Research

  • Fluorescent Probes/Sensors

Fluorescent small molecules are commonly used as labels or probes in modern bioresearch. Efficient in vivo sensing or imaging relies to a great extent on the physicochemical properties of the chromophore used. Depending on the nature of the biological process to be visualized or the molecular species that needs to be sensed it is possible to choose among a large variety of fluorescent probes that exhibit specific structural and spectroscopic properties. Most suitable labels for in vivo applications are biocompatible, stable and soluble in relevant media or fluids, and also conveniently excitable (>500nm to avoid auto-fluorescence of biological tissues and fluids). Ideally, they will also be very bright (high fluorescent quantum yield) under relevant conditions. Within the wide selection of fluorescent dyes available, those belonging to the Cyanine dye (Cy) and Acridine families have been developed extensively because of their biocompatibility and high molar absorptivity. They also offer the advantage of covering a very broad spectral range, from blue to near infra-red.

In our lab, we are designing and engineering original fluorescent (or fluorogenic) small molecules that are then being used as chemical probes for cellular imaging, pH and DNA sensing, among other applications.

References:

  1. T. Mahmood, Y. Wu, D. Loriot, M. Kuimova, S. Ladame. Chem. Eur. J., 2012, DOI: 10.1002/chem.201200802. Closing the Ring to Bring Up the Light: Synthesis of a Hexacyclic Acridinium Cyanine Dye.
  2.  K. Meguellati, M. Spichty, S. Ladame. Mol. BioSyst.,2010, 6, 1694-1699. Synthesis, Spectroscopic and DNA Alkylating Properties of Malondialdehyde (MDA) bis-Imine Fluorescent Adducts.
  3. T. Mahmood, A. Paul, S. Ladame. J. Org. Chem., 2010, 75, 204-207. Synthesis and spectroscopic and DNA-binding properties of fluorogenic acridine-containing cyanine dyes.
  4. K. Meguellati, M. Spichty, S. Ladame. Org. Letters, 2009, 11, 1123-1126. Reversible Synthesis and Characterization of Dynamic Imino Analogues of Trimethine and Pentamethine Cyanine Dyes.
  • Oligonucleotide Templated Reactions (OTRs)

In recent years, copying nature has become a trendy approach, one that many scientists have taken in their research. When it comes to find efficient ways to synthesise molecules, turning to Nature for inspiration is often a sensible and rewarding approach. Using DNA as a template to increase the efficiency of chemical reactions was first applied to the synthesis of oligonucleotide or oligonucleotide analogues and has notably given rise to the concept of PCR. Since then, a large diversity of OTRs have been developed that go well beyond the formation of phosphodiester bonds (as in oligonucleotides) and range from the Staudinger reaction to the reactions of carbon-carbon bond formation (e.g. Henry, Michael, Wittig…). But how far can we push the boundaries of OTRs?

In our lab, we have developed Peptide Nucleic Acid (PNA) fluorogenic probes that can be used for sensing nucleic acids in a sequence and/or structure-specific manner. The system is designed so that the appearance of a characteristic fluorescent signal (resulting from an oligonucleotide-templated reaction of cyanine dye formation) is directly associated to the presence of the oligonucleotide of interest.

References:

  1. G. Koripelly, K. Meguellati, S. Ladame. Bioconjugate Chem., 2010, 21, 2103-2109. Dual Sensing of Hairpin and Quadruplex DNA Structures Using Multicolored Peptide Nucleic Acid Fluorescent Probes.
  2. K. Meguellati, G. Koripelly, S. Ladame. Angew. Chem. Int. Ed., 2010, 49, 2738-2742. DNA-Templated Synthesis of Trimethine Cyanine Dyes: A Versatile Fluorogenic reaction for Sensing G-Quadruplex Formation.
  • G-quadruplex Nucleic Acids

G-rich nucleic acid sequences have a capacity to form highly stable four-stranded structures (termed quadruplexes) in vitro in the presence of physiological cations, notably K+ and Na+. The formation of intramolecular DNA G-quadruplexes at the end of telomeres or in the promoter region of genes is now well-documented in the literature and these DNA secondary structures have been validated as very attractive therapeutic (e.g. anti-cancer) targets. Extensive work has been carried out to specifically target such structures and few ligands are now available that strongly discriminate between quadruplex and duplex DNA and that also show biological activity in vivo. Surprisingly, RNA quadruplexes have received so far much less attention than their DNA analogues although there is now growing evidences that RNA quadruplexes are involved in key biological processes. Moreover, RNA quadruplexes are also more likely to form in vivo than their DNA analogues given the evidence that RNA is single stranded and thus does not necessarily need to compete with complementary strands to fold into a quadruplex.

As part of our studies, we are developing original tools (e.g. fluorescent probes) for investigating the formation of quadruplex structures in non-coding areas of the genome at the DNA and RNA level, both in vitro and in the context of a cell. We are also interested in the molecular recognition of these structures by engineered (or naturally occurring) proteins and small molecules in order to (1) better understand the biological function(s) of DNA and RNA quadruplexes in vivo and (2) validate these structures as suitable targets for chemical intervention with synthetic ligands.

References:

  1. A. Paul, P. Sengupta, Y. Krishnan, S. Ladame. Chem. Eur. J., 2008, 14, 8682-8689. Combining G-Quadruplex Targeting Motifs on a Single Peptide Nucleic Acid Scaffold: A Hybrid (3+1) PNA-DNA Bimolecular Quadruplex.
  2. V.A. Soldatenkov, A.A. Vetcher, T. Duka, S. Ladame. ACS Chem. Biol., 2008, 3, 214-219. First Evidence of a functional interaction between DNA quadruplexes and poly(ADP-ribose) polymerase-1.
  • Cellular uptake of Peptide Nucleic Acids

It has been a major goal within biological science to achieve controlled intervention and manipulation of cellular events by strategies that target nucleic acids and control gene expression. Synthetic oligonucleotides were first developed to inhibit RNA translation via either formation of a steric block or via RNase H-mediated cleavage of the duplex formed. However, the efficacy of these oligonucleotides in vivo is limited by their rather poor cellular uptake and their metabolic instability. In order to overcome these intrinsic limitations, uncharged oligonucleotide analogues with improved pharmacological properties have been developed that include Peptide Nucleic Acids (PNAs). Although these neutral analogues proved metabolically stable in cells and tissues, while retaining a very good affinity and sequence-specificity for their RNA target, they do not penetrate cells more easily than negatively charged oligonucleotides.

As part of our studies, we are exploring new ways to efficiently deliver PNAs inside a target cell. Improving the cellular uptake of this class of compounds could indeed see the development of nucleic acid-based therapeutics flourish over the coming years.