Tamara Šmidlehner’s research while affiliated with Rudjer Boskovic Institute and other places

Two novel isosteric conjugates of guanidiniocarbonyl-pyrrole and 6-bromo-TO (thiazole orange) were prepared, differing only in linker connectivity to cyanine (benzothiazole nitrogen vs. quinoline nitrogen). The quinoline analog was significantly more susceptible to aggregation in an aqueous medium, which resulted in induced circular dichroism (ICD; λ = 450-550 nm) recognition between A-T(U) and G-C basepair containing polynucleotides. The benzothiazole-isostere showed pronounced (four-fold) fluorimetric selectivity toward ds-RNA in comparison to any ds-DNA, at variance to its quinoline-analogue fluorescence being weakly selective to GC-DNA. Preliminary screening on human tumor and normal lung cell lines showed that both dyes very efficiently enter living cells and accumulate in mitochondria, causing moderate cytotoxic effects, and thus could be considered as lead compounds toward novel theragnostic mitochondrial dyes.

The core of the tau fibrils in Alzheimer disease is a hexapeptide sequence organized in paired helical filaments (PHF). This sequence AcPHF6 can be used as tau fibrils model for the fast screening of potential therapeutic inhibitors of fibril formation or their disruption. The assay is usually performed by monitoring the fluorescence increase of Thioflavin T (ThT), well-known reporter dye for fibrillation. However, the ThT assay is not faultless, and here we present novel fluorescent dye, cyanine attached to amino acid side-chain (Cy-aa) that shows several advantages over ThT. The fibrillation kinetics of AcPHF6 was monitored via Cy-aa at twenty times lower concentration compared to ThT and successfully reported the presence of fibrillation inhibitor by Cy-aa fluorescence decrease. Additionally, spectral properties of Cy-aa are red-shifted in comparison to ThT allowing screening of a wider range of potential fibrillation inhibitors. Moreover, in the mixture with the pre-formed fibrils, Cy-aa shows strong fluorescence light-up proportional to fibrils concentration. We also successfully coupled this fluorescent amino acid to PHF in order to completely avoid the possibility of dye displacement with screening compound, and this newly designed conjugate showed to be a reliable intrinsic fluorescent probe for monitoring fibrillation kinetics of amyloid peptides.

Study of seven new guanidiniocarbonylpyrrole (GCP)–fluorophore conjugates interactions with dipeptidyl peptidase III (DPP III) showed that all compounds bind strongly (Ks ≈ µM) to enzyme active site, but with very different fluorimetric response (varying from quenching to strong increase), dependent on the fluorophore type and intramolecular pre-organisation of molecule. Positively charged lysine side chain improved significantly compound solubility but diminished fluorescence increase upon DPP III binding and completely abolished inhibitory effect on DPP III activity, whereas linker-neutral analogues showed stronger emission increase and were efficient enzyme inhibitors. By far the best fluorimetric response and inhibitive properties showed cyanine–GCP analogue, thus being promising lead compound for both enzyme sensing and bio-activity inhibiting (theragnostic) studies of DPP III in the future. Communicated by Ramaswamy H. Sarma

Small molecule spectrophotometric probes for DNA/RNA and proteins are of the utmost importance for diagnostics in biochemical and biomedical research. Both, naturally occurring and synthetic probes, often include peptide sequence responsible for the selectivity toward the particular target; however, commercially available dyes are restricted to single point attachment to the peptide (having one reactive group). Here presented are our recent advances in the development of novel amino acid-fluorophore probes, with the unique characteristic of free N- and C-terminus available for incorporation at any peptide backbone position. Intriguingly, already monomeric amino acid-fluorophores showed recognition among various DNA/RNA, whereby steric impact and contribution of halogens is systematically studied. Moreover, some dyes revealed intracellular mitochondria specificity. Further, several hetero-dimeric chromophore systems were prepared, demonstrating that synergistic effect can lead to simultaneous DNA, RNA and protein fluorimetric recognition, combined with enzyme inhibition. Also, homodimeric cyanines equipped with chlorine revealed intriguing DNA/RNA selectivity in respect to well-known parent TOTO and YOYO dyes.

Here designed and prepared cyanine-guanidinocarbonyl-pyrrole conjugate (Cy-GCP), intrinsically non-fluorescent, revealed fluorescent switch-on recognition of various secondary structures of ds-DNA and ds-RNA. Moreover, at the same submicromolar concentrations, DNA/RNA recognition was observed by selective induced (I)CD pattern in the visible range. Preliminary results showed that Cy-GCP strongly interacted with DPP III enzyme, switching-on the fluorescence upon binding and inhibiting enzyme action with efficiency comparable to the best-known inhibitor tynorphin. Graphical abstract Open image in new window

