VII Reunión de Ácidos Nucleicos y...

VII Reunión de Ácidos Nucleicos y Nucleósidos

Escuela Universitaria de Jaca (Huesca)

Organizing committeeCarlos González Ibáñez

Douglas V. Laurents

Irene Gómez Pinto

Jorge Pedro López Alonso

Miguel Ángel Pardo Cea

Nerea Martín-Pintado Zugasti

Fernando Diez García

AcknowledgmentsThe organizers wish to thank Consejo Superior de Investigaciones Científicas

generous support, and Instituto de Química Fïsica Rocasolano and Real Sociedad

Española de Química for their help in the organization of th

Likewise, we want to thank

Orozco, M. José Camarasa, Enrique Pedroso and Federico Gago

chairs in the different meeting sessions.

1


10 - 12 de junio de 2009 Escuela Universitaria de Jaca (Huesca)

committee

Pintado Zugasti

Acknowledgments The organizers wish to thank Consejo Superior de Investigaciones Científicas


Española de Química for their help in the organization of the meeting.

Likewise, we want to thank Anna Grandas, Ramon Eritja, José Gallego, Modesto

marasa, Enrique Pedroso and Federico Gago for their help acting as

chairs in the different meeting sessions.


The organizers wish to thank Consejo Superior de Investigaciones Científicas for their


e meeting.

José Gallego, Modesto

for their help acting as

3

Programme

Wednesday 10th June

19:00-21:00 Registration

21:00 Welcome reception

Thursday 11th June

9:15-9:30 Opening of the VII RANN

Session I Chair: Anna Grandas

9:30-10:00 Effect of CpG Methylation in DNA Flexibility Alberto Pérez Antoñanzas, Life Science, Barcelona Supercomputing Center

10:00-10:30 Template PPRHs as a Tool to Knock Down the Expression of Genes Involved in Cell Proliferation

María Cristina de Almagro García, Departamento de Bioquímica y Biología

Molecular, Facultad de Farmacia, Universidad de Barcelona

10:30-11:00 Conformationally Restricted Purine Nucleosides: Stabilization through an Intramolecular Cation-Pi Interaction

Elena Casanova Malpica, Departamento de Quimioterapia, Instituto de Química

Médica, CSIC, Madrid

11:00-11:30 Solution Structure and Stability of FANA–RNA and ANA–RNA Hybrid

Duplexes

Nerea Martín-Pintado Zugasti, Departamento de Espectroscopía y Estructura

Molecular, Instituto de Química Física Rocasolano, CSIC, Madrid

11:30-12:00 Coffee Break

Session II Chair: Douglas V. Laurents

12:00-12:30 Use of DNA-Hairpins Carrying Photolabile Groups in Fabrication of Patterned Surfaces by Photolithography

Brendan J. Manning, Departamento de Nanotecnologia, Instituto de Química

Avanzada de Cataluña, CSIC, Barcelona

4

12:30-13:00 Indirect Effects Modulating the Interaction between DNA and a Cytotoxic Bis-naphthalimide Unravel Two Superposed Duplex Binding and Intercalation Processes

Luis González Bulnes, Departamento de Química Médica, Centro de Investigación

Príncipe Felipe, Valencia

13:00-13:30 Síntesis de conjugados aminoglicósido-dinucleótido como posibles ligandos específicos de RNA

Javier Alguacil Blanco, Departamento de Química Orgànica, Universitat de

Barcelona

14:00-16:00 Lunch at the Residence

Session III Chair: Ramon Eritja

16:00-16:30 The Structure and Dynamics of Triplex DNA in Gas Phase Annalisa Arcella, Institut de Recerca Biomèdica, Barcelona

16:30-17:00 Structural Insights into the Mechanism of GTPase Activity in β-tubulin and its Inhibition by Binding of the Antitumour Alkaloid Vinblastine

Claire Coderch Bou, Departamento de Farmacología, Universidad de Alcalá

17:00-17:30 Biocatalysis Applied to Separation of Mixtures β-D/L-Nucleosides Saúl Martínez Montero, Departamento de Química Orgánica e Inorgánica, Universidad de Oviedo

17:30-18:00 Synthesis of Oligonucleotide Conjugates by Diels-Alder Cycloaddition Samuel Ortega Carrillo, Departamento de Química Orgànica, Universitat de Barcelona


Session IV Chair: José Gallego

18:30-19:00 Using DNA Physical Properties to Determine Biological Markers Carles Fenollosa Bielsa, Nodo INB GN4-PCB, Parc Científic de Barcelona

19:00-19:30 Efficient Generation of Molecular Complexity from a Common Nucleosidic Precursor

María Cruz Bonache de Marcos, Departamento de Quimioterapia, Instituto de Química Médica, CSIC, Madrid

19:30-20:00 Was RNA Alone in the RNA World? Putative Ribonucleases Form under Plausible Prebiotic Earth Conditions

Jorge Pedro López Alonso, Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física Rocasolano, CSIC, Madrid

5

Friday 12th June

Keynote Conference Chair: Carlos González

9:30-10:30 Targeting the HIV-1 Genome: RNA Structure-Based Drug Discovery Thomas L. James, Department of Pharmaceutical Chemistry, University of California, San Francisco

Session V Chair: Modesto Orozco

10:30-11:00 Comparative Structural Study on DNA and RNA Flexibility Ignacio Faustino Plo, Departamento de Bioquimica y Biologia Molecular, Universitat de Barcelona

11:00-11:30 Modified siRNAs and Biocompatible Gold Nanoparticles Álvaro Somoza Calatrava, IMDEA-Nanociencia, Madrid


Session VI Chair: María José Camarasa

12:00-12:30 pH-Modulated Watson-Crick Duplex - Quadruplex Equilibria of Guanine-Rich and Cytosine-Rich DNA Sequences Upstream of the c-kit Transcription Initiation Site

Raimundo Gargallo Gómez, Departamento de Química Analítica, Universitat de Barcelona

12:30-13:00 A Loop-Loop RNA Tertiary Interaction Motif Shared by Most Natural Hammerhead Ribozymes

José Gallego Sala, Centro de Investigación Príncipe Felipe, Valencia

13:00-13:30 Carbohydrate-Oligonucleotide Conjugates: How Sugars Other Than (Deoxy-)Ribose Affect DNA Stability and Allow to Study Intermolecular Forces

Juan Carlos Morales Sánchez, Departamento de Química Bioorgánica, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla

14:00-16:00 Lunch at the Residence

6

Session VII Chair: Enrique Pedroso

16:00-16:30 Antiviral and Biological Properties of Glucose-Conjugated Antisense Oligodeoxynucleotides that Inhibit HIV-1 Replication in Cell Culture

Jose Antonio Reyes Darias, Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Granada

16:30-17:00 Structural Basis for the Binding of the New Antitumor Drug Zalypsis® to Select DNA Triplets Using Molecular Dynamics

