Estudi de la interacció de tres complexos de Pt amb proteïnes i oligonucleòtids mitjançant ESI-MS

En aquest treball s’investiga la reactivitat de tres complexos de Pt, cisplatí, complex 1, [Pt(dmba)(aza-N1)(dmso)], complex 2, i [Pt(dmba)(N9-9AA)(PPh3)]+, complex 3; els quals presenten activitat antitumoral, amb diferents proteïnes i oligonucleòtids mitjançant espectrometria de masses (ESI-TOF MS). ). L’estudi del cisplatí, 1, droga molt coneguda i emprada avui en dia pel tractament de molts tipus de càncer, permet comparar la reactivitat dels nous complexos d’estudi, 2 i 3, els quals presenten un IC50 més baix que el cisplatí en algunes línies de cèl·lules tumorals.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

Síntesi de siRNAs (short interfering RNAs) modificats amb nucleobases 5-metil i 5-propinil pirimidíniques i avaluació de la seva activitat in vitro en cultius cel•lulars i de la seva estabilitat en sèrum humà. Disseny de siRNAs dirigits a la inhibició de l'expressió de mutacions de l'oncogen K-ras.

Projecte de recerca elaborat a partir d’una estada a la Stanford University, EEUU, entre 2007 i 2009. El present projecte es basa 1) en la síntesi de cadenes d'ARN dirigides a la inhibició de l'expressió gènica per un mecanisme d'ARN d'interferència (siRNAs o short interefering RNAs) i 2) en l'avaluació de l'activitat in vitro d'aquests oligonucleòtids en cultius cel•lulars. Concretament, la meva recerca ha estat enfocada principalment a l'estudi de cadenes de siRNA modificades amb nucleobases 5-metil i 5-propinil pirimidíniques. Es tractava d'avaluar l'efecte que exerceixen els factors estèrics en el major groove (solc major) dels siRNAs sobre la seva activitat biològica. En aquest sentit, he dut aterme síntesi de fosforamidits de nucleòsis pirimidínics modificats a la posició C-5 de la nucleobase. A continuació he incorporat aquestes unitats nucleosídiques en cadenes d'ARN emprant un sintetitzador d’ADN/ARN i he estudiat l'estabilitat dels corresponents dúplexs d'ARN mitjançant experiments de desnaturalització tèrmica. Finalment he dut a terme experiments d'inhibició de l'expressió gènica en cèl.lules HeLa per tal d'avaluar l'activitat biològia d'aquests siRNAs modificats. Els resultats d'aquests estudis han posat de manifest que la presència de grups voluminosos com el propinil a l'extrem 5' del dúplex de siRNA (definit per la cadena guia o antisense) influeix de forma molt negativa en la seva activitat biològica. En canvi, grups menys voluminosos com el metil hi influeixen positivament, de manera que algunes de les cadenes sintetitzades han resultat ser més actives que els corresponents siRNAs naturals (wild type siRNAs). A més, aquest tipus de modificació contribueix positivament en l'estabilitat de cadenes de siRNA en sèrum humà. Aquest treball ha estat publicat (Terrazas, M.; Kool, E.T. "Major Groove Modifications Improve siRNA Stability and Biological Activity" Nucleic Acids Res. 2009, in press).