Two novel conjugates of pyrene and cyanine were constructed by linking them with the rigid triazole-peptide linker. New probes bind very strongly (with 0.1 µM affinity) to both, ds-DNA(RNA) and proteins (BSA), giving significantly different fluorimetric response: strong pyrene emission change highly selective for protein and “switch-on” of cyanine fluorescence highly selective for DNA(RNA). Moreover, new probes yield induced CD bands only with DNA/RNA, but not with BSA, which allowed independent check of DNA presence in DNA/protein mixtures. Furthermore, probes contain FRET pair of chromophores, whereby FRET is silent in a free molecule solution and activated by binding small molecule to biomacromolecular target. The efficiency of the FRET is to some extent related to the secondary structure of DNA/RNA and only for one of the probes FRET is activated in protein. Two probes show distinctively different induced CD pattern in 400-600 nm range (attributed to a different position of linker attachment on cyanine core), allowing differentiation between various secondary structures of DNA or RNA, showed to be additionally enhanced by combining pyrene and cyanine in one molecule. Due to low cytotoxicity and efficient cellular uptake, probes are good candidates for further biological studies.

By a simple click-CuAAC procedure several cyanine dye analogues were attached to the amino acid side chain, yielding chromophore-amino acid conjugates, with potential of fluorescence upon binding to target. Due to the amino acid C- and N-termini available for peptide coupling, these conjugates are suitable for easy incorporation into a peptide backbone. Moreover, "click" procedures also allow post-peptide synthesis attachment of the dye. Novel amino acid dyes, although intrinsically non-fluorescent, give rise to strong fluorimetric response upon binding to double-stranded (ds)-DNAs or ds-RNA, whereby selectivity in emission response to various polynucleotide secondary structures is controlled either by linker length or halogen atom located on the cyanine part of the molecule. Molecular modeling confirmed the mode of binding to different polynucleotides, which was responsible for recognition. Interestingly, cell localisation experiments show that the dyes were specifically localised in mitochondria at variance with the localisation of parent dyes, which accumulate in cell nuclei, pointing out that the amino acid tail (containing triazole ring) might function as a novel mitochondria-directing appendage

The structural characterization of non-covalent complexes between nucleic acids and small molecules (ligands) is of a paramount significance to bioorganic research. Highly informative methods about nucleic acid/ligand complexes such as single crystal X-ray diffraction or NMR spectroscopy cannot be performed under biologically compatible conditions and are extensively time consuming. Therefore, in search for faster methods which can be applied to conditions that are at least similar to the naturally occurring ones, a set of polarization spectroscopy methods has shown highly promising results. Electronic circular dichroism (ECD) is the most commonly used method for the characterization of the helical structure of DNA and RNA and their complexes with ligands. Less common but complementary to ECD, is flow-oriented linear dichroism (LD). Other methods such as vibrational CD (VCD) and emission-based methods (FDCD, CPL), can also be used for suitable samples. Despite the popularity of polarization spectroscopy in biophysics, aside several highly focused reviews on the application of these methods to DNA/RNA research, there is no systematic tutorial covering all mentioned methods as a tool for the characterization of adducts between nucleic acids and small ligands. This tutorial aims to help researchers entering the research field to organize experiments accurately and to interpret the obtained data reliably.

One-pot tandem synthesis was for the first time applied to attach fluorophore to the amino acid side chain, yielding amino acid ready for peptide coupling at the N-terminus, and also upon activation at the C-terminus. Two new compounds differing only in fluorophore-linker length showed exceptional fluorimetric and CD recognition between DS-RNA and DS-DNA, thus being promising beacons for versatile peptide incorporation.

Monomethine cyanine dyes are well known molecular probes with a great increase in fluorescence efficiency upon binding to nucleic acid while nonfluorescent in the free form. Here are presented two cyanine-amino acid conjugates, synthesized by a Copper-catalysed azide-alkyne cycloaddition (CuAAC) method with copper wire as a catalyst source. Novel compounds differ in only one substituent on cyanine part (chlorine and fluorine substituent) and are suitable for peptide incorporation on both C- and N-end. Circular dichroism (CD) titrations revealed that both compounds cause strong distortion of DNA backbone (decrease of the band between 240-290 nm) combined with pronounced induced CD (ICD) band in the range where only cyanine dye absorbs (420-550 nm). While dye with chlorine atom, 2a, intercalates (negative ICD signal) between alternating GC-DNA base pairs, fluorine analogue 2b aggregates along GC-DNA backbone (bisignate ICD bands) in P-configured helicity. Both dyes bind into the minor groove of the alternating AT-DNA sequence. Recognition of AU-RNA sequence based on one-atom-difference of dyes is observed with fluorimetric titration and thermal experiments. 2b shows doubly enhanced fluorescence emission with pApU and moderate thermal stabilization, at variance to 2a, which does not show any stabilization of the same polynucleotide. Presented fluorescent amino acids show recognition of secondary structure of DNA and RNA based on one-atom-difference and in future prospects should be easily incorporated into DNA/RNA targeting peptides.