Juan Antonio Bueren Calabuig, Departamento de Farmacología, Universidad de Alcalá

17:00-17:30 Structure and Function of the Leader Transcription-Regulating Sequence of Transmissible Gastroenteritis Virus

David Dufour Rausell, Departamento de Química Médica, Centro de Investigación Príncipe Felipe, Valencia


Session VIII Chair: Federico Gago

18:00-18:30 Screening for Small Molecules Interacting With RNAs by NMR Irene Gómez Pinto, Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física Rocasolano, CSIC, Madrid

18:30-19:00 Effects of Major Groove Substitution on RNA Interference Montserrat Terrazas Martínez, Departamento de Nanotecnologia, Instituto de Química Avanzada de Cataluña, CSIC, Barcelona

19:00-19:30 General Discussion and Closing of the VII RANN

21:30 Meeting Banquet

7

Abstracts

Session I – 1

9

Effect of CpG methylation in DNA flexibility

Alberto Perez1 , Chiara Castellazzi2, Montse Soler2, Modesto Orozco1,2,3

1Joint IRB-BSC programme. Barcelona Supercomputing Center (BSC)

2 Institut de Recerca Biomedica de Barcelona (IRBB)

3 Universitat de Barcelona (UB)

The analysis of CpG basepair steps in humans reveals that it is unique in many ways: for one thing its frequency is much lower than statistically expected1 at the genome level (1 every 100 bp), they are also special in the sense that in these steps dC can be methylated at position 5. There are some parts in the genome where this is not the case: the frequency is around 10 times higher and Cytosines in this regions are usually not methylated. These regions are called CpG islands, and the total C+G content is also much higher than the genome average (67% compared to 41% in genome average1). CpG islands are found in promoter regions of ~60% of all human genes, including all the housekeeping genes and around half of the tissue specific ones. Hypermethylation of these CpG islands is correlated with gene silencing2 and cancer. In this study we use experimental and computational tools to study how the presence of a methyl group in the 5’ position of cytosines can affect the properties of DNA. In particular, we want to explore how the flexibility pattern changes with methylation and how these changes influence the ability of DNA to bind to different proteins.

References 1. Antequera, F. Cell. Mol. Life. Sci. 2003, 60, 1647-1658 2. Kristensenm L.S., Mikeska, T., Krypuy, M., Dobrovic, A. Nucleic Acids Research 2008, 36, e42 1-13.

Session I – 2

10

Template PPRHs as a tool to knock down the expression of genes involved in cell proliferation

MªCristina de Almagro, Véronique Noé, Carlos J. Ciudad

Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Av. Diagonal 643, E-08028 Barcelona, Spain.

PPRHs are DNA hairpins formed by two antiparallel polypurine-domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target sequence, forming triplex structures1. Their target sequence is a polypyrimidine stretch, preferably lacking purine interruptions, in the Template DNA strand. We reported that they bind specifically to their DNA target sequence decreasing gene transcription2. Some of the advantages of PPRHs in front of other types of knocking-down molecules are that they are non–modified DNA molecules easy to synthesize and inexpensive, with a noticeable half-life of around 5 days. In this study we explored the ability of Triplex-forming antiparallel purine-hairpins (PPRHs) to be used as therapeutic tools by targeting genes involved in cell proliferation. We used different types of target genes that could produce a decrease in cell viability if inhibited: Survivin and BCL-2 (apoptosis), Telomerase and c-Myc (proliferation), Topoisomerase (DNA topology), mdm2 (check point status) and Her2 (Oncogene receptor). We tested the ability of these PPRHs to cause cytotoxicity in three different breast cancer cell lines (SKBR3, MCF7 and MDA-MB 468). We found that they were already active at concentrations of 100nM, whereas the negative controls Hp-Sc (Scrambled) and Hp-WC (Watson-Crick containing intramolecular bonds, that cannot bind their polypyrimidine target sequence) scarcely produced changes in cell survival. The genes that showed a greater response when targeted with PPRHs were Survivin, Telomerase, c-Myc, Topoisomerase, and mdm2, causing a 90% of cytotoxicity. We also measured the mRNA levels of each target gene when cells were transfected with PPRHs, observing a decrease that was specific, as it was not caused by using the PPRHs negative controls. Thus, we conclude that PPRHs are a powerful tool to be used to decrease specifically gene transcription of genes with therapeutic interest.

Acknowledgements: SAF08-043 and RETICC RD06/0020/0046

References

1. Coma, S., Noe, V., Eritja, R., and Ciudad, C. J. (2005) Oligonucleotides 15, 269-283 2. de Almagro, MC ; Coma, S ; Noé, V ; Ciudad, CJ ; J Biol Chem. 2009 Apr 24; 284 (17):11579-89

Session I – 3

Conformationally restricted

an intramolecular cation

Elena Casanova1, María Luisa Jimeno

1 Instituto de Química Médica (CSIC),

2 Centro de Química Orgánica Lora

The cation-Pi interaction has been proposed asthe molecular recognition in biochemical processescomplexes in supramolecular chemistry.applied in asymmetric synthesis, since the introduction of a positive charge in a pyridine ring implies the change from a free rotational system to a conformationally restricted one with a higher control in the regio and stereoselectivity.3

In the course of our studies in nucleoside derivativesisopropylideneinosines with a benzyl group (Figure 1, 1-3). The 1H NMR spectra of Therefore, the conformational properties of these nucleosides in solution were studied by performing different 1H NMR experiments, application of PSEUROT and molecular modeling. The data obtainedpointed towards a major conformation (Figure 2) where the benzyl at the 5´imidazolium ring of the base, probably stabilized by an intramolecular cationthat this interaction was the driving force for thypothesis we studied by NMR and by molecular dynamics simulations the conformation of the 1,5’dibenzyl-2’,3’-O-isopropylideneinosine compound 4 is quite flexible in solution, when protonation takes place at N7 as in towards restricted conformations characterized by the disposition of the base and the benzyl group shown in Figure 2 and where the distance between both frTherefore, these inosine derivatives represent one of the few examples described where the conformation is restricted through an intramolecular cation

N

N

O

OO

O

R1

1 R1= Bn R2= H X2 R1= Me R2= H3 R1= Me R2= Me5 R1= H R2= H

X-R2

Figure 1

Acknowledgements: E. C. acknowledges the Comunidad de Madrid and the FSE for a predoctoral grant. E-M.P. has a CSIC contract from the I3P programme finasupported by the Spanish CICYT (SAF2006 References 1. Ma, J. C.; Dougherty, D. A. Chem. Rev.2. Meyer, E. A.; Castellano, R. K.; Diederich, F3. Yamada, S. Org. Biomol. Chem. 2007,

11

Conformationally restricted purine nucleosides: stabilization

an intramolecular cation−−−−Pi interaction

María Luisa Jimeno2, Eva María Priego1, María José Camarasaand María Jesús Pérez Pérez1

