Eric Defrancq

Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation.

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.

Label-free impedimetric thrombin sensor based on poly(pyrrole-nitrilotriacetic acid)-aptamer film

A label-free and highly sensitive impedimetric aptasensor was developed based on electropolymerized film for the determination of thrombin. The first step is the electrogeneration of a poly(pyrrole-nitrilotriacetic acid) (poly(pyrrole-NTA)) film onto the surface of electrodes followed by complexation of Cu(2+) ions. Then, the histidine labeled thrombin aptamer was immobilized onto the electrode through coordination of the histidine groups on the NTA-Cu(2+) complex. The aptamer sensor was applied for the detection and quantification of thrombin via impedimetric detection without a labeling step. A linear quantification of thrombin was obtained in the range 4.7×10(-12)-5.0×10(-10) mol L(-1) with a sensitivity of 2838 Ω/log unit (R(2)=0.9984). The impedance modulus at 0.3 Hz as a function of thrombin concentration was used to elaborate a similar linear relationship from 4.7×10(-12) to 5×10(-10) mol L(-1). In addition, aptamer-poly(pyrrole-NTA) electrodes incubated for 40 min in aqueous solutions of bovine serum albumin (BSA), lysozyme and IgG (5×10(-7) mol L(-1)) did not exhibit non-specific adsorption of proteins. Moreover, it has been demonstrated that the selective sensor can be regenerated several times with a good reproducibility.

Plasmon-induced CD response of oligonucleotide-conjugated metal nanoparticles

Non-chiral metal nanoparticles conjugated with chiral oligonucleotide molecules demonstrated a circular dichroism (CD) at the plasmonic wavelengths due to aggregation effects.

Template-assembled synthetic G-quadruplex (TASQ): a useful system for investigating the interactions of ligands with constrained quadruplex topologies.

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular-like G-quadruplex motif 1 (parallel G-quadruplex conformation), an intramolecular G-quadruplex 2, and a duplex DNA 3 have been designed and developed. The method is based on the concept of template-assembled synthetic G-quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G-quadruplex conformation. Various known G-quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a pi-stacking binding mode showed a higher binding affinity for intermolecular-like G-quadruplexes 1, whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2. In addition, the present method has also provided information about the selectivity of ligands for G-quadruplex DNA over the duplex DNA. A numerical parameter, termed the G-quadruplex binding mode index (G4-BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G-quadruplex 1 against intramolecular G-quadruplex 2. The G-quadruplex binding mode index (G4-BMI) of a ligand is defined as follows: G4-BMI=K(D)(intra)/K(D)(inter), where K(D)(intra) is the dissociation constant for intramolecular G-quadruplex 2 and K(D)(inter) is the dissociation constant for intermolecular G-quadruplex 1. In summary, the present work has demonstrated that the use of parallel-constrained quadruplex topology provides more precise information about the binding modes of ligands.

A Photoreactive Ruthenium(II) Complex Tethered to a Guanine‐Containing Oligonucleotide: A Biomolecular Tool that Behaves as a “Seppuku Molecule”

The design of specific DNA or RNA damaging agents may be achieved by anchoring a reactive species to an oligonucleotide (ODN) probe which, by duplex or triplex formation, should direct the irreversible chemical modification towards the targeted sequence. This approach is certainly interesting in the context of gene silencing, particularly for the development of anticancer agents. However, the achievement of a high level of selectivity remains a challenge because many side reactions such as interaction with proteins can occur in biological systems. In this context, the tethering of a transition metal complex to an ODN strand and subsequent activation by light is an attractive strategy. Indeed, the reactivity of the metallic compound towards the genetic material can be 1) triggered by light to act on specific tissues and 2) directed towards a DNA or RNA target sequence to cause damage. We have prepared oligonucleotides labeled with ruthenium(II) complexes (Ru-ODNs) that are able to photo-cross-link with their complementary strand under visible irradiation (Figure 1 a). The different parameters that govern the photo-cross-linking efficiency have been extensively studied, 6] these conjugates could be promising as anticancer agents based on gene silencing. Indeed, this sequence-specific photo-cross-linking process has been shown to inhibit in vitro DNA polymerase with a high efficiency. The prerequisites for this photochemical reaction are 1) the presence of at least two p-deficient polyazaaromatic ligands such as 1,4,5,8-tetraazaphenanthrene (tap) in the tethered metallic complex and 2) the presence of at least one guanine base (G) in the target sequence, in the vicinity of the tethered complex after hybridization. The mechanism involves a photoinduced electron transfer (PET) from the G base towards the oxidizing excited Ru compound with a recombination of the produced radicals, which covalently links one of the tap ligands to the G unit [Figure 1b, Eqs. (1)–(3)]. The structure of the photoadduct (Figure 1b) has been determined by ESI mass spectrometry and NMR spectroscopy. This strategy was thought to be restricted to target sequences without cytosine in order to avoid the presence of a guanine in the Ru-ODN probe, which would be unfavorable for the photo-cross-linking. In spite of these limitations, we decided to investigate the effect of the presence of a G base in the Ru-ODN probe sequence on the photoreactivity of such systems. Herein, we report the unique photochemical behavior of a new generation of photoreactive G-containing Ru-ODN probes that can selfinhibit in the absence of their specific target strands. We have named these molecules “seppuku molecules” because their photochemical behavior reminds us of the suicide of someone who has not accomplished his attributed duty in antic Japanese society. The Ru complexes contain a phenanthroline ligand derivatized by a linker, namely phen’’ (phen’’= N-(2-(1,10phenanthrolin-5-ylamino)-2-oxoethyl)-2-(aminooxy)acetaFigure 1. a) Photo-cross-linking processes that occur between an ODN probe labeled with a suitable Ru complex and the complementary G-containing ODN strand and b) formation and structure of the photoadduct produced upon illumination of [Ru(tap)3] 2+ in the presence of a guanine residue in GMP (guanosine-5’-monophosphate) or DNA after acid hydrolysis.

