in CAFs, nanomedicine seems to be a promising approach (Truffi et al. 2019). Here, we first prepared navitoclax (Nav) loaded H-ferritin nanocages (HFn, HNav). Nav is an experimental pro-apoptotic drug, whose clinical development is limited by strong systemic toxicity and hydrophobicity. Its nanoformulation could help to circumvent these limitations.

To load Nav in the nanocages we complexed the drug with CuSO4 and followed the metal ions affinity approach, as already described by our group (Bonizzi et al. 2019). Loading was verified by UPLC-MS/MS. Then, we functionalized the nanocages with anti-fibroblast activation protein (FAP) antibody fragments using two different NHS-PEG-Mal molecules (5 and 10 kDa MW) as heterobifunctional linkers. FAP is indeed consid- ered a reliable CAF marker (Sitia et al. 2021). The properties of the nanocages were characterized by Dynamic Light Scattering, Transmission Electron Mi- croscopy and Raman Spectroscopy. Then, we tested interactions with FAP+ CAFs and FAP- tumor cells, finding that the 10 kDa induced the highest preferential binding only in FAP+ cells, while no difference was observed in FAP- cells. Cytotoxicity studies allowed us to find that Nav mechanisms of action (PARP cleavage and BAX activation) were maintained after encapsulation and that the activity in CAFs was significantly higher when us- ing functionalized HFn (HNav-FAP) as compared to non-functionalized nanocages. By UPLC MS/MS we verified that the higher activity was correlated with a higher intracellular Nav uptake only in FAP+ cells, confirming the efficacy of our functionalization strategy. Finally, biodistribution and CAF targeting were studied by in vivo and ex vivo imaging in a mouse model of triple negative breast cancer, finding that the HFn-FAP reach the tumor after intravenous administration and are able to target and accumulate in CAFs. In conclusion, we proved that functionalized HFn promote selective drug delivery into CAFs. Further nanodrug optimization and in vivo efficacy studies, could open the way to the development of innovative anticancer ther- apeutic strategies aimed at targeting the tumor microenvironment.

75 International Symposium on SupraBiomolecular Systems 2021

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76 International Symposium on SupraBiomolecular Systems 2021

￿-Galactosidase Enzyme Nanoformulated in Multifunctional Nanoliposomes for Fabry Disease Treatment

Wednesday, 12th May - 14:45: Oral Session 2 - Oral - Abstract ID: 72

Ms. Judit Tomsen-Melero 1, Dr. Elisabet González-Mira 2, Mr. Josep Merlo-Mas 3, Dr. Edgar Cristóbal-Lecina 4, Dr. Vanessa Díaz-Riascos 5, Dr. Albert Font 6, Mr. Marc Moltó-Abad 5, Ms. Natalia García-Aranda 5, Dr. José Luis Corchero 7, Ms. Aida Carreño 2, Dr. Jannik Nedergaard Pedersen 8, Dr. Jeppe Lyngsø 8, Dr. Ionita Inbal 9, Dr. Daniel Pulido 4, Dr. Santi Sala 3, Prof. Jaume Veciana 2, Dr. Simó Schwartz, Jr. 5, Prof. Dganit Danino 9, Prof. Jan Skov Pedersen 8, Dr. Andreu Soldevila 6, Dr. Miriam Royo 4, Dr. Alba Córdoba 3, Dr. Ibane Abasolo 5, Dr. Nora Ventosa 2 1. Institut de Ciència de Materials de Barcelona, ICMAB-CSIC, 08193 Bellaterra, Spain; Centro de Investigación Biomédica en Red - Bioingeniería, Biomateriales y Nanomedicina, CIBER-BBN, 28029 Madrid, Spain; Nanomol Technologies SL, 08193 Bellaterra, Spain, 2. Institut de Ciència de Materials de Barcelona, ICMAB-CSIC, 08193 Bellaterra, Spain; Centro de Investigación Biomédica en Red - Bioingeniería, Biomateriales y Nanomedicina, CIBER-BBN, 28029 Madrid, Spain, 3. Nanomol Technologies SL, 08193 Bellaterra, Spain, 4. Institut de Química Avançada de Catalunya, IQAC-CSIC, 08034 Barcelona, Spain; Centro de Investigación Biomédica en Red - Bioingeniería, Biomateriales y Nanomedicina, CIBER-BBN, 28029 Madrid, Spain, 5. Drug Delivery and Targeting and Functional Validation and Preclinical Research, CIBBIM-Nanomedicine, Vall d’Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; CIBER-BBN, Madrid, Spain, 6. Leanbio SL, 08028 Barcelona, Spain, 7. Departament de Genètica i de Microbiologia, Institut de Biotecnologia i de Biomedicina, IBB, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; CIBER-BBN, Madrid, Spain, 8. Interdisciplinary Nanoscience Center, iNANO, and Departament of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark, 9. CryoEM Laboratory of Soft Matter, Faculty of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, 32000 Haifa, Israel