Instituto de Química Médica (CSIC),Juan de la Cierva 3, 28006 Madrid, Spain

ica Orgánica Lora-Tamayo (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

n has been proposed as one of the most relevant noncovalent interactions in the molecular recognition in biochemical processes,1 as well as in the stabilization of hostcomplexes in supramolecular chemistry.2 Recently, the intramolecular cationapplied in asymmetric synthesis, since the introduction of a positive charge in a pyridine ring implies the hange from a free rotational system to a conformationally restricted one with a higher control in the regio

In the course of our studies in nucleoside derivatives, we have performed the benzyl group at the 5’ position and a benzyl, or a methyl group at N7

H NMR spectra of 1-3 seemed to indicate conformational restrictions in solution. Therefore, the conformational properties of these nucleosides in solution were studied by performing

NMR experiments, application of PSEUROT and molecular modeling. The data obtainedpointed towards a major conformation (Figure 2) where the benzyl at the 5´imidazolium ring of the base, probably stabilized by an intramolecular cation-Pi interaction. We postulated that this interaction was the driving force for the conformational restrictions observed. To prove this hypothesis we studied by NMR and by molecular dynamics simulations the conformation of the 1,5’

isopropylideneinosine 4 and its N7 protonated analogue 5. We could show that while is quite flexible in solution, when protonation takes place at N7 as in

towards restricted conformations characterized by the disposition of the base and the benzyl group shown in Figure 2 and where the distance between both fragments is in the range of a cationTherefore, these inosine derivatives represent one of the few examples described where the conformation is restricted through an intramolecular cation-Pi interaction.

N

N

O

X= BrX= IX= IX= TFA

Figure 1 Figure 2

E. C. acknowledges the Comunidad de Madrid and the FSE for a predoctoral P. has a CSIC contract from the I3P programme financed by the FSE.

supported by the Spanish CICYT (SAF2006-12713-C02-01)

Rev. 1997, 97, 1303-1324. . Meyer, E. A.; Castellano, R. K.; Diederich, F. Angew. Chem. Int. Ed. 2003, 42, 1210-1250.

, 5, 2903-2912.

stabilization through

José Camarasa1

28006 Madrid, Spain

28006 Madrid, Spain

one of the most relevant noncovalent interactions in as well as in the stabilization of host-guest

Recently, the intramolecular cation-Pi interaction is being applied in asymmetric synthesis, since the introduction of a positive charge in a pyridine ring implies the hange from a free rotational system to a conformationally restricted one with a higher control in the regio

performed the synthesis of 2’,3’-O-and a benzyl, or a methyl group at N7

seemed to indicate conformational restrictions in solution. Therefore, the conformational properties of these nucleosides in solution were studied by performing

NMR experiments, application of PSEUROT and molecular modeling. The data obtained pointed towards a major conformation (Figure 2) where the benzyl at the 5´-position is facing the

Pi interaction. We postulated he conformational restrictions observed. To prove this

hypothesis we studied by NMR and by molecular dynamics simulations the conformation of the 1,5’-O-. We could show that while

is quite flexible in solution, when protonation takes place at N7 as in 5, there is a switch towards restricted conformations characterized by the disposition of the base and the benzyl group shown

agments is in the range of a cation-Pi interaction. Therefore, these inosine derivatives represent one of the few examples described where the

Figure 2

E. C. acknowledges the Comunidad de Madrid and the FSE for a predoctoral nced by the FSE. This work has been

Session I – 4

12

Structure and stability of 2’F-ANA/RNA and ANA/RNA hybrid duplexes

Nerea Martín-Pintado1, Jonathan Watts2, Irene Gómez-Pinto1, Guillem Portela3, Modesto Orozco3, Carlos González1, and Masad Damha2

1Instituto de Química Física “Rocasolano” Consejo Superior de Investigaciones Científicas, Serrano 119,

E-28006, Madrid, Spain

2 Department of Chemistry, McGill University, Montreal, Canada

3 IRB and Department of Biochemistry, University of Barcelona, Spain.

Arabinonucleic acid (ANA) and, especially, its 2’-fluoro-arabino analog (FANA) are interesting compounds for their potential applications in antisense and interference RNA therapy. When hybridized to their complementary RNA, the resulting complex is a substrate of the enzyme RNase H, which cleaves the RNA strand of DNA/RNA hybrids, but is not active against pure RNA duplexes. FANA/RNA and ANA/RNA hybrids are also able to activate RNA interference pathways. These features have attracted the interest of biophysical and structural studies of ANA and FANA derivatives. However, until now only the structure of a small quimeric ANA-RNA and FANA-RNA hairpin had been determined in solution.1,2 In this communication, we report the three-dimensional structure of two hybrid decamers, with the sequence X(GCTATAATGG).r(CCAUUAUAGC), and compare them to the unmodified DNA/RNA hybrid. The structure of the hybrid is intermediate between A- and B-families of double helical structures. Whereas the RNA strand retains most of the features of A-form RNA duplexes, with riboses in the C3’-endo conformation, the sugars in the modified strand adopt conformation between O4’-, and C2’-endo, more typical of B-form duplexes. Although similar to the structure of the control DNA/RNA duplex, FANA and ANA hybrids appear to be less dynamic. Interestingly, FANA substitutions stabilize the duplex structure, whereas ANA substitutions tend to destabilize it. In light of the determined solution structures of these molecules and state-of-the-art molecular dynamics calculations, we can rationalize the effect of these substitutions on the thermodynamics of these molecules.

References 1. Denisov et al.. Nucleic Acids Research 2001, 29, 4284 2. Trempe et al.. J. Am. Chem. Soc 2001, 123, 4896

Session II – 1

13

Use of DNA-hairpins carrying photolabile groups in fabrication of patterned surfaces by

photolithography

Brendan J. Manning1, Simon Leigh2, Sónia Pérez1, Roger Ramos1, Anna Aviñó1, Jon A. Preece2, Ramón Eritja1

1Institute for Research in Biomedicine, Institute for Advanced Chemistry of Catalonia (IQAC), CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine. Helix Building, Baldiri Reixac 15,

E-08028 Barcelona, Spain. 2Nanoscale Chemistry Laboratory. School of Chemsitry. The Universty of Birmingham, B15 2TT, United

Kingdom.