Synthesis of fluorescent probes for the detection of abasic sites in DNA

Abstract The three luminescent molecules 1–3 were prepared as highly sensitive and specific probes for the quantification of a major DNA lesion, the abasic site. These molecules incorporate in their structure the reactive oxyamino function linked to the Dansyl or Lissamine-Rhodamine fluorophores through amido or polyether chains.

Oxime bond formation for the covalent attachment of oligonucleotides on glass support

The chemical attachment of oligonucleotides on glass slides has been achieved using oxime bond formation. This method has been shown very efficient by comparison with the attachment of amino-oligonucleotides via reductive amination.

Efficient preparation of carbohydrate–oligonucleotide conjugates (COCs) using oxime bond formation

Abstract Chemoselective ligation of carbohydrates to oligonucleotides was accomplished by oxime bond formation. The conjugation was performed by reacting an oxyamino sugar with an oligonucleotide containing an aldehyde moiety at the 5′-end. The carbohydrate–oligonucleotide conjugates (COCs) were obtained in good yield without the need of a protection strategy and under mild aqueous conditions.

A new fluorescent probe for sensitive detection of carbonyl compounds

A new method for the convenient and sensitive detection of aldehydes and ketones in environmental samples was developed. This method is described in detail for the measurement of carbonyls in water samples, but it is also potentially applicable to their determination in air. Derivatization of carbonyls is performed using N-(5-dimethylamino-1-naphtalenesulphonamido)-3-oxapentane-1,5-dioxyamine (dansyloxyamine, DNSOA). The resulting oxime ethers are separated by using reversed phase liquid chromatography and fluorescence detection. Parameters influencing both reactivity and sensitivity are discussed. The oxyamino function allows efficient chemical derivatization in water without the use of a catalyst. The dansyl fluorophore and the reactive oxyamino group are linked by an etheral chain which leads to very similar fluorescence responses for formaldehyde, acetaldehyde and acetone derivatives. The detection limit of this method compares favourably with classical methods. Furthermore, the analysis procedure is easily set up and can be directly applied to environmental water samples.

Photocrosslinking in ruthenium-labelled duplex oligonucleotides.

The formation of a photoadduct between a [Ru(1,4,5,8‐tetraazaphenanthrene)24,7‐diphenylphenanthroline]2+ complex chemically attached to a synthetic oligonucleotide, and a guanine moiety in a complementary targeted single‐stranded DNA molecule was studied for ten 17‐mer duplexes by denaturing gel electrophoresis. This photoadduct formation leads to photocrosslinking of the two strands. The percentage quenching of luminescence of the complex by electron transfer was compared to the resulting yield of photocrosslinked product. This yield does not only depend on the ionisation potential of the guanine bases, which are electron donors, but also on other factors, such as the position of the guanine bases as compared to the site of attachment of the complex. The photocrosslinking yield is higher when the guanine moieties are towards the 3′ end on the complementary strand as compared to the tethering site. Computer modelling results are in agreement with this preference for the 3′ side for the photoreaction. Interestingly, the photocrosslink is not alkali labile. Moreover, a type III exonuclease enzyme is blocked at the position of photocrosslinking.