Introduction Lysosomal storage disorders (LSD) are a group of rare diseases currently without definitive cure. In Fabry LSD Disease (FD), the deficiency in α-Galactosidase (GLA) enzyme activity results in the cellular accumulation of neutral glycosphingolipids (mainly Gb3), with particular affectation to kidneys, heart, and nervous system. The current treatment is the Enzyme Replacement Therapy (ERT), in which free GLA recombinant protein is admin- istered intravenously to patients. ERT shows several drawbacks, including poor biodistribution, low stability, limited efficacy, high immunogenicity, and low capacity to cross biological barriers, such as cell membranes and BBB. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating enzymes.1 Methods GLA-loaded nanoliposomes (nanoGLA) functionalized with Arginine-Glycine-Aspartic acid (RGD) peptide were prepared by a one-step, green, and easy scalable method based on compressed CO2, named DELOS-susp, fol- lowed by a TFF procedure. A deep physicochemical and biological characterization of nanoGLA was done com- bining several techniques (e.g. DLS, SAXS, cryoTEM, HPLC) with biological assays. The efficacy of nanoGLA to metabolize Gb3 accumulations was compared to free GLA, both in vitro cell culture and in vivo in a Fabry mouse model, characterized by the lack of endogenous GLA. Results Nanoliposomes functionalized with RGD-peptide have already emerged as a good platform to protect and de- liver GLA to endothelial cells.2 Consequently, in the last years we have continued advancing the development of this nanoconjugate in the frame of a European project (Smart-4-Fabry). Small and uniform nanoGLA lipo- somes, functionalized with targeting-peptides, were successfully prepared by DELOS-susp, showing high GLA entrapment efficiency, enhanced enzymatic activity, and superior in vitro efficacy.3 Moreover, in vivo, nanoGLA showed improved Gb3 levels in several tissues (e.g. plasma, liver, spleen, lung, heart, kidney, skin, and brain) compared to the effects of the free enzymes (included the commercially available Replagal®).4

77 International Symposium on SupraBiomolecular Systems 2021

Discussion Results confirm the suitability of DELOS-susp for the preparation of nanoconjugates with controlled charac- teristics at nanoscopic and supramolecular level. Deep characterization analysis allowed the identification of nanoGLA’s critical quality attributes, to ensure its consistent quality for further regulatory purposes. Together with an improved efficacy in vivo, our results provide evidences that support a clinically relevant advantage of the nanoGLA versus authorized ERTs. Based on it, the EMA has recognized the significant benefit that nanoGLA can offer to Fabry disease patients and has granted the Orphan Drug Designation. 1Solomon,Adv.Drug Deliv.Rev.(2017) 2Cabrera,Adv.HealthcareMater.(2016) 3Tomsen-Melero, ACS Appl.Mater.Interfaces(2021); Merlo-Mas, J.of SupercriticalFluids(Inpress) 4WO2014/001509;EP21382062.4 Acknowledgment: European Commission (Smart-4-Fabry ID720942, Phoenix ID953110)

