Pharmacophore Nanoarrays on DNA Origami Substrates as a Single-Molecule Assay for Fragment-Based Drug Discovery.

The rational combination of techniques from the fields of nanotechnology, single molecule detection, and lead discovery could provide elegant solutions to enhance the throughput of drug screening. We have synthesized nanoarrays of small pharmacophores on DNA origami substrates that are displayed either as individual ligands or as fragment pairs and thereby reduced the feature size by several orders of magnitude, as compared with standard microarray techniques. Atomic force microscopy-based single-molecule detection allowed us to distinguish potent protein-ligand interactions from weak binders. Several independent binding events, that is, strong, weak, symmetric bidentate, and asymmetric bidentate binding are directly visualized and evaluated. We apply this method to the discovery of bidentate trypsin binders based on benzamidine paired with aromatic fragments. Pairing of benzamidine with the dye TAMRA results in tenfold enhancement of the trypsin binding yield.

[1]  T. Fournier,et al.  Alpha-1-acid glycoprotein. , 2000, Biochimica et biophysica acta.

[2]  David R. Liu,et al.  DNA-Templated Organic Synthesis and Selection of a Library of Macrocycles , 2004, Science.

[3]  Samu Melkko,et al.  On the magnitude of the chelate effect for the recognition of proteins by pharmacophores scaffolded by self-assembling oligonucleotides. , 2006, Chemistry & biology.

[4]  P. Rothemund.  Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[5]  Masahiko Hirota,et al.  The role of trypsin, trypsin inhibitor, and trypsin receptor in the onset and aggravation of pancreatitis , 2006, Journal of Gastroenterology.

[6]  Christoph E. Dumelin,et al.  Isolation of high-affinity trypsin inhibitors from a DNA-encoded chemical library. , 2007, Angewandte Chemie.

[7]  Hao Yan,et al.  Self-Assembled Water-Soluble Nucleic Acid Probe Tiles for Label-Free RNA Hybridization Assays , 2008, Science.

[8]  Hao Yan,et al.  Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding. , 2008, Nature nanotechnology.

[9]  Christoph E. Dumelin,et al.  Isolation of potent and specific trypsin inhibitors from a DNA-encoded chemical library. , 2010, Bioconjugate chemistry.

[10]  Chunhai Fan,et al.  A DNA-Origami chip platform for label-free SNP genotyping using toehold-mediated strand displacement. , 2010, Small.

[11]  David R. Liu,et al.  Small-molecule discovery from DNA-encoded chemical libraries. , 2011, Chemical Society reviews.

[12]  Masayuki Endo,et al.  Single-molecule analysis using DNA origami. , 2012, Angewandte Chemie.

[13]  Arivazhagan Rajendran,et al.  Einzelmolekülanalysen mithilfe von DNA‐Origami , 2012 .

[14]  Michael A. Stravs,et al.  Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library. , 2012, Chemistry.

[15]  K. Gothelf,et al.  Electron-induced damage of biotin studied in the gas phase and in the condensed phase at a single-molecule level , 2013 .

[16]  Y. Liu,et al.  Photoaffinity labeling of small-molecule-binding proteins by DNA-templated chemistry. , 2013, Angewandte Chemie.

[17]  Jürgen Popp,et al.  Single virus detection by means of atomic force microscopy in combination with advanced image analysis. , 2014, Journal of structural biology.

[18]  Adrian Keller,et al.  Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy , 2014, Molecules.

[19]  Dario Neri,et al.  DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries. , 2014, Accounts of Chemical Research.

[20]  Zitian Chen,et al.  Selection of DNA-encoded small molecule libraries against unmodified and non-immobilized protein targets. , 2014, Angewandte Chemie.

[21]  Masayuki Endo,et al.  State-of-the-art high-speed atomic force microscopy for investigation of single-molecular dynamics of proteins. , 2014, Chemical reviews.

[22]  V. Uzunova,et al.  Characterization of DNA-conjugated compounds using a regenerable chip. , 2015, Analytical chemistry.

[23]  Nicolas Winssinger,et al.  Identifizierung von niedermolekularen kovalenten Bromodomäne‐Bindern aus einer DNA‐kodierten Bibliothek , 2015 .

[24]  Nicolas Winssinger,et al.  Identification of Covalent Bromodomain Binders through DNA Display of Small Molecules. , 2015, Angewandte Chemie.

[25]  C. Zambaldo,et al.  PNA-encoded chemical libraries. , 2015, Current opinion in chemical biology.

[26]  F. Reddavide,et al.  DNA-Encoded Dynamic Combinatorial Chemical Libraries. , 2015, Angewandte Chemie.

[27]  J. Daguer,et al.  DNA display of fragment pairs as a tool for the discovery of novel biologically active small molecules† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc01654h , 2014, Chemical science.

[28]  Z. Li,et al.  Nanoscale patterning of self-assembled monolayers using DNA nanostructure templates. , 2016, Chemical communications.

[29]  J. Daguer,et al.  Novel PTP1B inhibitors identified by DNA display of fragment pairs. , 2016, Bioorganic & medicinal chemistry letters.

[30]  Georg Krainer,et al.  Structural stability of DNA origami nanostructures in the presence of chaotropic agents. , 2016, Nanoscale.

[31]  W. Chiu,et al.  Designer nanoscale DNA assemblies programmed from the top down , 2016, Science.

[32]  Casey Grun,et al.  Programmable self-assembly of three-dimensional nanostructures from 104 unique components , 2017, Nature.

[33]  Hendrik Dietz,et al.  Biotechnological mass production of DNA origami , 2017, Nature.

[34]  Maximilian T. Strauss,et al.  Quantifying absolute addressability in DNA origami with molecular resolution , 2018, Nature Communications.