Protease-like Oligonucleotides, Functionalization and Catalysis – PrOLIFiC

FuON librairies could be obtained following the phosphoramidite approach by positioning at precise coordinates up to three modifications (alcohol/imidazole/carboxylate) along the FuON backbone. After the incorporation of 5'-convertible nucleotides after solid phase synthesis, the resulting FuON are conjugated. If three modifications on only one FuON are needed, the functionnalized approach based on already 5'-modifed phosphoramidites is used.
FuON are then organized into unpaired secondary structures such as bulges by hybridation with a complementary strand. Those complementary strand could be modified by a chromogenic ester or amide substrate to bring in close proximity the two partners to evaluate the FuON amidolytic properties.

Using the convertible approach, a tripod already bearing the three modifications is conjugated to the FuON via ‘click chemistry’.
The FuON are then structured following two strategies, first with an umodified complementary strand to screen for different topologies or with a modified one. In order to increase the effective molarity, as the FuON do not present an artificial binding pocket for the peptid substarte, a covalent approach was envisaged to bring all components in close proximity, one FuON bearing the tripode and a second one bearing a chromogenic amide substrate.

In order to increase the chemical diversity of FuON to screen for a functional catalyst, the convertible and the functionnalized approach are combined to developp a new family of bulged shaped DNA catalysts.

Using argininamide-DNA aptamers as a starting point, thymidines could be switched to 5'-convertible nucleotides to test the ability of those functionalized aptazymes to hydrolyse arginine-derived amides. In particular, using the convertible approach the tripod could be introduced to evaluate its impact.

The catalytic evaluation of each synthetized and shaped FuON is in progress. The incremental step is the introduction of such catalytic FuON into 3D tetraedrons, nanostructures well fitted for in vivo biological applications.

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This project aims at the rational design of functionalized oligonucleotides (FuON) covalently modified to expand their catalytic repertoire towards biomimetic reactions to give access to protease-like DNA catalysts. Those nucleic mimics would be designed to perform the hydrolysis of the amide bond which is realised in Nature by the serine proteases thanks to a catalytic triad of three cooperative amino acids, serine (Ser), histidine (His) and aspartate (Asp). Libraries of protease-like DNA catalysts could be obtained by the phosphoramadite approach by positioning amino acid side chains-like residues at precise coordinates along the oligonucleotide backbone. FuON would then be folded into different secondary structures thank to the perfectly controlled folding of DNA with the goal of reproducing the active site of the serine proteases. A toolbox of convertible or already functionalized phosphoramidites with different linkers (triazole, amide or amino) at the 5'-position bearing suitably protected alcohol, imidazole and carboxylate functions for (Ser), (His), (Asp) respectively, could be used to expand the diversity of the DNA catalysts libraries. With careful design sequence, FuON could be folded into different flexible secondary structures (bulge, hairpin, 3-way junction…) by the use of a pertinent modified (or not) complementary strand. The combinations of functionalized nucleotide phosphoramidites coupled to the choice of secondary structures would lead to heterogeneous libraries with skeletal and topological variety increasing the chances to screen for a functional catalyst. The catalytic properties of those DNA catalysts will then be evaluated on chromogenic ester or amide substrates. The most promising 1D DNA mimics will then be self-assembled in nucleic networks thanks to DNA hybridization properties. The organization into the 2D DNA lattices could increase the numbers of catalytic sites and the protease-like catalysts could also be organized in 3D nanostructures like DNA tetrahedrons. This is a shift of paradigm with DNA architecture use as it is the only one functionalized scaffold able to organize at the local and supramolecular level the multiple catalytic triads to achieve hydrolysis of the amide bond.