There is a large interest in the use of the self-assembly properties of biomolecules for electronic or biological or sensing applications. Among the biomolecules, oligonucleotides have been captured a large part of this interest1,3. This is due to the existence of a robust method for the preparation of oligonucleotides that allows the production of these compounds carrying reactive groups needed to anchor these molecules to surfaces. Recently it has been shown that is possible to modify a specific region of a surface introducing chemical functionality to direct the adsorption of particulate species4. As example self-assembled monolayers (SAMs) carrying 4-nitrophenoxy head-groups can be converted to 4-aminophenoxy groups by electron-beam and X-ray irradiation5. Selective deposition of citrate-passivated gold nanoparticles (NPs) to the chemically patterned surfaces can subsequently be achieved due to the affinity of negatively charged gold NPs to protonated amino groups at the surface. In the present communication we study the replacement of the electrostatic recognition system for a DNA recognition system that may provide more diversity of patterns. To this end, a method for the fabrication of patterned surfaces using hairpin oligonucleotides carrying photolabile groups is described. A photolabile group has been introduced at the loop of an intramolecular oligonucleotide hairpin. The photolabile oligonucleotide was immobilized on glass and SiO2 surfaces. Photolysis results on the formation of areas carrying single-stranded DNA sequences that direct the deposition of the complementary sequence at the photolyzed sites.

References 1. Aldaye, F. A., Palmer, A.L., Sleimar, H.F. Science 2008, 321, 1795-1799. 2. Seeman, N. C., Lukeman, P. S. Rep. Prog. Phys. 2005, 68, 237-270. 3. Gothelf, K.V., LaBean, T.H. Org. Biomol. Chem. 2005, 3, 4023-4037. 4. Mendes, P. M., Preece, J. A. Curr. Opinion Coll. Inter. Sci. 2004, 9, 236-248 5. Mendes, P. M., Jacke, S., Critchley, K., Plaza, J., Chen, Y., Nikitin, K., Palmer, R.E., Preece, J.A., Evans, S.D., Fitzmaurice, D. Langmuir 2004, 20, 3766-3768

Session II – 2

14

Indirect effects modulating the interaction between DNA

and a cytotoxic bis-naphthalimide unravel two superposed duplex binding and

intercalation processes

Luis González and José Gallego

Centro de Investigación Príncipe Felipe. Avda. Autopista del Saler 16-3 46012 Valencia, Spain.

The structural and dynamic characteristics of double-helical DNA influence the sequence-specific recognition of this polymer by proteins and drugs through indirect sequence effects. Using NMR spectroscopy, surface plasmon resonance, isothermal titration calorimetry and UV thermal denaturation experiments, we have investigated how sequences not making direct contact with the drug modulate the interaction between the cytotoxic agent elinafide and its preferred bisintercalation sites on double-helical DNA. Our combined data are consistent with two superposed interactions, one process involving ligand binding to the DNA duplexes with nanomolar dissociation constants, and another process of ring intercalation characterized by faster dissociation rates and substantially lower affinity in some cases. The sequence of the base pairs flanking the bisnaphthalimide binding tetranucleotides influence both events through indirect readout effects, but these effects appear to be particularly relevant for the intercalation process. The most unfavorable sequences contain specifically oriented A-tracts that oppose DNA-intercalation of the naphthalimide rings, as reflected by strikingly different thermal stability and thermodynamic binding profiles. The complexes of elinafide with these sequences are characterized by weaker intra- and inter-molecular stacking interactions and by increased dynamics of the ligand´s intercalated rings and of the base-pairs forming the tetranucleotide binding site. References 1. González Bulnes, L.; Gallego, J. J. Am. Chem. Soc. 2009 (in press)

Session II – 3

Síntesis de conjugados aminoglicósido

Javier Alguacil1Departamento de Química Orgánica, Universitat de Barcelona, Martí i Franquès 1

Los aminoglicósidos son una gran familia de productos naturales con propiedades

se caracterizan por contener un anillo de 2funcionalizados con grupos amino. La mayor parte de estos compuestos deben su actividad antibiótica a la gran afinidad que presentan por una zona dedisminuyendo la fidelidad en el proceso de traducción y biosíntesis de proteínas y, en consecuencia, provocando la acumulación de proteinas no funcionales que desembocan en la muerte celular.

Sin embargo, el uso terapéutico de los aminoglicósidos como neomicina y paromomicina se ve limitado por una elevada toxicidadRNA com muy escasa selectividad.

Una posible solución a la escasa especificidad dderivatización con elementos de reconocimiento adicional, con el objeto de aumentar su selectividad por el RNA. Relacionado con ello, nuestro interés se ha centrado en evaluar los conjugados aminoglicósidooligonucleótido y aminoglicósidoestos derivados aminoglicosídicos se ha puesto a punto una nueva metodología que combina la cicloadición dipolar de Huisgen catalizada por Cu(I), más conocida como con microondas, que precisa de un mínimo número de etapas intermedias de purificación y que rinde los productos con relativa eficacia. Se expondrán los resultados preliminares de evaluar la afinidad de los conjugados así sintetizados por un modelo del RNA del lugar A bacteriano mediante experimentos de fusión por UV y CD y valoraciones de calorimetría isotérmica (ITC).

References 1. D.P. Arya, “Aminoglycoside-Nucleic Acid Interactions: The case for Neomycin” en Verlag 2005, 263, 149-178. 2. K.B. Sharpless, V.V. Fokin, et al., Angew. Chem. Int. Ed.

15

Síntesis de conjugados aminoglicósido-dinucleótido como posibles ligandos

específicos de RNA

Javier Alguacil1, Sira Defaus1, Ana Claudio1 y Jordi RoblesDepartamento de Química Orgánica, Universitat de Barcelona, Martí i Franquès 1

Los aminoglicósidos son una gran familia de productos naturales con propiedades se caracterizan por contener un anillo de 2-desoxiestreptamina unido a diversos glicósidos funcionalizados con grupos amino. La mayor parte de estos compuestos deben su actividad antibiótica a la gran afinidad que presentan por una zona del RNA ribosomal bacteriano conocida por lugar A, disminuyendo la fidelidad en el proceso de traducción y biosíntesis de proteínas y, en consecuencia, provocando la acumulación de proteinas no funcionales que desembocan en la muerte celular.

uso terapéutico de los aminoglicósidos como neomicina y paromomicina se ve limitado por una elevada toxicidad1, producto de una elevada afinidad a muy diversos segmentos de RNA com muy escasa selectividad.

Una posible solución a la escasa especificidad de los aminoglicósidos podría consistir en la derivatización con elementos de reconocimiento adicional, con el objeto de aumentar su selectividad por el RNA. Relacionado con ello, nuestro interés se ha centrado en evaluar los conjugados aminoglicósido

nucleótido y aminoglicósido-PNA como potenciales ligandos específicos de RNA. Para preparar estos derivados aminoglicosídicos se ha puesto a punto una nueva metodología que combina la cicloadición dipolar de Huisgen catalizada por Cu(I), más conocida como “química click” con microondas, que precisa de un mínimo número de etapas intermedias de purificación y que rinde los productos con relativa eficacia. Se expondrán los resultados preliminares de evaluar la afinidad de los

sintetizados por un modelo del RNA del lugar A bacteriano mediante experimentos de fusión por UV y CD y valoraciones de calorimetría isotérmica (ITC).

