Systematic evolution of split aptamers by exponential enrichment from fragment libraries

• Applicant: Professorin Dr. Sabine Müller
• Subject Area: Biological and Biomimetic Chemistry ; Organic Molecular Chemistry - Synthesis and Characterisation
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545729536

Disease diagnosis and therapy require molecular recognition of target structures with high specificity. Nucleic acid folding motifs are at the forefront of the fight against new or newly discovered diseases, and the ease and speed with which such folding motifs can be identified makes the difference between failure and success. In vitro selection is the method of choice for the successful discovery of such folding motifs (aptamers). The selection of oligonucleotide fragments for the assembly of aptamers, rather than the selection of full-length strands, is a new and innovative concept, but a method that is not yet available. Only when dynamic covalent bonds are formed during fragment assembly on a target, can the required level of affinity and selectivity be locked in place. The development of such an on-target dynamic covalent assembly method is the aim of this research project. The chosen method exploits the unique properties of boronic acid and RNA in a synergistic manner. The intrinsic properties of boronate esters, based on the reversible covalent bond-forming interactions between boronic acids and diols in aqueous media, have led to a wide range of applications including RNA sensing, separation and purification, and siRNA delivery. Our consortium has already demonstrated that boronate esters, formed by the reaction of an oligonucleotide bearing a 5'-boronic acid moiety with the 3'-terminal cis-diol of another oligonucleotide, support the assembly of functional nucleic acid architectures. In particular, we have shown that templated formation of boronate esters is able to restore the activity of split DNA and RNA enzymes, as well as a split fluorescent light-up aptamer. The main objective of the SPLITEX project is to develop and demonstrate an innovative methodology for the direct selection of split systems from a library of short 5'-boronic acid modified oligonucleotides. The ease of reversible boronate ester formation makes it the chemistry of choice for the problem at hand: to aid the assembly of fragments with modest individual affinity and weld them into a covalent structure that binds with exceptional selectivity. The selection of split aptamers against new targets is the culmination of our project, which will provide essential and complementary elements to the selection approaches conventionally used, while allowing the use of shorter sequences with fewer negative charges. The success of this project depends on the contribution of both partners, combining expertise in the synthesis and characterisation of boronic acid-based dynamic assemblies (French partner) with expertise in RNA engineering and aptamer and ribozyme design (German partner).

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Professor Dr. Michael Smietana