78 International Symposium on SupraBiomolecular Systems 2021

A plug-and-play RNA detection platform for sensing cell physiology and phenotype

Wednesday, 12th May - 15:00: Oral Session 2 - Oral - Abstract ID: 24

Dr. James Chappell 1 1. Assistant Professor of Biosciences, Rice University

Cells respond to environmental and intracellular signals by modulating the levels of a myriad of proteins that ultimately determine cell physiology and phenotype. Regulatory mechanisms that operate at the transcriptional level (RNA)represent a major control point and provide blueprints to translate instructions contained in the genome into proteins. Detection of RNA levels thus provides a unique and critical readout of a cell’s physiologic status and phenotype. Moreover, integration of RNA sensing capabilities in the context of synthetic genetic programs, would enable advanced cellular programming in which engineered functions can be dynamically coupled with cell state to achieve feedback control to improve overall performance. However, technologies that link the detection of cellular RNAs in living cells, to the control of biomolecules are currently lacking. To address this, we present a genetically encoded plug-and-play RNA detection platform that can be programmed to sense user-specified cellular RNAs and in response, produce user-defined biomolecular outputs (i.e.RNAs or proteins). The basis for this system is a synthetically split-splicing ribozyme that has been engineered to be non-functional when present alone. However, in the presence of a user-specified RNA input, the ribozyme is able to bind to this RNA and reconstitute a functional ribozyme, resulting in splicing of the attached RNA exons (Figure 1A). RNA exons can be designed so that splicing results in formation of a mature mRNA, resulting in protein production upon RNA detection. We have demonstrated the plug-and-play nature of this technology (Figure 1B) allowing for facile exchange of new inputs and outputs. This talk will highlight our work in creating and optimizing this technology by harnessing concepts and technical approaches from protein engineering, to engineer a large and structurally complex catalytic RNA.

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79 International Symposium on SupraBiomolecular Systems 2021

Pushing the limits of the inhibitory multivalent effect in glycoscience

Wednesday, 12th May - 15:50: Plenary 2 - Oral - Abstract ID: 114

Mr. Philippe Compain 1 1. University of Strasbourg

TBD

80 International Symposium on SupraBiomolecular Systems 2021

Metal-Modified DNA: Merging Bioinorganic Chemistry with Supramolecular Chemistry

Wednesday, 12th May - 16:35: Plenary 2 - Oral - Abstract ID: 118

Prof. Jens Müller 1 1. University of Münster

Due to their predictable self-assembly and their ease of modification, nucleic acids represent an important build- ing block in nanotechnology.[1] As a result of their polyanionic nature, they always occur in combination with metal ions. A site-specific functionalization of nucleic acids with (transition) metal ions by using ligand-based nucleosides enables the formation of artificial nucleic acids with metal-based functionality and thereby extents their applicability significantly. The resulting metal-mediated base pairs (see figure) are at the focus of our research in this field of bioinspired supramolecular coordination chemistry.[2] This lecture will summarize our efforts in the previous decade to establish metal-mediated base pairs and to understand better the principles of their formation, starting with the imidazole–Ag(I)–imidazole pair,[3] but also encompassing dinuclear metal-mediated base pairs[4] and the light-triggered formation of metal-mediated base pairs.[5] References [1] E. Stulz, G. H. Clever, DNA in supramolecular chemisty and nanotechnology, John Wiley & Sons, Chichester, 2015. [2] a) S. Naskar, R. Guha, J. Müller, Angew.Chem. Int. Ed. 2020, 59, 1397; b) J. Müller, Coord. Chem.Rev. 2019, 393, 37; c) Y. Takezawa, J. Müller, M. Shionoya, Chem. Lett. 2017, 46, 622. [3] a) S. Johannsen, N. Megger, D. Böhme, R. K. O. Sigel, J. Müller, Nat. Chem. 2010, 2, 229; b) K. Petrovec, B. J. Ravoo, J. Müller, Chem. Commun. 2012, 48, 11844; c) X. Tan, S. Litau, X. Zhang, J. Müller, Langmuir 2015, 31, 11305. [4] S. Mandal, M. Hebenbrock, J. Müller, Angew. Chem. Int. Ed. 2016, 55, 15520. [5] a) S. Naskar, J. Müller, Chem. Eur. J. 2019, 25, 16214; b) S. Naskar, M. Hebenbrock, J. Müller, Inorg. Chim. Acta 2020, 512, 11985.