Nucleic Acid Interactions: The case for Neomycin” en Topics in Current Chemistry

Angew. Chem. Int. Ed., 2002, 41, 14, 2596-2598. Miller, S. L. Science

dinucleótido como posibles ligandos

y Jordi Robles1 Departamento de Química Orgánica, Universitat de Barcelona, Martí i Franquès 1-11, Barcelona 08028

Los aminoglicósidos son una gran familia de productos naturales con propiedades antibióticas que desoxiestreptamina unido a diversos glicósidos

funcionalizados con grupos amino. La mayor parte de estos compuestos deben su actividad antibiótica a l RNA ribosomal bacteriano conocida por lugar A,

disminuyendo la fidelidad en el proceso de traducción y biosíntesis de proteínas y, en consecuencia, provocando la acumulación de proteinas no funcionales que desembocan en la muerte celular.

uso terapéutico de los aminoglicósidos como neomicina y paromomicina se ve , producto de una elevada afinidad a muy diversos segmentos de

e los aminoglicósidos podría consistir en la derivatización con elementos de reconocimiento adicional, con el objeto de aumentar su selectividad por el RNA. Relacionado con ello, nuestro interés se ha centrado en evaluar los conjugados aminoglicósido-

PNA como potenciales ligandos específicos de RNA. Para preparar estos derivados aminoglicosídicos se ha puesto a punto una nueva metodología que combina la

“química click” 2, y la irradiación con microondas, que precisa de un mínimo número de etapas intermedias de purificación y que rinde los productos con relativa eficacia. Se expondrán los resultados preliminares de evaluar la afinidad de los

sintetizados por un modelo del RNA del lugar A bacteriano mediante experimentos de

Topics in Current Chemistry,Springer-

Science 1953, 117, 528-529.

Session III – 1

16

The Structure and Dynamics of Triplex DNA in gas phase

Annalisa Arcella1, Alberto Pérez1, Modesto Orozco1

1Institut de Recerca Biomèdica, Parc Científic de Barcelona, C/ Baldiri Reixac 10 , Barcelona 08028, Spain

In the last years studies of triplex DNA have been paid much more attention because of its importance as a tool for DNA sequencing, gene control and terapeutic application. The specific character of triplex formation offers viable biochemical, pharmacological and therapeutic applications by acting as represors at the trascriptional (antigene) level and also holds strong promise in the areas of genome mapping. Properties of the Triplex DNA have been studied experimentally using electrospray soft ionization technics. Where DNA undergoes a transition from solvent to vacuo changing its ionization pattern, the process is too fast to follow the structural changes with resolution. Here is where computational technics come in handy. By performing simulations in environments that simulate conditions before and after the ionization we can better understand the nature of these triplexes and how we might be able to improve this sequences for antigene therapy. Intrinsic properties of Triplex are studied exploring its configuartional space through Molecular Dynamic simulation in gas phase. Gas phase trajectrories are compared with solution ones. I'm considering differents structures, such as (GCC⁺)x12mer/18mer, (GCC⁺-ATT)x6mer/9mer and (ATT)x12mer/18mer, where C⁺ stands for protonated cytosine and simulations of ATT structures are performed adding an amino group NH2 at the carbon C8 of adenine. In water simulation treplexes can diffuse though a polyedric box and AMBER8 and TIP3P force field are used. Long range interaction are dealt with PME method, SHAKE algorithm is used for bonds constraint. In gas phase simulations no cutoff was used for non bonded interactions, SHAKE and 1fs time step were used. A delicate decision for MD simulation in gas phase is the assignment of charge state of the triplexes, since control simulations of fully charged and fully neutral triplexes yield completely unfold structuters and coil conformations showing no helicity. According to electrospray experiments1 a net charge of -6/-9 is assigned to the 12mer/18mer triplexes respectively. Since there is no information about the location of these charges, two neutralization protocols were considered. First, the total charge was equally distributed along all phosphates by appropriate scaling of their charges. In the second protocol a set of 6/9 phosphates which minimize the coulombic potential is chosen, while the rest of the phosphates are neutralized by protonating phosphate groups. Both distributed and localized charge methods are performed at 300K and 372K, with this latter temperature usually used in electrospray experiments. Unrestrained MD simulations at constant pressure and temperature in water yields stable structures, as noted in the RMSd values. Hydrogen bonds are fully preserved along the intire trajectory. After 100 ns MD simulations, the structures of the triplex in vacuo are distorted. During the dynamic in gas phase helices fold and the whole structure appear more compacted as shown by cross section and gyration radius time behavior. In general hydrogen bonds are not preserved unlike stacking interactions which instead appear not so different from in solution one as aspected from DNA behavior in gas phase2. Hoogsteen duplexes which compose the triplexes look like more stable than Watson-Crick one probably because of presence of positive charge of the protoneted cytosine. References 1. Wan C., Guo X., Liu, Z. And S. J. Mass spectrom. 2008, 43,164-172. 2. Rueda M., KalKo G.S., Luque F.J., Orozco M. J.Am. Chem. Soc. 2003, 125, 8007-8014

Session III – 2

Structural insights into the mechanism of GTPase activity in

inhibition by binding of the antitumour alkaloid Vinblastine

1 Departmento de Farmacología, Universidad de Alcalá, 28871 Alcalá de Henares, Spain

The binding, hydrolysis and exchange of guanine nucleotides have been identified as central to the conformational flexibility that tubulin exhibits and disassembly of microtubules (MT). The stochastic switching between these two antagonistic processes at the ends of MT results in dynamic instability, which is crucial for many cellular functionsincluding formation of the mitotic spindle during cell division. MT assemble from protofilaments made up of tubulin GTP molecule. But while the GTP bound to the bound to the β subunit is hydrolyzable and, in unassembled tubulin heterodimers, exchangeable. The site for GTP hydrolysis in the β subunit is completed by residues from the glutamate at position 254) following the headGTP hydrolysis, GDP is sequestered in MT at the nucleotide exchangeable site (see Figure). The GTPase activity of tubulin can be enhanced by the binding of colchicine and inhibited by the binding of antitumour Vinca alkaloids such as vinblastine. The 4.1 Å resolution Xcolchicine-vinblastine-stathmin-like domain complex (PDB code 1Z2B) givebinding site, which is located at the interface between two heterodimers. To gain further insight in atomic detail about the structural determinants of both vinblastine binding and the mechanism of GTP hydrolysis, two differGDP/GTP-bound β subunit and the presence and one in the absence of vinblastine. The model templates were, respectively, PDB entries1Z2B, which is curved, and 1JFF,induced tubulin sheets stabilized with taxol. These complexes were refined and simulated by means of molecular dynamics in explicit solvent using the Awill be compared with those obtained for a true GTP-binding site.

