Sylvain Ladame

DNA‐Templated Synthesis of Trimethine Cyanine Dyes: A Versatile Fluorogenic Reaction for Sensing G‐Quadruplex Formation

It has been known for several decades that G-rich nucleic acid sequences have a propensity to fold into highly stable four-stranded structures in vitro in the presence of physiological cations, notably K and Na. Such structures, termed quadruplexes, have had their biological significance demonstrated for a number of processes. For example, it has been shown that the single-stranded 3’-end of telomeric DNA could adopt a quadruplex conformation under near physiological conditions, which has implications on telomere maintenance mechanisms. More recently, a number of DNA Gquadruplex sequences have been identified in the promoter region of genes that have been proposed to act as regulatory elements for gene expression at the transcriptional level. Amongst the 43% of human genes that contain a putative quadruplex forming sequence in their promoter, specific oncogenes have received particular attention. These include the c-myc, bcl-2, K-ras, and c-kit genes. Although there exist increasing evidences for the formation of Gquadruplexes at telomere ends in vivo, the possible existence of promoter quadruplexes in vivo is still subject to debate. Recent studies using small molecule approaches have demonstrated that quadruplex formation within the nuclease hypersensitive element of the c-myc gene or within the promoter of the c-kit gene upstream were coupled to a significant inhibition of c-myc and c-kit expression at the transcriptional level in various cell lines. However, whilst the 3’-overhang of telomeric DNA is single-stranded, and therefore is free to adopt any stable secondary structure, quadruplex formation within a promoter would require at least a local and temporary opening of the DNA double-helix, despite the high stability of Watson-Crick G-C base pairs. Recent studies using fluorescence resonance energy transfer (FRET) or fluorescent probes have demonstrated that quadruplexes could potentially form, even when in competition with a thermodynamically more stable duplex form. Moreover, it is well established that doublestranded DNA transiently becomes single-stranded during key biological processes like DNA replication, transcription or even recombination, thus allowing the folding of each DNA strand into alternative (i.e. non B-DNA) structures. Herein, we were interested in designing sensitive fluorescent biosensors that would be highly specific for unique G-quadruplexes in the genome. The general strategy consists in targeting simultaneously the quadruplex structure itself but also its two flanking regions in a sequence specific manner. Briefly, two short peptide nucleic acids (PNAs) complementary to both quadruplex flanking regions are functionalised with two non-fluorescent components A and B of a fluorogenic reaction (i.e. reaction between non-fluorescent derivatives A with B leads to the formation of fluorescent entity C). The system can be designed in such a way that, upon hybridization of the PNA probes to their complementary DNA sequences through Watson-Crick base pairing, A and B will be in close enough proximity to react with each other when the DNA sequence between both PNAs is folded into a quadruplex structure only, while they will be kept separated if the DNA remains singlestranded (Figure 1).

Recognition and discrimination of DNA quadruplexes by acridine-peptide conjugates

We have explored a series of trisubstituted acridine-peptide conjugates for their ability to recognize and discriminate between DNA quadruplexes derived from the human telomere, and the c-kit and N-ras proto-oncogenes. Quadruplex affinity was measured as the peptide sequences were varied, together with their substitution position on the acridine, and the identity of the C-terminus (acid or amide). Surface plasmon resonance measurements revealed that all compounds bound to the human telomeric quadruplex with sub-micromolar affinity. Docking calculations from molecular modelling studies were used to model the effects of substituent orientation and peptide sequence. Modelling and experiment were in agreement that placement of the peptide over the face of the acridine is detrimental to binding affinity. The highest degrees of selectivity were observed towards the N-ras quadruplex by compounds capable of forming simultaneous contacts with their acridine and peptide moieties. The ligands that bound best displayed quadruplex affinities in the 1-5 nM range and at least 10-fold discrimination between the quadruplexes studied.

Combining G-Quadruplex Targeting Motifs on a Single Peptide Nucleic Acid Scaffold: A Hybrid (3+1) PNA-DNA Bimolecular Quadruplex

We describe the first G-quadruplex targeting approach that combines intercalation and hybridization strategies by investigating the interaction of a G-rich peptide nucleic acid (PNA) acridone conjugate 1 with a three-repeat fragment of the human telomere G 3 to form a hybrid PNA-DNA quadruplex that mimicks the biologically relevant (3+1) pure DNA dimeric telomeric quadruplex. Using a combination of UV and fluorescence spectroscopy, circular dichroism (CD), and mass-spectrometry, we show that PNA 1 can induce the formation of a bimolecular hybrid quadruplex even at low salt concentration upon interaction with a single-stranded three-repeat fragment of telomeric DNA. However, PNA 1 cannot invade a short fragment of B-DNA even if the latter contains a CCC motif complementary to the PNA sequence. These studies could open up new possibilities for the design of a novel generation of quadruplex ligands that target not only the external features of the quadruplex but also its central core constituted by the tetrads themselves.

