Ligand-templated assembly of aptamers and ribozymes from short borono-based nucleic acids sequences

• Applicant: Professorin Dr. Sabine Müller
• Subject Area: Biological and Biomimetic Chemistry ; Biochemistry ; Organic Molecular Chemistry - Synthesis and Characterisation
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 316800023

• Final Report Year: 2021

Final Report Abstract

Over the past two decades, a large number of aptamers, DNAzymes and ribozymes with potential for biotechnological application have been developed and characterized. Although being very attractive tools, such architectures are not free of limitations. The number of negative charges present in the nucleic acid strands and the sometimes extended length limit their use. It is in this context that split architectures, consisting of two or more short nucleic acid strands, have emerged as attractive alternatives that are easier to synthesize and carry less negative charges per strand. These split architectures allow the design of new tools for the development of e.g. innovative biosensors. In this context, the use of architectures divided into a series of two or more independent and non-functional fragments, capable of selectively assembling in the presence of the cognate target is particularly attractive. The stability of the assembled structure can be further enhanced by decorating the individual strands with chemical functions that, upon folding, lead to covalent end-joining of the fragments. The assembly brings the two reactive functionalities into close proximity and thus allows, eventually by chemical activation, the formation of an additional natural or modified internucleosidic bond. In the current project, several split systems including DNAzyme 10-23, the hairpin ribozyme as well as a series of aptamers (FMN, Spinach and Mango) were designed and studied. The special feature of these split systems was the formation of a reversible boronic acid ester linkage at the split site between individual fragments, owing to functionalization of fragments with a 5'-boronic acid moiety and a 2',3'-cis diol. This way, the functional architecture was stabilized as compared to the non-modified control split structures and the functionality of the split system upon assembly was preserved. In conclusion, the potential of the boron chemistry for the controlled reversible assembly of functional nucleic acids has been clearly demonstrated. In addition to studying the functional assembly of the chosen split architectures, work in the project has led to the development of new oxazaborolidine-type internucleoside linkages, to novel support-based conjugation methods for incorporation of boronic acid functions in nucleic acid sequences, and to initial results on self-assembly properties of 5'-boronoribonucleosides as well as on enzymatic synthesis of cDNAs from 5'-boronylated RNA templates.

Publications

• RNA-based boronate internucleosidic linkages: an entry into reversible templated ligation and loop formation. Org Biomol Chem. 2018, 16, 8824-8830
  A. Gimenez-Molina, I. Barvik, S. Müller, J.-J. Vasseur, M. Smietana
  
• Selective chemical modification of DNA with boronic acids by on-column CuAAc reactions. Synthesis 2020, 52, 2962-2969
  M. Debiais, J.-J. Vasseur, S. Müller, M. Smietana
  
• Splitting Aptamers and Nucleic Acid Enzymes for the Development of Advanced Biosensors. Nucleic Acids Res. 2020, 48, 3400-3422
  M. Debiais, A. Lelievre, M. Smietana, S. Müller
  
• Boronic acid-mediated activity control of split 10-23 DNAzymes. Chem. Eur. J. 2021, 27, 1138-1144
  M. Debiais, A. Lelievre, J.-J. Vasseur, S. Müller, M. Smietana