Aptasensores impedimétricos para la detección de trombina

En el presente trabajo se ha desarrollado el primer aptasensor (biosensor de aptámero) en nuestro grupo de investigación, Grup de Sensors i Biosensors de la Universidad Autònoma de Barcelona. En concreto se han desarrollado dos aptasensores para la detección de la proteína trombina, uno basado en la inmovilización de aptámeros por adsorción física, y otro basado en la inmovilización de aptámeros por enlace covalente mediante la reacción EDAC-NHS. El aptasensor utiliza la afinidad específica de la cadena de DNA (aptámero) por la proteína con la que interacciona. Los cambios de carga y estéricos del complejo aptámero proteína alteran la capacidad y la resistencia de transferencia interfacial de electrones en la superficie del electrodo. El principio de detección se basa en la detección de cambios de estas propiedades de interfase del electrodo con el marcador redox [Fe(CN)6]3- / [Fe(CN)6]4-, utilizando mediciones de Espectroscopia Electroquímica de Impedancia. El aptasensor basado en adsorción física del aptámero mostró una respuesta lineal a trombina en el rango de 7.5 a 75 pM y un límite de detección de 5pM, después de optimizar todas las condiciones experimentales. Posteriormente se estudió la especificidad del sistema respecto proteínas potencialmente interferentes presentes en suero sanguíneo, obteniendo cierta interferencia por parte de fibrinógeno e inmunoglobulina G, pero no por parte de albúmina. El sensor demostró ser regenerable mediante la ruptura del complejo formado entre el aptámero y la trombina con una solución de NaCl 2.0 M, aumento de la temperatura y agitación. El segundo aptasensor, basado en enlace covalente del aptámero mostró una respuesta lineal a trombina y limite de detección mejor que el anterior sensor; de 2.5 a 100 pM y 1.5 pM respectivamente. Aunque cabe destacar que este aptasensor está siendo optimizado actualmente.

A minimal i-motif stabilized by minor groove G:T:G:T tetrads

The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

A minimal i-motif stabilized by minor groove G:T:G:T tetrads

The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

Photocontrolled DNA Binding of a Receptor-Targeted Organometallic Ruthenium(II) Complex

A photoactivated ruthenium(II) arene complex has been conjugated to two receptor-binding peptides, a dicarba analogue of octreotide and the Arg-Gly-Asp (RGD) tripeptide. These peptides can act as"tumor-targeting devices" since their receptors are overexpressed on the membranes of tumor cells. Both ruthenium-peptide conjugates are stable in aqueous solution in the dark, but upon irradiation with visible light, the pyridyl-derivatized peptides were selectively photodissociated from the ruthenium complex, as inferred by UV-vis and NMR spectroscopy. Importantly, the reactive aqua species generated from the conjugates, [(η6-p-cym)Ru(bpm)(H2O)]2+, reacted with the model DNA nucleobase 9-ethylguanine as well as with guanines of two DNA sequences, 5′dCATGGCT and 5′dAGCCATG. Interestingly, when irradiation was performed in the presence of the oligonucleotides, a new ruthenium adduct involving both guanines was formed as a consequence of the photodriven loss of p-cymene from the two monofunctional adducts. The release of the arene ligand and the formation of a ruthenated product with a multidentate binding mode might have important implications for the biological activity of such photoactivated ruthenium(II) arene complexes. Finally, photoreactions with the peptide-oligonucleotide hybrid, Phac-His-Gly-Met-linker-p5′dCATGGCT, also led to arene release and to guanine adducts, including a GG chelate. The lack of interaction with the peptide fragment confirms the preference of such organometallic ruthenium(II) complexes for guanine over other potential biological ligands, such as histidine or methionine amino acids.

Conjugation reactions involving maleimides and phosphorothioate oligonucleotides

Phosphorothioate diester oligonucleotides proved to be fully compatible with maleimides in the context of two different conjugation reactions: (a) reaction of 5′diene-[phosphorothioate oligonucleotides] with maleimido-containing compounds to afford the Diels-Alder cycloadduct; (b) conjugation of 5′maleimido-[phosphorothioate oligonucleotides] with thiol-containing compounds. No evidence of reaction between phosphorothioate diesters and maleimides was found in any of these processes. Importantly, in the preparation of 5′maleimido-[phosphorothioate oligonucleotides] from [protected maleimido]-[phosphorothioate oligonucleotides], which requires the maleimide to be deprotected by retro-Diels-Alder reaction (heating for 3-4 h in toluene at 90 °C), no addition of phosphorothioate diester to the maleimide was found either. Finally, maleimide-[phosphorothioate monoester] conjugation was also explored for comparison purposes.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.