PyMOL-generated figure of a growing protofilament fragment made up of two tubulin References 1. Nogales, E. et al. Nature 2005, 435, 9112. Nogales, E. et al. J Cell Sci 2002, 115, 33. Knossow, M. et al. Biochemistry 2007

4. Case, D.A. J Comput Chem 2005, 26, 16685. Nogales, E. et al. J Mol Biol 2001, 313, 1045

17

Structural insights into the mechanism of GTPase activity in


Claire Coderch1 & Federico Gago1

Departmento de Farmacología, Universidad de Alcalá, 28871 Alcalá de Henares, Spain

The binding, hydrolysis and exchange of guanine nucleotides have been identified as central to the conformational flexibility that tubulin exhibits when it polymerises and depolymerises during the assembly and disassembly of microtubules (MT). The stochastic switching between these two antagonistic processes at the ends of MT results in dynamic instability, which is crucial for many cellular functionsincluding formation of the mitotic spindle during cell division.1,2 MT assemble from protofilaments made up of tubulin αβ heterodimers in which each subunit binds a

GTP molecule. But while the GTP bound to the α subunit is nonhydrolyzable and nonexchangsubunit is hydrolyzable and, in unassembled tubulin heterodimers, exchangeable. The site

subunit is completed by residues from the α subunit (including the conserved glutamate at position 254) following the head-to-tail assembly of two heterodimers. Consequently, after GTP hydrolysis, GDP is sequestered in MT at the nucleotide exchangeable site (see Figure).

ubulin can be enhanced by the binding of colchicine and inhibited by the binding alkaloids such as vinblastine. The 4.1 Å resolution X-ray structure of the tubulin

like domain complex (PDB code 1Z2B) gives evidence of the vinblastine binding site, which is located at the interface between two heterodimers. To gain further insight in atomic detail about the structural determinants of both vinblastine binding and

the mechanism of GTP hydrolysis, two different molecular models of a tubulin heterodimer (comprising a subunit and the α subunit from the following heterodimer) were built, one in the

presence and one in the absence of vinblastine. The model templates were, respectively, PDB entries1Z2B, which is curved, and 1JFF,5 which is straight and contains the 3.5 Å resolution structure of zincinduced tubulin sheets stabilized with taxol. These complexes were refined and simulated by means of molecular dynamics in explicit solvent using the AMBER4 force field. The results from these simulations will be compared with those obtained for a true αβ tubulin heterodimer enclosing

generated figure of a growing protofilament fragment made up of two tubulin

, 435, 911-915. , 115, 3-4. 2007, 46, 10595-10602. , 26, 1668-1688. , 313, 1045-1057.

Structural insights into the mechanism of GTPase activity in ββββ-tubulin and its


Departmento de Farmacología, Universidad de Alcalá, 28871 Alcalá de Henares, Spain

The binding, hydrolysis and exchange of guanine nucleotides have been identified as central to the when it polymerises and depolymerises during the assembly

and disassembly of microtubules (MT). The stochastic switching between these two antagonistic processes at the ends of MT results in dynamic instability, which is crucial for many cellular functions

in which each subunit binds a subunit is nonhydrolyzable and nonexchangeable, that

subunit is hydrolyzable and, in unassembled tubulin heterodimers, exchangeable. The site subunit (including the conserved

tail assembly of two heterodimers. Consequently, after GTP hydrolysis, GDP is sequestered in MT at the nucleotide exchangeable site (see Figure).2

ubulin can be enhanced by the binding of colchicine and inhibited by the binding ray structure of the tubulin-

s evidence of the vinblastine

To gain further insight in atomic detail about the structural determinants of both vinblastine binding and ent molecular models of a tubulin heterodimer (comprising a

subunit from the following heterodimer) were built, one in the presence and one in the absence of vinblastine. The model templates were, respectively, PDB entries

which is straight and contains the 3.5 Å resolution structure of zinc-induced tubulin sheets stabilized with taxol. These complexes were refined and simulated by means of

force field. The results from these simulations heterodimer enclosing a nonhydrolysable

generated figure of a growing protofilament fragment made up of two tubulin heterodimers.

Session III – 3

18

Biocatalysis Applied to Separation of Mixtures ββββ-D/L-Nucleosides

Saúl Martínez-Montero,1 Susana Fernández,1 Yogesh S. Sanghvi,2 Vicente Gotor,1 Miguel Ferrero1

1Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006-Oviedo (Asturias), Spain. 2Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA

Nucleoside analogues are a promising class of compounds in drug development for the treatment of diseases like cancer, fungal, bacterial and viral infections.1 For a long period of time it was thought that only nucleosides possessing the natural β-D configuration could present biological activity. However, since the discovery of a β-L-nucleoside, Lamivudine (L-2’,3’-dideoxy-3’-thiacytidine, 3TC) as a drug for the treatment of hepatitis B virus (HBV), the interest in L-nucleosides has drastically increased. A series of simple unnatural L-nucleosides that inhibit HBV replication had been discovered: β-L-2’-deoxythimidine (LdT) and β-L-2’-deoxycytidine (LdC) are examples of this kind of compounds. It is of note that L-nucleosides present lower toxicity while maintaining and antiviral activity comparable to and sometimes greater than their D-counterparts, and higher metabolic stability. The tremendous therapeutic potential as antiviral and antitumor agents of L-nucleosides has stimulated interest in their synthesis,2 however formation of a mixture of D- and L-nucleosides is commonly obtained. This results in a challenging separation of the racemic mixtures.

On the other hand, enzymatic transformations in nucleosides have been recognized as potent strategy to synthesize analogues orthogonally protected in 3’ or 5’ positions.3 Although biocatalysts are frequently used for preparation of enantiopure compounds, they have not been fully exploited as a separation tool in racemic mixtures of nucleosides. Pseudomonas cepacia Lipase (PSL-C) shows total selectivity in acylation toward 3’ position of β-D-2’-deoxynucleosides, however opposite selectivity is shown with β-L-2’-deoxynucleosides acylating the 5’ position. The different behavior showed by PSL-C let us the separation of the β-D/L-thymidine racemic mixture.4 This presentation will describe the extension of this resolution to others β-D/L-2’-deoxynucleosides (Scheme 1) and β-D/L-ribonucleosides. In addition, molecular modeling was performed to explain the selectivity showed by PSL-C in acylation of β-L-nucleosides, which clearly contrasts with the behavior observed in β-D-nucleosides.5

Scheme 1

References

1. a) Rachakonda, S.; Cartee, L. Curr. Med. Chem. 2004, 11, 775-793. b) De Clercq, E. J. Clin. Vir. 2004, 30, 115-133. 2. For a recent review: Mathé, C.; Gosselin, G. Antiviral Res. 2006, 71, 276-281. 3. a) García, J.; Fernández, S.; Ferrero, M.; Sanghvi, Y. S.; Gotor, V. J. Org. Chem. 2002, 67, 4513-4519. b) García, J.;

Fernández, S.; Ferrero, M.; Sanghvi, Y. S.; Gotor, V. Tetrahedron: Asymmetry 2003, 14, 3533-3540. 4. García, J.; Fernández, S.; Ferrero, M.; Sanghvi, Y.S.; Gotor, V. Org. Lett. 2004, 6, 3759-3762. 5. Lavandera, I.; Fernandez, S.; Magdalena, J.; Ferrero, M; Grewal, H.; Saville, C. K.; Kazlauskas, R. J.; Gotor, V.

