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Chapter 8 APPENDIX Modified oligonucleotides for triple helix studies and for the obtention of structures with biomedical and technological interest Margarita Alvira Torre ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. La consulta de esta tesis queda condicionada a la aceptación de las siguientes condiciones de uso: La difusión de esta tesis por medio del servicio TDR (www.tdx.cat) ha sido autorizada por los titulares de los derechos de propiedad intelectual únicamente para usos privados enmarcados en actividades de investigación y docencia. No se autoriza su reproducción con finalidades de lucro ni su difusión y puesta a disposición desde un sitio ajeno al servicio TDR. No se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al resumen de presentación de la tesis como a sus contenidos. En la utilización o cita de partes de la tesis es obligado indicar el nombre de la persona autora. WARNING. On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tdx.cat) service has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized neither its spreading and availability from a site foreign to the TDX service. Introducing its content in a window or frame foreign to the TDX service is not authorized (framing). This rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. Tesis Doctoral Modified oligonucleotides for triple helix studies and for the obtention of structures with biomedical and technological interest Margarita Alvira Torre Departamento de Química Orgánica Facultad de Química Universidad de Barcelona 2010 Memoria presentada por Margarita Alvira Torre para optar al grado de Doctor Europeo por la Universidad de Barcelona Departamento de Química Orgánica Programa de doctorado de Química Orgánica Bienio 2005-2007 Dirigida y revisada por: Dr. Ramon Eritja Casadellà Tutora: Dra. Anna Grandas Sagarra Barcelona, Septiembre de 2010 A mis padres y mi hermana A mis abuelos A Alejandro Acknowledgements En primer lugar quiero agradecer al Dr. Ramon Eritja, por aceptarme en su grupo y dirigir mi tesis doctoral, por la confianza depositada, la ayuda prestada, la motivación inculcada y sus sabios consejos. Voy a estar siempre agradecida. También a Anna Grandas, por aceptar ser mi tutora de tesis y estar ahí siempre que la he necesitado. El haber disfrutado tanto durante mi etapa predoctoral se lo debo en gran parte a las personas con las que he tenido la suerte de trabajar durante estos últimos años: Marta (aunque coincidimos poco tiempo lo pasé muy bien), Sandra (¡suerte con las patentes!), Roger (que máster tan diver..) Anna (contigo he compartido toda mi etapa de tesis, desde que empecé. Mil gracias siempre por tu ayuda y tus consejos), Clara (estoy deseando ver a tu baby..), Brendan (por enseñarnos tantas “nano”cosas), Alejandra (ché, animo con los geles boludos), Rubén (la alegría del laboratorio, siempre son una sonrisa-y una risa tambien. De hecho la estoy oyendo mientras escribo esta página), Montse (una “crack” de la orgánica, ¡espero que sigas publicando tanto! echaré de menos las ‘meriendetas’), Carme (por fin te podré hacer el seminario, jeje), Álvaro (por tantas charlas existenciales a horas intempestivas), Santi (el rey de los siRNAs y el delivery), María (la benjamina del labo..), Sonia (¡ánimo con los TTF!), Stefi (¡¡eres parte de este grupo!! y siempre tendrás una familia en Barcelona). Y no me quiero dejar a los compañeros del CSIC, Antonio, Raul, Jose Antonio, Jaume, Miquel Pons, con quienes compartí esos buenos ratos a la hora de la comida el primer año de mi tesis en el CSIC. Durante mi tesis doctoral he realizado dos estancias breves en el extranjero, durante las cuales he aprendido muchísimo, tanto a nivel profesional como personal. Quiero agradecer al Dr. Morten Grøtli y su grupo, por acogerme en su laboratorio y hacer que mi estancia en Suecia fuera tan agradable, tack så mycket! Al Dr. David Schiffrin por dejarme investigar en su laboratorio y abrirme las puertas al mundo de la electroquímica. Gracias también a la gente del departamento y de su grupo, especialmente a Jose y Laura, por su ayuda con las voltametrías y porque hicieron de mi experiencia en Liverpool un recuerdo inolvidable. Al Dr. Carlos González, por su ayuda con los experimentos de RMN con los quadruplexes, y