Summary

Biomimetic “funnel” and “bowl” supramolecular systems for the selective recognition of DNA/RNA

The development of new molecular systems able to recognize specific portions of nucleic acid sequences is a subject of uttermost importance for the development of new drugs, biosensors or tools for basic biochemical and biological studies. Nature regulates the nucleic acid activity (e.g. transcription) with specific proteins. The affinity and selectivity of the recognition process by proteins relies on the interaction of well-structured small protein sequences with the DNA or RNA, mostly through the major groove. This interaction is supported by plethora of non-covalent interactions. On the other hand, the number of low molecular weight ligands that have been found to interact with the major groove is rather small. Such an underinvestigated niche offers a remarkable potential for development of novel molecular agents for DNA/RNA recognition.

We have, therefore, succesfully proposed to the Croatian Science Foundation, this project, based on the development of new biomimetic compounds capable of recognizing specific sequences of DNA or RNA, similarly as proteins do. Our systems have a calixarene- or resorcinarene-based scaffold of the right size, featuring a flexible “funnel” or more rigid “bowl” shape, respectively. These macrocycles are equipped at both extremities by functional groups such as imidazole, amines or ammonium ions attached through flexible linkers, to mimic the amino-acid residues (e.g. His, Lys, Arg) of a protein. They are also capable of efficient binding of a metal cation, which rigidifies the structure (Figure S1).

The project envisages following research activities:

• design and synthesis of the series od supramolecular biomimetic binders (SBBs),
• recognition and binding capacity studies of SBBs to various nucleic acids,
• structural characterization of the formed assemblies by single crystal X-ray crystallography, and
• in vitro bioassays of the interactions between our biomimetic binders and nucleic acids in an environment provided by the cell.

The project is structured as an iterative research process, where, based on the results of structural and functional studies obtained in the previous iteration, the structures of the binders shall be fine-tuned for the next iteration, in order to come up, at the end, with a set of promising supramolecular biomimetic binders (SBBs) capable of selective recognition of defined structural elements within the nucleic acid chains, and hence with the clear and imminent applicative potential in drug design and biosensor research (Figure S2).

Our SBBs are constructed by optimized procedures of grafting three (or four) nitrogen-containing (methylimidazole, pyridine or amine) coordination arms to one rim of the macrocycle. The opposite rim contains six (in the case of calix[6]arenes) or four (in the case of resorcinarenes) “legs” (C5-alkyl, tBu, amino, ammonium-containing, pyridinium-containing, OMe or their combinations) (Figure S1). These unmodified SBBs make up the generation Zero (genZ) set of the SBBs. In the course of the project, their “legs” will be modified by fluorophoric amino acids in the frame of creation of the first (gen1) and second generation (gen2) of SBBs. Fluorophoric amino acids to be grafted to the SBB macrocycles are very sensitive to the microenvironment of DNA/RNA binding site. Binding of multichromophoric SBB to any particular DNA or RNA sequence would depend on steric/interaction compatibility of binding site and SBB’s steric adaptability. Consequently, intrinsic relations between chromophores would be re-adjusted, reporting it back by spectrophotometric alterations. Newly prepared fluorophoric SBBs will be characterised in aqueous medium by UV/Vis, fluorescence spectrophotometry and circular dichroism spectropolarimetry (for chiral SBBs). For the recognition and binding studies between SBBs and DNA/RNA, standard thermal denaturation procedures for ds-DNA/RNA: viscometry measurements, polarized spectroscopy techniques and microcalorimetric (ITC, nanoDSC) experiments will be used. The targeted poly- or oligonucleotides (double stranded DNA) will be chosen according to the properties of their minor and major grooves. Interactions of our SBBs with single stranded (ss-) DNA and RNA will also be studied. Crystallization assays on the most promissing SBB-oligonucleotide complexes (hanging and sitting drop techniques) will be performed in combination with automated crystallization screenings. Data collections will be performed on the home single crystal diffractometer and at the synchrotron beamline (Elettra SF, ESRF Grenoble). X-ray structures will be solved by classical ab initio structure solution methods (MAD, MIR and SIR), based, when applicable, on the presence of metals in our SBBs (metal complexes). Alternatively, bromine and iodine atoms, routinely attachable to uracil, provide outstanding anomalous diffraction signals. Molecular replacement analyses in cases where the isomorphous structures are already deposited in the PDB/CSD will be employed.