ChemBioChem 2006, 7, 693-698.

Session III – 4

19

Synthesis of oligonucleotide conjugates by Diels-Alder cycloaddition

Samuel Ortega, Vicente Marchán, Anna Grandas

Departament de Química Orgànica, IBUB, Universitat de Barcelona

Martí i Franquès 1-11, 08028 Barcelona

In the field of oligonucleotides, the Diels-Alder cycloaddition has proved to be very useful for the introduction of reporter groups, to cross-link oligonucleotide chains, and for the preparation of oligonucleotide conjugates.

Our group has pioneered the preparation of peptide-oligodeoxyribonucleotide conjugates taking advantage of the Diels-Alder reaction.1 Peptides were derivatized with a maleimide group at the N-terminal end, and an acyclic diene was attached to the 5’ end of oligodeoxyribonucleotides. The two biomolecules reacted smoothly in water to afford the target conjugate in good yield and high purity. No sequence or composition restriction was imposed, provided that the side chain of cysteine was protected during the conjugation reaction. Conjugates in which the oligonucleotide was replaced by a peptide nucleic acid (PNA) analog were also obtained, though the cycloaddition reaction proceeded more slowly.

In the subsequent step, this methodology was successfully extended to the preparation of peptide-oligoribonucleotide conjugates2 and carbohydrate-oligodeoxynucleotide conjugates. Replacement of maleimide by acryloyl and of the diene by furan, as well as conjugation to cholesterol, were also examined.

More recently, our work has been focused on the synthesis of conjugates in which the desired molecule is attached to the 3’ end of the oligonucleotide chain, which should increase the stability of oligonucleotides to the most ubiquitous 3’-exonucleases.

An overview of the results and problems encountered in the development of the Diels-Alder conjugation methodology will be presented.

References

1. Marchán, V.; Ortega, S.; Pulido, D.; Pedroso, E.; Grandas, A. Nucleic Acids Res., 2006, 34, e24. 2. Marchán, V.; Grandas, A. In Current Protocols in Nucleic Acid Chemistry, chapter 4, unit 4.32; Beaucage, S.L.; Bergstrom, D.E.; Herdewijn, P.; Matsuda, A. Editors (E.W.Harkins, Series Editor); John Wiley & Sons, Inc. USA (2007).

Session IV – 1

20

Using DNA physical properties to determine biological markers

Carles Fenollosa1

1Instituto Nacional de Bioinformática, Parc Científic de Barcelona. Josep Samitier 1-5, 08028 Barcelona.

[email protected]

DNA sequencing projects have started the race to fully annotate complete genomes, including the human one. Despite that, little is known about genetic regulation, the mechanisms that control where and when the genes are expressed. An increasing number of studies have been focused on the DNA molecule and its structure. This has lead to a set of physical properties which can be computed from mathematical models, and describe some aspects of this molecule. Genetic regulation is highly dependant on the DNA structure and, as such, its physical properties seem to hide information which is not actually linked to the sequence but its 3D structure. This talk will first present DNAlive1, a platform to calculate DNA physical properties, which has a friendly interface to view the results in for genetic researchers, plus an API to launch automated calculations if required. Then, some practical examples will be presented which show how relevant and useful is the analysis of this data. Many software promoter detectors use physical properties to locate the position of the promoter and the TSS in using a wide range of descriptors2,3. Furthermore, this data is not restricted to humans, as a detailed analysis drops useful data used to locate regulators in bacteria (work in progress).

References 1. Goñi JR, Fenollosa C, Pérez A, Torrents D, Orozco M. Bioinformatics 2008, 24, 1731-1732 2. Abeel T, Saeys Y, Rouze P, Van de Peer Y. Bioinformatics 2008, 24, i24-i31 3. Goñi JR, Pérez A, Torrents D, Orozco M. Genome Biology 2007, 8, R263

Session IV – 2

21

Efficient Generation of Molecular Complexity from a Common Nucleosidic Precursor

María-Cruz Bonache1, Alessandra Cordeiro1, Ernesto Quesada1, María-José Camarasa1, María-Luisa Jimeno2 and Ana San-Félix1

1Instituto de Química-Médica (C.S.I.C.), Juan de la Cierva, 3, Madrid

2Centro de Química Orgánica “Manuel Lora-Tamayo (C.S.I.C.),Juan de la Cierva, 3, Madrid

One of the goals of modern drug discovery is to increase the diversity and number of small molecules available for biological screening.1 To achieve this goal one approach involves the generation of structural, functional and stereochemical complexities in an efficient manner. Reactions in tandem are some of the ways to achieve efficiency and molecular complexity.2

We have recently discovered that the hydroxy pyrrolidine tricyclic nucleoside 1, (efficiently obtained from xylose) reacted with ketones (acetone, 2-butanone, methylvinyl ketone and 2,4-pentanodione) in a short and efficient fashion to afford a highly diverse set of compounds of unusual and complex architecture. For the synthesis of these compounds a tandem reaction involving iminium/enamine intermediates have been proposed. Details about the synthesis of these compounds and a plausible mechanism to explain their formation will be presented.

References 1.- Schreiber, S. L. Science, 2000, 287, 1964-1969. 2.-(a) Tietze, L.F. Chen. Rev. 1996, 96, 115-136. (b) Hudlicky, T. Chem. Rev. 1996, 96, 3-30. (c) Winkler, J.D. Chem. Rev. 1996,

96, 167-176.

Session IV – 3

22

Was RNA Alone in the RNA World?

Putative Ribonucleases Form under Plausible Prebiotic Earth Conditions

Jorge P. López-Alonso1, Miguel A. Pardo-Cea1, Irene Gómez-Pinto1, Avijit Chakrabartty2, Carlos González1, and Douglas V. Laurents1*

1Instituto de Química Física “Rocasolano” Consejo Superior de Investigaciones Científicas, Serrano 119,

E-28006, Madrid, Spain

2 Departments of Medical Biophysics and Biochemistry, University of Toronto, Toronto, ON M5G-2M9, Toronto, Canada

RNAs are generally thought to have served as the genetic material and as the chief catalysis in the evolution of life.1 Proteins would have gradually replaced many catalytic RNAs at the twilight of the RNA World. Some amino acids, including Lys, Ile, Ala, Gly and His form under the same putative prebiotic conditions2,3, whereas different ones are needed for producing Tyr, Phe and Trp. Here, we report the 3D structure of a peptide called KIA7H which is composed of only Lys, Ile, Ala, Gly and His. This 20 mer peptide adopts a four helix bundle with a specifically packed hydrophobic core. Therefore, “one pot” prebiotic proteins with well defined structures might have arisen early, even at the dawn of the RNA World. The Trp variant, KIA7W, was also studied. It adopts a 3D structure similar to that of KIA7H and also its Tyr and Phe variants studied previously4, but is remarkably more stable. When tested for ribonucleolytic activity, KIA7H, KIA7W and even short, unstructured peptides rich in His and Lys, in combination with Mg2+, Mn2+ or Ni 2+ but not Cu2+, Zn2+ or EDTA, specifically cleave the single stranded region in a RNA stem loop. This suggests that prebiotic peptide – divalent cation complexes with ribonucleolytic activity might have co-inhabited the RNA World. References 1. Gilbert, W. Nature 1986, 319, 618 2. Miller, S. L. Science 1953, 117, 528-529. 3. Matthews, C. N. and Moser, R. E. Nature 1967, 215, 1230-1234. 4. López de la Osa, J., Bateman, D. A., Ho, S., González, C., Chakrabartty, A. and Laurents, D. V. PNAS 2007, 104, 14941-

14946.