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81 International Symposium on SupraBiomolecular Systems 2021

Authors Index

Abasolo, I. 77 Bonizzi, A. 75 Abdin, B. 19 Borodina, T. 55 Abdulraheem, A. 19 Bourgeade-Delmas, S. 47 About, I. 26 Boye, S. 68 Albertazzi, L. 3, 9 Brunel, F. 26 Aleksiev, M. 65 Bukreeva, T. 55 Alies, B. 17, 32 Burmistrov, I. 55 Allevi, R. 75 Burrato, J. 71 Anaya-Plaza, E. 10 Burrows, C. 35 Aparicio, F. 4 Appelhans, D. 68 Cabanas-Danes, J. 46 Arosio, P. 6 Cabrita, E. 28 Arpin, C. 39 Camplo, M. 26 Atieh, D. 19 Carrara, S. 26 Aubry, C. 39 Carreño, A. 77 Auditto, S. 26 Chamorro, P. 4 Chamorro, R. 4 Baillet, J. 52 Chappell, J. 79 Barthélémy, P. (Equipe ChemBioPharm, INSERM Chesnokova, N. 54 U1212, UMR 5320 CNRS-Université de Chieffo, C. 60 Bordeaux) 8, 17, 32, 39 Collie, G. 71 Barthélémy, P. (Université de Bordeaux, INSERM, Collin, S. 24 U1212, CNRS UMR 5320, ARNA, ARN : Compain, P. 80 Régulations Naturelle et Artificielle, Corchero, J. 77 ChemBioPharm, 33076 Bordeaux, France) Corsi, F. 75 5, 52 Coste, M. 40 Bartocci, A. 43 Courtine, C. 13 Battu, S. 17 Crauste-Manciet, S. 8 Bayer, P. 59 Cretich, M. 49 Beer, P. 23 Cristóbal-Lecina, E. 77 Benoit-Marquié, F. 13 Cruz, C. 28, 29, 31 Bereau, T. 30 Cunha, A. 5 Bergamaschi, G. 49–51 Córdoba, A. 77 Berrocal, J. 9 Bettotti, P. 50 Danino, D. 77 Beuck, C. 59 De Borggraeve, W. 11 Beznos, O. 54 De Bournonville, S. 11 Bickerton, L. 23 de la Escosura, A. 65 Blüggel, M. 59 Dehay, B. 5 Bohr, S. 44 Dessane, B. 8 Bolla, J. 26 Desvergnes, V. 8 Bonduelle, C. 47 Duarte, F. 23

82 International Symposium on SupraBiomolecular Systems 2021

Dumont, E. 43 Imbert, L. 28 Dupuy, B. 47 Inbal, I. 77 Dutta, A. 30 Ives, R. 58 Díaz-Riascos, V. 77 Jeanneau, C. 26 El Hamoui, O. 17 Jensen, S. 44 Elbeltagy, M. 19 JI, H. 56

Fayolle, D. 60 Karmacharya, S. 24 Figueiredo, J. 29 Kauffmann, B. 8 Fiore, M. 60 Kauss, T. 39 Font, A. 77 Kaygisiz, K. 14, 30 Frigerio, R. 49 Kerckhofs, G. 11 Fuentes, E. 3, 9 Kirupakaran, A. 59 Kjems, J. 37 Gabelica, V. 71 KLUFTS-EDEL, A. 8 Gacanin, J. 16 Klyachko, N. 55 Gadre, S. 25 Konstantinovsky, D. 33 García-Aranda, N. 77 Korczak, P. 39 Gaubert, A. 5, 52 Kost, O. 54 Gaudin, K. (ARN : Régulations Naturelles et Kostiainen, M. 10 Artificielles, Inserm U1212, UMR 5320 CNRS, Université de Bordeaux) 17 Langenegger, S. 42 Gaudin, K. (University of Strasbourg) 57 Langton, M. 23, 70 Gerth, M. 9 Largy, E. 31 Gillet, N. 43 Latxague, L. 5, 52 Gonzalez-Rodriguez, D. 4 Lauth de Viguerie, N. 13 González-Mira, E. 77 Le Coustumer, P. 17 Gori, A. 49–51 Lecommandoux, S. 47 Gorostiza, P. 9 Lederer, A. 68 Grignani, E. 75 Lespes, G. 17 Gryko, D. 61 Liu, K. 36 Gudeangadi, P. 41 Lopez, A. 60 Guichard, G. 71 Loquet, A. 8 Guisán, S. 65 Lorenzo, C. 13 Lyngsø, J. 77 Haener, R. 42 Löffler, P. 44 Halma, M. 46 Hammes-Schiffer, S. 33 M. Nau, W. 24 Hamoud, A. 8 Mackereth, C. 71 Hatzakis, N. 44 Malle, M. 44 Hayduk, M. 59 Marcos Sanz, M. 65 Hedegård, P. 44 Matera, C. 9 Heller, I. 46 Matile, S. 38 Hennig, A. 24, 72 Mazzucchelli, S. 75 Herrmann, A. 1 McGonigal, P. 58 HEUZE, K. 56 Mergny, J. 29 Merlo-Mas, J. 77 Iacovache, I. 42 Meyer, H. 59

