Carme Fàbrega

DNA Origami as a DNA Repair Nanosensor at the Single-Molecule Level†

The folding of DNA molecules by DNA origami is used in a nanosensor to analyze enzymatic DNA repair activity of hAGT. The method uses conformational changes that condition α-thrombin interaction with DNA aptamers, and illustrates the use of DNA origami as a proteinrecognition biosensor.

Thioctic acid derivatives as building blocks to incorporate DNA oligonucleotides onto gold nanoparticles

Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold surfaces. However, the stability of such nucleic acid–gold nanoparticle conjugates in certain conditions may be a limitation due to premature break of the thiol-gold bonds followed by aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides carrying thioctic acid moiety based on the use of several thioctic acid-l-threoninol derivatives containing different spacers, including triglycine, short polyethyleneglycol, or aliphatic spacers. The novel thioctic-oligonucleotides were used for the functionalization of gold nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols than gold nanoparticles prepared with commercially available thiol-oligonucleotides. Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B present in endosomes. In this way this derivative may be interesting for the cellular delivery of therapeutic oligonucleotides as these results provides the basis for a potential endosomal escape.

Development of a Novel Fluorescence Assay Based on the Use of the Thrombin Binding Aptamer for the Detection of O6-alkylguanine–DNA Alkyltransferase Activity

Human O6-alkylguanine-DNA alkyltransferase (hAGT) is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA). The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein) and a quencher (dabsyl) attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group.

Synthesis and properties of oligonucleotides containing 8-bromo-2'-deoxyguanosine.

The preparation of oligonucleotides containing 8-bromo-2′-deoxyguanosine is described. Substitution of G by 8-bromoguanine on an alternating CG decamer stabilizes the Z-form in such a way that the B-form was not observed. Melting temperatures showed that duplexes in which 8-bromo-2′-deoxyguanosine paired with natural bases were much less stable.

Preparation of Oligonucleotides Containing 5-Bromouracil and 5-Methylcytidine.

Abstract A previously described side reaction on 5-bromouracil during standard oligonucleotide deprotection conditions has been studied in detail. The side product, 5-amino-2′-deoxyuridine, is isolated and characterized. The use of several 5-methylcytidine protected derivatives for the preparation of oligonucleotides containing 5-bromouracil and 5-methylcytidine free of 5-amino-2′-deoxyuridine is discussed.

Synthesis and properties of oligonucleotides containing the mutagenic base O4-benzylthymidine.

The preparation of synthetic oligodeoxynucleotides containing O4-benzylthymidine (Tbn) is described. The use of standard and t-butylphenoxyacetyl amino protecting groups is compared. The thermal stabilities of duplexes containing Tbn paired with adenine and guanine have been measured.

Gold-Coated Superparamagnetic Nanoparticles for Single Methyl Discrimination in DNA Aptamers

Au- and iron-based magnetic nanoparticles (NPs) are promising NPs for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core puts together the benefits from adding the magnetic properties to the robust chemistry provided by the thiol functionalization of gold. Here, the use of Au-coated magnetic NPs for molecular detection of a single methylation in DNA aptamer is described. Binding of α-thrombin to two aptamers conjugated to these NPs causes aggregation, a phenomenon that can be observed by UV, DLS and MRI. These techniques discriminate a single methylation in one of the aptamers, preventing aggregation due to the inability of α-thrombin to recognize it. A parallel study with gold and ferromagnetic NPs is detailed, concluding that the Au coating of FexOy NP does not affect their performance and that they are suitable as complex biosensors. These results prove the high detection potency of Au-coated SPIONs for biomedical applications especially for DNA repair detection.

Synthesis of oligonucleotides carrying fluorescently labelled O6-alkylguanine for measuring hAGT activity

O(6)-alkylguanine-DNA-alkyltransferase (hAGT) activity provides resistance to cancer chemotherapeutic agents and its inhibition enhances chemotherapy. We herein present the development of a novel fluorescence assay for the detection of hAGT activity. We designed a dsDNA sequence containing a fluorophore-quencher pair, where the fluorophore was attached to an O(6)-benzylguanine. This precursor was synthesized using the Mitsunobu reaction to introduce the benzyl group. The alkyl-fluorophore group is transferred to the active site during the dealkylation, producing an increase in fluorescence which is correlated to hAGT activity. This assay can be used for the evaluation of potential inhibitors of hAGT in a straightforward manner.

Preparation of Oligonucleotides Containing Non-Natural Base Analogs.

Abstract The preparation of oligonucleotides containing 5-amino-2′-deoxyuridine, 5-Nacetamido- 2′-deoxyuridine, 5-aza-2′-deoxycytidine and N2-substituted guanosine derivatives is described. In each case selection of the appropriate protective group, synthesis and deprotection conditions is discussed.

AS1411-decorated niosomes as effective nanocarriers for Ru(III)-based drugs in anticancer strategies

Niosomes are self-assembled vesicles made up of single chain non-ionic surfactants combined with appropriate amounts of cholesterol or other lipids, exploited as carriers for hydrophilic or lipophilic drugs. Compared to liposomes, niosomes are typically more stable, less expensive and, being generally obtained from synthetic surfactants, more easily derivatizable, providing vesicular structures with a higher versatility and chemical diversity. Herein, we investigated the physico-chemical and biological properties of niosomes loaded with two active ingredients, i.e. the nucleolipidic Ru(iii)-complex HoThyRu, selected as an anticancer agent, and the nucleolin-targeting AS1411 aptamer, allowing selective recognition of cancer cells. The morphology, average size, zeta potential, electrophoretic mobility, and stability over time of the functionalized niosomes were analyzed using different biophysical techniques. These formulations, tested on both cancer and normal cells, showed promising antiproliferative activity on HeLa cells, with a higher efficacy associated with the nanosystems containing both AS1411 and HoThyRu with respect to the controls. In all the tested cell lines, AS1411 proved to markedly enhance the bioactivity of the Ru(iii)-containing niosomes.