a todo su grupo, por las atenciones brindadas durante mi corta estancia en Madrid. Quiero agradecer especialmente a la Dra. Irene Pinto y al Dr. Douglas V. Laurents su ayuda con los experimentos de dicroísmo circular. A la Dra. Irene Fernández y la Dra. Marta Vilaseca, de la Universidad de Barcelona, por su ayuda con la espectrometría de masas en los servicios científico-técnicos, especialmente en la primera parte de mi tesis. Quiero agradecer también al Ministerio de Educación y Ciencia (ahora MICINN), por la concesión de una beca predoctoral dentro del programa de ayuda para la Formación de Personal Investigador (Programa FPI), sin la que este trabajo no se hubiera podido realizar. Por último, me gustaría dar mi más sincero agradecimiento a mi familia, a quienes les dedico este trabajo. A mis padres (¿como podría agradeceros todo lo que hacéis por mí?), mi hermana, mis abuelitos, a Marian, a la ita, a mis tíos, a mis primos. Gracias a todos por ser como sois. ¡Os quiero mucho! Y sobre todo a Alejandro, mi marido, por ser siempre un apoyo incondicional y por recorrer juntos este camino tan bonito que es la vida. INDEX Abstract………………………………………………………………………………………………………………………….i Resumen..………………….…………………………………………………………………………………………………iii List of Abbreviations………………………………………………………………………………….…………………v CHAPTER 1: “Introduction” 1.1. DNA: structure and function………………………………………………………………………………..1 1.2. Oligonucleotides and their applications……………………………………………………………….4 1.2.1. Oligonucleotides as therapeutic agents………………………………………………………4 1.2.2. Oligonucleotides as tools in DNA-templated synthesis……………………………..6 1.2.3. Oligonucleotides as useful tools in nanotechnology...……………………………….6 1.2.4. Oligonucleotides and chemical modifications…………………………………….....….8 1.3. Objectives……………………………………………………………………………………………………………12 1.4. References……………………………………………………………………….………………………….……. 13 CHAPTER 2: “Triplex-Stabilizing Properties of Parallel Clamps Carrying LNA Derivatives” 2.1. DNA triplexes.…………...…………………………………………………………….………………………..18 2.2. Oligonucleotide hairpins and triplex.……………………………………………..……………….…19 2.3. Triplex modifications..…………………………………………………………………………………….….19 2.3.1. LNA…………………………………………………………………………………………………………….21 2.4. Objectives…………………………………………………………………………………………………………..22 2.5. Design of the sequences………………………………………………………………………………….…22 2.6. Synthesis of oligonucleotides..…………………………………………………….……….….……….23 2.7. Thermal denaturation studies………………………………………………………………………..….25 2.7.1. Evaluation of triplex stability by thermal melting curves……………………..…25 2.7.2. Thermal stability of the Hoogsteen parallel duplexes………………………………27 2.7.3. Thermal stability of triplexes formed by parallel clamps and their DNA polypyrimidine target strand……………………………………………………………………….………30 2.7.4. Thermal stability of triplexes formed by parallel clamps and their RNA polypyrimidine target strand…………………………………………………………………………….…32 2.8. Conclusions…………………………………………………………………………………………………………34 2.9. References……………………………………………………………………………………………………….…35 CHAPTER 3: “Synthesis of Oligonucleotides Carrying 5’-5’ Linkages Using Copper-Catalyzed Cycloaddition Reactions” 3.1. Click chemistry……………………………………………………………………………………………………40 3.1.1. The Cu (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction……….41 3.1.2. Applications of the CuAAC reaction………………………………………………………….44 3.2. Aim of the work……………………………………………………….…………………………………………48 3.3. Design of oligonucleotide sequences…………………………………………………………………49 3.4. Synthesis of oligonucleotides carrying alkynyl groups…………………………………….51 3.4.1. Synthesis of 5’-alkyne-ODNs from 5’-amino-ODNs………………………………..51 3.4.2. Synthesis of 5’-alkyn-ODNs from 5’-carboxy-ODNs……………………………….53 3.4.3. Synthesis of 5’-alkyn-ODNs with an alkyne-containing phosphoramidite…………………………………………………………………………………………………..56 3.5. Synthesis of oligonucleotides carrying azido groups…………………………………………57 3.5.1. Synthesis of 5’-azido-ODNs from 5’-amino-ODNs……………………………………58 3.5.2. Synthesis of 5’-azido-ODNs from 5’-iodo-ODNs……………………………………..59 3.5.3. Synthesis of 5’-azido-ODNs with a bromine-containing phosphoramidite………………………………………………………………………………………....……..61 3.6. Cu(I)-catalyzed cycloaddition of azido- and alkynyl- oligonucleotides….........64 3.6.1. Preliminary
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