83 International Symposium on SupraBiomolecular Systems 2021

Mikheev, A. 55 Royo, M. 77 Mingotaud, A. 13 Sadeghi, N. 25 Miranda, A. 28, 31 Sala, S. 77 Moltó-Abad, M. 77 Salas-Ambrosio, P. 47 Morasso, C. 75 Salgado, G. 28 Moreno, S. 68 Salman, A. 19 Morvan, E. 71 Santos, T. 28, 31 Musicò, A. 49–51 Sayde, T. 17 Müller, J. 81 Schrader, T. 21, 25, 59 Münch, J. 30 Schwartz, Jr., S. 77 Nedergaard Pedersen, J. 77 Sen, D. 36 Negri, S. 75 Sergent, M. 26 Nilam, M. 24 Serpell, C. 41 Nogal, N. 65 Serrano, Á. 25 Severac, C. 13 O’Reilly, R. 2 Sevieri, M. 75 Sheikh-Yasin, K. 19 Pantos, D. 63, 74 Shvetsova, A. 60 Panzarasa, G. 69 Silva, F. 75 Parac-Vogt, T. 11 Since, M. 47 Pasco, M. 71 Sitia, L. 75 Peng, L. 64 Skov Pedersen, J. 77 Perets, E. 33 Sletfjerding, M. 44 Peterman, E. 46 Soldevila, A. 77 Peters, M. 25 Sorrentino, L. 75 Pizzi, A. 51 Sottani, C. 75 Popova, E. 54 Sterling, A. 23 Prosperi, D. 75 Stigliani, J. 47 Prévot, G. 8 Strada, A. 49–51 Pudney, C. 74 Strazewski, P. 60 Puente, A. 65 Synatschke, C. 14, 16, 30 Pujals, S. 3, 9 Pulido, D. 77 T. Ly, H. 11 Tikhomirova, V. 54 Qattan, D. 19 Tomsen-Melero, J. 77 TRON, T. 56 Raimundo, J. 26 Tronnet, A. 47 Rasadean, D. 74 Truffi, M. 75 Rauch, L. 30 Trushina, D. 55 Redondo, V. 21 Reithofer, M. 41 Ulrich, S. 40 Risgaard, N. 44 Rizzuto, M. 75 van den Boom, J. 59 Rosu, F. 71 van Oijen, A. 46 Rothenbuehler, S. 42 Vangrunderbeeck, S. 11 Rouvier, F. 26 Vax, A. 47 Roy, A. 71 Veciana, J. 77 Royes, J. 13 Vela, S. 65

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Ventosa, N. 77 Wilson, A. 67 Verget, J. 52 Wuite, G. 46 Verhaghe, P. 47 Veselov, M. 55 Xu, L. 46 Vialet, B. 39 Yan, E. 33 Voets, I. 9 Yoo, S. 71 Vogel, S. 44 Yu, H. 36 Voit, B. 68 Voskuhl, J. 59 Zhang, M. 44 Weil, T. 14, 16, 30 Zuber, B. 42

85 THANK YOU !