Modulation of Integrase Activity by Means of Conformationaly Constrained Nucleotides – MIAM CoCoNuts

This project relies on a heavy synthetic work for the preparation of the constrained nucleotide units and their incorporation within nucleic acids strands at defined position in order to modulate the geometry of the secondary structure. The synthetic pathway is a multi-step process and the modified nucleic acid strands will be prepared by automated synthesis that requires rather large amounts of each modified units. Finally, it will be necessary to determine the ability of the protein to interact with the modified substrate. The last achievement would be to determine a 3D structure of the complex by mean of X-ray diffraction analysis.

Alpha,beta-dioxaphosphorinnane CNA phosphoramidites featuring either canonical or non-canonical constrains have been synthesized at a scale allowing their use in automated DNA synthesis to prepare two modified Holiday junction arms in a ten-micromole scale. Each of them features a define and different geometry allowing the construction of preorganized structures useful for protein interaction studies. Moreover, we have developed new thiodioxaphosphorine analogues of alpha, beta, gamma-CNA and incorporated them within nucleic acids secondary structures.
Int4 integrase has been produced and purified and will be used in complex formation. First pairing assays of Wt Holiday junction with 3' or 5' extra bases were successful. They can be produced at large scale and combined for crystallography studies.

At this stage, our perspectives are those proposed earlier, the determination of the integrase ability to recognise its substrate in a conformational state fixed and thanks to crystallography study of this transient state to be able to design new inhibitors of this key enzyme.

1. Dioxaphosphorinane- Constrained nucleic acid Dinucleotides as tools for Sructural Tuning of Nucleic acids by D. A. Catana, B-L. Renard, M. Maturano, C. Payrastre, N. Tarrat, J.-M. Escudier, J. Nucleic Acids, 2012, 2012, 1-17.
2. Differential Effects on Allele Selective Silencing of Mutant Huntingtin By Two Stereoisomers of a,ß-Constrained Nucleic Acid. M. Østergaard, B. Gerland, J-M Escudier, E. E. Swayze, P. P. Seth ACS Chem Biol. 2014, 9, 1975-1979.
3. Stabilization of hairpins and bulged secondary structures of nucleic acids by single incorporation of a,ß-D-CNA featuring a gauche(+) alpha torsional angle, B. Gerland, P. Millard, C. Dupouy, B.L. Renard and J.-M. Escudier, RSC Advances, 2014, 4(90), 48821-48826.

Synthesis of conformationaly constrained dinucleotides building blocks by introduction of a six-membered dioxaphosphorinane or phostone ring (D- or P-CNA: Dioxaphosphorinane- or Phostone- Constrained Nucleic Acids) should allow to propose structural units in which the geometrical parameters of the sugar/phosphate backbone of nucleic acids will be controlled and locked into canonical values, or not, of the double helical standard structure. Constrains along the sugar/phosphate backbone are introduced on all the torsional angles at preselected positions and will provide access to a tool box of sixteen different building units. We already have available diastereoisomeric alpha, beta constrained thymidine dinucleotides units defined as B-type mimics or able to stabilize unpaired region of hairpin structures. In that context, from the chemist’s point of view, this project aims first to prepare existing CNA structures with all different base combinations, second to synthesize new CNA in 2'-deoxyribo (or ribo) series with epsilon and dzeta torsional angles constrained to B-type canonical values or not and finally to develop this approach towards the preorganization of DNA structures. The concept is to try to translate the entropic benefits obtained by the chemist by synthesising "pre-structured" dinucleotides to the modulation of protein/DNA complexes formation and activity.
The Holliday junction is a key constrained DNA structure that exists in nature with 4 double stranded arms connected at a geometric center. This architecture which is central to DNA recombination and repair is an ideal biological target for demonstrating the potential of CNAs to modulate protein activity. The integron integrase produce this structure which then awaits the replication machinery for resolution into integrated products. We plan to monitor the impact of the geometric constraints introduced by –NN– steps at the central cross-over region on the recombinase activity by biochemical and x-ray crystallographic approaches. This will be a first insight into pre-transition state recognition and processing by recombination enzymes.