Methodology

Preparation of supramolecular biomimetic binders (SBBs)

Syntheses of our funnel and bowl SBBs, as well as their basic chemical characterizations will be conducted in the Reinaud lab (RL) at the Université de Paris by B. Colasson, O. Reinaud and a PhD student who will be employed in the frame of this project.

Synthetic procedures for dozens of promising candidate molecules, both funnel and bowls, are already optimized and many of them reported.¹ The project will require modification of candidate structures, mostly at the rim opposite to the imidazole arms, i.e. modification of the scaffold “legs“. These legs have been traditionally composed of hydrophobic tBu or C5 alkyl chains, and recently replaced by cationic hydrophilic legs in water soluble structures.² However, for the purpose of this project, we have envisaged grafting the fluorophoric amino acids to the calixarene/resorcinarene scaffolds of the zero generation, which will create the first generation of modified SBBs (gen1 SBBs) within the second project period, when the Reinaud group will be joined by the PhD student (Figure M1).

In the Piantanida group at the RBI, currently, we have a large library of unnatural amino acids (Figure M2). Unnatural fluorescent amino acids available are ideally suited for attachment to our SBBs and DNA/RNA targeting since they combine three essential characteristics: (i) by themselves show affinity toward nucleic acids, (ii) are excellent chromophores/fluorophores and (iii) most of them are water soluble.

Finally, the preparation of the gen2 SBBs will include directed chemical modifications of the gen1 SBBs in order to further enhance their ability to specifically recognize targeted portions of the DNA/RNA sequences.

Study of interactions SBB vs. DNA/RNA

Newly prepared SBBs will be characterised in aqueous medium by UV/Vis and fluorescence spectrophotometry, allowing fast and accurate determination of solubility, stability and aggregation properties, which is essential for subsequent study of their interactions with DNA/RNA targets. Based on the screening results, the crystallization setup will be planned to obtain suitable single crystals for X-ray diffraction analysis of the most promising SBB-oligonucleotide complexes. In parallel, when possible, NMR characterization in water will be performed.

Aside spectrophotometric methods mentioned above, for the recognition and binding studies between the selected set of SBBs and DNA/RNA we will use standard thermal denaturation procedures for ds-DNA/RNA,³ viscometry measurements,⁴ as well as various polarized spectroscopy methods.⁵ For the direct determination of thermodynamic parameters of binding, as well as for particular samples with inadequate spectrophotometric properties (colloid, turbid), we will use microcalorimetric experiments: ITC for titrations and nanoDSC for denaturation/renaturation experiments. When applicable, NMR and AFM techniques will be employed, for the most intriguing multichromophore SBB-DNA/RNA systems. The targeted poly- or oligonucleotides (double stranded DNA) will be chosen according to the properties of their minor groove (width, occupation by substituents),^6,7 which is directly related to the secondary structure of the double helix and partly to the basepair composition.

Interactions of our SBBs with single stranded (ss-) DNA and RNA will also be studied. When applicable, NMR and X-ray diffraction techniques will be employed using specifically synthesized short oligonucleotides, mostly for previously described systems (oligonucleotide-polynucleotide) which showed promising recognition.

Molecular modelling calculations are envisaged on selected combination of SBBs and nucleic acids in order to further elucidate the complex structures and the correlate it with the observed fluorimetric responses and binding affinities.

Biological assays

Our SBBs target DNA/RNA, which makes them potentially bioactive in living systems. In the scope of this project we have envisaged the study of chosen (based on methods described above) novel compounds in human cell lines. Firstly, antiproliferative activity will be screened by MTT test. Secondly, these SBBs will be studied for the cell uptake and intracellular distribution by CLSM, taking advantage of their fluorimetric properties upon binding to the target (previously characterised in isolated systems with only DNA/RNA target present). Particularly interesting will be determining the correlation between intracellular localisation and bioactivity, and also detailed characterisation of fluorescent derivatives which accumulate specifically in cell organelles with no cytotoxic effect (potential novel probes).

single crystal X-ray structural studies

Laboratory of the Chemical and Biological Crystallography (LCBC) at the RBI in Zagreb currently has an Oxford Diffraction Xcalibur Nova diffractometer equipped with the cryo-cooling system, suitable for macromolecular data collection studies, while the funding for the purchase of a diffractometer of the newest generation is secured and envisaged for 2021. In addition to the existing and secured home instrumentation for the data collection, crystallographic data will be, when needed, collected at the synchrotron facilities (Elettra Trieste and/or ESRF Grenoble).

The true bottleneck in the contemporary single crystal X-ray diffraction study is the production of the good quality single crystals. Despite all the progress made, to this day, to the best of our knowledge, there is no single structure in PDB accounting for the DNA/RNA complex with a calixarene or resorcinarene based binders, yet there is a number of reports dealing with this intriguing interaction.⁸ Crystallization assays (by means of classical hanging and sitting drop techniques) will be performed in the LCBC and conducted by A. Višnjevac and PhD student who will be employed as a part of this project scheme. When needed, crystallization screenings will be performed in the Gruber group in Graz, where the use of the automated equipment (crystallization robot) will be possible. X-ray diffraction patterns from oligonucleotide crystals will be analyzed, and structure solved by different phasing methods, depending on the characteristics of each sample, and their proneness to undergo chemical modifications needed (introduction of heavy atoms into the structure).

references

1. (a) N. Le Poul, Y. Le Mest, I. Jabin, O Reinaud, Acc. Chem. Res., 2015, 48, 2097–2106; (b) J.-N. Rebilly, B. Colasson, O. Bistri, D. Over, O. Reinaud, Chem. Soc. Rev. 2015, 44, 467-489; (c) R. Lavendomme, S. Zahim, G. De Leener, A. Inthasot, A. Mattiuzzi, M. Luhmer, O. Reinaud, I. Jabin, Asian J. Org. Chem. 2015, 4, 10–722.
2. (a)  O. Bistri and O. Reinaud, Org. Biomol. Chem., 2015, 13, 2849–2865; (b) S. Collin, N. Giraud, E. Dumont, O. Reinaud, Org. Chem. Front., 2019, 6, 1627–1636.
3. J. L. Mergny, L. Lacroix, Oligonucleotides 13 (2003) 515-537.
4. M. Wirth, O. Buchardt, T. Koch, P. E. Nielsen, B. Norden, J Am Chem Soc. 110  (1988) 932-939.
5. T. Šmidlehner, I. Piantanida, G. Pescitelli, Beil. J. Org. Chem. 14 (2018) 84–105.
6. C.R. Cantor and P.R. Schimmel, Biophysical Chemistry, vol. 3. WH Freeman and Co., San Francisco, 1980, 1109-1181.
7. W. D. Wilson, Y.-H. Wang, C. R. Krishnamoorthy, J. C. Smith, Biochemistry 24(1985) 3991–3999.
8. See, for example : (a) Y. Tauran et al Scientific Reports 8, 1226(2018) 1 – 8; (b) M. Giuliani, I. Morbioli, F. Sansone, A. Casnati, Chem. Commun. 51 (2015) 14140; (c) W. Hu, C. Blecking, M. Kralj, L. Šuman, I. Piantanida, T. Schrader, Chemistry, Eur. J., 18 (2012), 3589–3597.