Aptamer templated peptide ligation - on the way to synthtic ligases

• Applicant: Dr. Sebastian Pomplun
• Subject Area: Biological and Biomimetic Chemistry ; Biochemistry ; Organic Molecular Chemistry - Synthesis and Characterisation
• Term: from 2018 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 412851589

The need and interest for bioorthogonal reactions such as site specific conjugation of proteins or condensation of biomolecules, has been growing in recent years. These reactions are important tools in chemical biology, for example in the development of antibody-drug-conjugates, PEG-stabilized proteins or for the chemical synthesis of proteins. The constraint of operating in physiological conditions limits the scope of many organic reactions. Moreover, the selective labelling of specific amino acid residues by chemical strategies is hard to achieve, because more than one copy of the same residue is typically present in proteins, and also several other potentially reactive groups can interfere. There are enzymes which recognize specific peptide sequences and can help overcome this problem, but the artificial development of enzymes with new recognition specificities is not practical and in addition they usually have to be produced by expensive and time-consuming methods. Herein we propose a novel approach, which combines the advantages of chemical and enzymatical bioconjugation techniques: two reaction partners are designed to be unreactive under normal bioconjugation conditions, but their reaction with each other is triggered in the presence of a template, which brings them in close proximity, enhancing dramatically their local concentration. The reactants in this case are two peptides, carrying one at the C-terminus and the other at the N-terminus "semi-reactive" functionalities that do not react spontaneously with each other. The template is designed to recognize the reaction product, thus combining the two peptides to trigger the ligation of the two fragments. This template should be a molecule which can be designed by an easy and effective evolution process. Highly attractive candidates for such a templated approach are DNA or RNA aptamers. Aptamers are short ribo- and deoxyribonucleic acids, which have the potential to bind to virtually any target. They are synthesized chemically and in an in vitro evolution-enrichment-selection process they are selected for a specific target.In this proposal we show strategies for the identification of suitable semireactive peptides and we outline methods for the evolution of aptamer templates that trigger specifically the reaction between the two moieties. The potential use of this methodology for the synthesis of stable anticancer antibody-drug-conjugates is depicted in a final example. Since one of the peptide fragments can be co-expressed directly in the protein (or antibody), this technology represents a practical new bioconjugation method.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Bradley L. Pentelute