Abstract

With our expertise in the chemical synthesis of constrained and/or functionalized nucleotides, we aim to finely tune the conformation of nucleic acids and expand their catalytic or fluorogenic recognition properties. 

Structural modulation of nucleic acids

The control of the α-ζ torsional angles of the sugar/phosphate backbone of nucleic acids can be achieved by introduction at selected position of the nucleic chain of preorganized dinucleotides steps (D-CNA: Dioxaphosphorinane Constrained Nucleic Acid) in which the torsional angles are stereocontrolled within a dioxaphophorinane ring.

In the α,β-D-CNA dinucleotides, an ethylene bridge connects one oxygen atom of the phosphate moiety P to the carbon C5' of the lower nucleotide in the P "R" and C5' "R" configurations locking the α and β angles into the canonical values of the B-DNA helix (Fig 1). Any other combinations of configurations of the P or C5' atoms lead to sets of values for torsional angles found into the non-canonical structures displayed by DNA secondary structures.

Obtained by a synthetic diverging approach, corresponding thio- and seleno- α,β-D-CNA derivatives along with additional α,β,γ-D-CNA members broaden the CNA family (Fig 2). α,β-P-CNAs (Phostone constrained Nucleic Acid) were also obtained by an intramolecular Arbuzov reaction. The structural studies demonstrated that a large chemical space is therefore covered (Fig 3).
 

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Fig 1. RX structure of a TT step from B-DNA (PDB 436D) (left) and the simulated structure of a α, β-D-CNA (right)


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Fig 2. Examples of synthetized D-CNAs

 

Fig 3. Superimposition of D-CNAs

The corresponding D-CNA phosphoramidites were incorporated by solid phase synthesis (SPS) into oligonucleotides (ODN) in order to evaluate:

  • the impact of a single D-CNA bearing a torsional angle α gauche(+) within hairpins or bulges on the stabilization of those DNA secondary structures bearing unpaired moieties.
  • the efficiency of both gauche(+) and (-) nucleotides during an antisens approach to modulate an allele selective silencing of the Huntingtin protein expression for the development of new therapeutics for Huntington's disease.
  • their ability to stabilize transient secondary structures such as 4-way Holliday junctions formed within the integron/integrase complex during a key step of the bacterial recombination process.

Nucleic acids functionalization

Depending on the projects (either the development of nucleic mimics of serine proteases or fluorogenic detections tools), convertible phosphoramidites (CvNs) bearing different linkers were synthetized from thymidine 5'-aldehyde (Scheme 1). After their efficient incorporation into precise positions along the ODN backbone, the introduction of the desired functionalities was achieved by click chemistry or nucleophilic substitution (Scheme 2). Moreover, the design and synthesis of functionalized phosphoramidites (FuNs) already bearing the suitably protected group compatible with the SPS process offer the possibility to introduce multiple modifications into a unique sequence. A combinatorial approach combining the convertible and functionalized strategy could lead to numerous DNA libraries with topological and chemical diversity.

nullScheme 1. Convertible and functionalized phosphoramidites

nullScheme 2. FuONs libraries

            1-Towards functionalized oligonucleotides as protease mimics

(Funded by ANR JCJC PrOLIFiC, starting date 01/2019)

The purpose is to design short oligonucleotides that could display catalytic activities by grafting at precise positions along the sugar-phosphodiester backbone chemical functions such as hydroxyl, imidazole and carboxylate groups. Those are reminiscent of the lateral chains of the three amino acids serine, histidine and aspartate that formed the catalytic triad of the serine proteases. The programmability and the reproducible folding of DNA could be used to incorporate at selected positions the desired functionality in order to mimic the active site of serine proteases and performing the cleavage of the amide bond.

Unlike other DNA catalysts developed in biomimetic chemistry, mostly based on the iconic double helix, we diverged by building a 3-way junction decorated at its core with three unpaired functionalized thymidines. 5'-C-Alkyne convertible phosphoramidites were incorporated by SPS then conjugated through CuAAC Click chemistry with the corresponding azides bearing the functional group of interest. ODN were then assembled in a stoichiometric way to access 3-way junctions in order to recreate an pseudo active site with a controlled topology (Scheme 3).

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Scheme 3. Click CuAAC synthesis of 5'-FuONs and their assembly into 3-way junctions

We now focus on a combinatorial approach to access librairies of DNA based-proteases presenting different secondary structures and linkers and assess their catalytic abilities to cleave an amide bond.

 

            2- Fluorogenic detection by ODN labelled by streptocyanines

In order to detect DNA secondary structures or small organic molecules, a method using streptocyanine-labelled ODN was developed, starting from non fluorescent hemicarboxonium salts and aminoalkyl-functionnalized ODN. Following the incorporation of a convertible 5'-bromopentenyle phosphoramidite, the resulting amino alkyl function formed after the final aminolysis attacks through a nucleophile substitution, a hemicarboxonium salt to give fluorescent ODN, further analyzed for their spectrophotometric properties (Scheme 4).

nullScheme 4. Fluorogenic detection

Functionalization and structuration of nucleic acids

Both approaches could be now combined towards the development of a new family of convertible and constrained nucleic acid (C2NA). Switching one of the pre-existing oxygens of the dioxophosphorinane ring by an atom of azote which could be further alkylated gives access to the newly synthetized oxaza-phosphorinane cycle while still maintaining constraints on the torsional angles of the sugar-phosphate backbone (Fig 4). The introduction of propargyl arm was not detrimental to the geometry of the C2NA as the values of α and β torsional angles were respected compared to their D-CNA analogues. Finally, the bioconjugaison of secondary structures such as hairpins with fluorescent dyes demonstrated that the propargyl group was still accessible to react through a click chemistry reaction with a fluorescent dye. This example highlighted the importance of the C2NA as those nucleotides could be needed when preorganization and functionalization may be needed at the same position.

nullFig 4. Left: α,β -C2NA nucleotides, right: superimposition of minimized structures.