Ein modularer Ansatz zur Selektion chemisch modifizierter Aptamere für bisher nicht zugängliche Zielstrukturen

• Antragsteller: Professor Dr. Günter Mayer
• Fachliche Zuordnung: Biologische und Biomimetische Chemie
• Förderung: Förderung von 2011 bis 2020
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 207227604

• Erstellungsjahr: 2019

Zusammenfassung der Projektergebnisse

We developed a versatile approach to generate nucleobase-modified aptamers, so called clickmers. Chemically modified DNA libraries are generated by a click chemistry reaction, using C5-ethynyl-deoxyuridine modified DNA and a specific azide. We showed that many azides can be used, e.g., indole or benzyl and are compatible with the PCR step of an in vitro selection scheme. We dubbed the process click-SELEX and its features makes it a modular and broadly applicable procedure to generate modified aptamers. Using this method, we explored a variety of different modifications and showed their principle compatibility with the selection process. The modularity by which modifications can be chosen enables a receptorguided design (RGD) strategy of modified DNA libraries, tailored towards specific recognition elements. In a proof-of-concept study we have demonstrated utility of the RGD approach in generating clickmers binding to D9-tetrahydrocannabinol (THC), a difficult target molecule which was not compatible for SELEX using canonical DNA. Within the course of the project, we demonstrate the click-SELEX approach being suitable to generate clickmers binding to small molecules (THC), proteins (C3-GFP, streptavidin, CXCL9) and cells (HEK293). We developed a generic method allowing access to nucleobase-modified DNA aptamers. As this approach does not require extensive synthesis of nucleotide triphosphates or phosphoramidites for DNA library generation it can be applied by many researchers. It allows access to aptamers for difficult target molecules for which canonical building blocks do not provide a solution. The interaction properties of clickmers can exceed those of naïve DNA in terms of slower off-rates and higher overall affinities.

Projektbezogene Publikationen (Auswahl)

• Click reaction on solid phase enables high fidelity synthesis of nucleobase-modified DNA. Bioconjug Chem. 2016 Feb 5
  Mayer G, Tolle F, Pfeiffer F, Rosenthal M
  
• Preparation of SELEX Samples for Next-Generation Sequencing. Methods Mol Biol. 2016; 1380:77-84
  Tolle F, Mayer G
  
• Customised nucleic acid libraries for enhanced aptamer selection and performance. Curr Opin Biotechnol. 2017 Dec; 48:111-118
  Pfeiffer F, Rosenthal M, Siegl J, Ewers J, Mayer G
  
• Identification and characterization of nucleobase-modified aptamers by click-SELEX. Nat. Protocols. 2018 May; 13(5):1153-1180
  Pfeiffer F, Tolle F, Rosenthal M, Brändle G, Ewers J, Mayer G
  
• A receptor‐guided design strategy for ligand identification. Angew Chem Int Ed. 2019 May 03
  Rosenthal M, Pfeiffer F, Mayer G
  
• Copper-Catalyzed Alkyne-Azide Cycloaddition on the Solid Phase for the Preparation of Fully Click-Modified Nucleic Acids. Methods Mol Biol. 2019; 1973:177-183
  Rosenthal M, Pfeiffer F, Mayer G