Synthesis and Spectroscopic and DNA-Binding Properties of Fluorogenic Acridine-Containing Cyanine Dyes

The synthesis of a new subclass of mono- and polymethine cyanine dyes that incorporate an acridinium moiety and that absorb in the orange to near-infrared region of the spectrum is reported. The mono-, tri-, and pentamethine dyes in particular exhibit promising fluorogenic properties. Their ability to aggregate in solution and to interact with B-DNA is also discussed.

New glycosidase substrates for droplet-based microfluidic screening.

Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30 °C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-d-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4-11) and with incubation times of up to 24 h in droplet-based microfluidic systems.

Tetrapeptides induce selective recognition for G-quadruplexes when conjugated to a DNA-binding platform

3,6-Bis-peptide acridine and acridone conjugates have been designed and synthesised to selectively interact with G-quadruplex DNA. The ligand properties are peptide sequence dependent, the highest discrimination being obtained with the FRHR tetrapeptide (up to >50-fold specificity). Molecular modeling studies have helped us rationalise the data and suggest that human telomeric quadruplex DNA can readily accommodate tetrapeptides, and furthermore that FRHR contributes to stabilization of the complex by non-bonded interactions within the TTA loop pockets of the quadruplex. These studies indicate that targeting distinct features of a G-quadruplex with hybrid molecules is a promising strategy for discriminating between quadruplex and duplex DNA.

9-Amino acridines undergo reversible amine exchange reactions in water: implications on their mechanism of action in vivo.

9-Amino substituted acridines undergo a reversible amine exchange reaction in water under near-physiological conditions via an unstable hemiaminal intermediate. This thermodynamically controlled reaction may have implications in understanding the mode of action of 9-aminoacridines in vivo and in the future design of drugs based on this scaffold.

Reversible Covalent Chemistries Compatible with the Principles of Constitutional Dynamic Chemistry: New Reactions to Create More Diversity

An approach to make chemical diversity space more manageable is to search for smaller molecules, or fragments, and then combine or elaborate these fragments. Dynamic Combinatorial Chemistry (DCC) is a powerful approach whereby a number of molecular elements each with binding potential can be reversibly combined via covalent or noncovalent linkages to generate a dynamic library of products under thermodynamic equilibrium. Once a target molecule has been added, the distribution of products can be shifted to favor products that bind to the target. Thus the approach can be employed to identify products that selectively recognize the target. Although the size of the repertoire of reversible covalent reactions suitable for DCC has increased significantly over the past 5-10 years, the discovery of new reactions that satisfy all the criteria of reversibility and biocompatibility remains an exciting challenge for chemists. Increasing the number of chemical reactions will enable the engineering of larger and more diverse DCLs, which remains a key step toward a broader use of DCC. In this review, we aim to provide a nonexhaustive list of reversible covalent reactions that are compatible with the concept of DCC, focusing mainly on the most recent examples that were reported in the literature in the past 5 years.

Red/NIR G‐Quadruplex Sensing, Harvesting Blue Light by a Coumarin–Naphthalene Diimide Dyad

A conceptually new light-up nucleic acid fluorescent probe resulting from the conjugation of a coumarin to a naphthalene diimide exhibits a single wavelength emission at 498 nm when free in solution and an additional red/NIR emission when bound to G-quadruplex DNA. The light-up response centred at 666 nm is highly specific for quadruplex DNA when compared to duplex DNA or to RNA quadruplexes.

Reversible synthesis and characterization of dynamic imino analogues of trimethine and pentamethine cyanine dyes.

A new family of unsymmetrical imine-based trimethine and pentamethine cyanine dye analogues is reported that can form under reversible and thermodynamically controlled conditions from non- or weakly emissive amine and aldehyde building blocks. These dynamic fluorophores show spectroscopic properties comparable to those of their parent cyanine dyes and are responsive to external effectors.