KIA7H structure

Keynote Conference

23

Targeting the HIV-1 Genome: RNA Structure-Based Drug Discovery

Thomas L. James

Department of Pharmaceutical Chemistry

University of California, San Francisco

With growing resistance of HIV-1 to current drugs, there is need for research on other HIV-1 targets. Although it may not be possible to devise a drug to which resistance will never emerge, some molecular targets are constrained by structure and function, and therefore may be more difficult for the virus to alter in order to acquire resistance. Due to cross-resistance, it is also mandatory to find new molecular targets. We are using structural biology to explore some functionally critical elements of the HIV-1 RNA genome, including structure determination, with the goal of using the information to develop drug leads. There has been little effort to discover drugs rationally using RNA structures, although there is precedent for RNA-binding drugs in that such drugs are commonly used for bacterial infections. Techniques of structure-based drug discovery applicable to proteins are not directly applicable to RNA, so we have been developing methods to find small molecule ligands that will bind with some specificity to selected RNA targets. Each retroviral particle contains two copies of HIV-1 RNA as a noncovalent dimer with strands aligned parallel and in register, contacting each other most stably at a highly conserved region called the dimer linkage site or the dimerization initiation site (DIS) within the 5' leader. The DIS is crucial for both HIV-1 strands to recognize one another and to transform into the linear form that will interact with the nucleocapsid protein in the mature virion. We have used NMR to determine three-dimensional structures and other features of DIS. We have also developed a strategy to discover ligands for RNA targets based on computational (or virtual) screening of large databases of commercially available compounds for binding, followed by NMR binding screens of computational “hits”. The results of some of these studies will be described.

Session V – 1

24

Comparative Structural Study on DNA and RNA Flexibility

Ignacio Faustino1, Alberto Pérez1,2,3, Modesto Orozco1,2,4,5

1 Joint IRB-BSC Program on Computational Biology, Institute of Research in Biomedicine, Parc Científic de Barcelona, Josep Samitier 1-5, Barcelona 08028. Spain and Barcelona Supercomputing Centre, Jordi Girona 31, Edifici Torre Girona. Barcelona 08034, Spain 2 Barcelona Supercomputing Centre, Jordi Girona 31, Edifici Torre Girona. Barcelona 08034 3 Department de Fisicoquímica. Facultat de Farmàcia. Universitat de Barcelona. Avgda Diagonal 643, Barcelona 08028, Spain. 4 National Institute of Bioinformatics, Parc Científic de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain 5 Departament de Bioquímica, Facultat de Biología, Avgda Diagonal 647, Barcelona 08028, Spain.

DNA and RNA molecules are known to have different helical flexibility properties1. We present the most extensive molecular dynamics (MD) study ever made using the latest versions of nucleic acids force fields (CHARMM27 and parmbsc0) using for this purpose four representative long duplexes (18-mer) which yield canonical B-DNA and A-RNA respectively, designed to contain several copies of each individual base pair step. This study, which extends and complements previous works in our group 2,3,4, highlights the main differences not only between parmbsc0 and CHARMM27 families of simulations but with X-ray and NMR data. CHARMM27 maintains the integrity of DNA duplex, but in RNA multiple base pairs experienced local opening events into the major groove on the nanosecond time scale. Parmbsc0 maintains the integrity of both DNA and RNA duplex allowing the full characterization of the structural and physical properties of both molecules. As a final output of our work a new mesoscopic model that allows the fast and accurate representation of flexibility in large DNA and RNA polymers.

References 1. Hagerman PJ. Annu. Rev. Biophys. Biomol. Struct., 1997, 20. 2. Pérez et al. Nucleic Acids Res., 2004, 32, 6144-6151. 3. Noy et al. Journal of Molecular Biology 2004, 343, 627-38. 4. Perez et al. Nucleic Acids Research 2008, 36 (7), 2379-2394.

Session V – 2

Modified siRNAs and Biocompatible Gold nanoparticles

Álvaro Somoza1, Brendan Manning

1IMDEA Nanociencia, Facultad de Ciencias, Avda Fco. Tomás y Valiente 7, Ciudad Universitaria de Cantoblanco

2 IRB Barcelona, Parc Cientific de Barcelona

RNA interference (RNAi) is a powerful tool that allows the inhibition

way.1This process is triggered by double stranded RNAs known as small interfering RNAs (siRNAs).During the last years many efforts have been focused on the use of this technology as targeted therapy, since it could be used for the treatment of diseases were the overas cancer or inflammation related diseases.to have long lasting effects, good biodistribution and high tissue, communication we present novel chemical modifications in siRNAs to increase serum stability and a novel biocompatible gold nanoparticle (NP) as a carrier.the gold NP´s yields a nanostructure that inhibits the gene expression of the luciferase gene in SHand HeLa cell lines.

References 1. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. 2. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. 3. Castanotto, D. & Rossi, J. J. Nature 2009

4. Kurreck, J. Angew. Chem. Int. Ed. 2009

5. Giljohann, D. A., Seferos, D. S., Prigodich, A. E., Patel, P. C. & Mirkin, C. A.

25


, Brendan Manning2, Monserrat Terrazas2, Ramón Eritja

IMDEA Nanociencia, Facultad de Ciencias, Módulo C-IX, 3ª Planta

Avda Fco. Tomás y Valiente 7, Ciudad Universitaria de Cantoblanco28049, Madrid

IRB Barcelona, Parc Cientific de Barcelona

Baldiri Reixac, 10 08028, Barcelona

RNA interference (RNAi) is a powerful tool that allows the inhibition of genes in a very specific This process is triggered by double stranded RNAs known as small interfering RNAs (siRNAs).

During the last years many efforts have been focused on the use of this technology as targeted therapy, r the treatment of diseases were the over-expression of genes are involved, such

as cancer or inflammation related diseases.3 In order to have an effective treatment it would be desirable to have long lasting effects, good biodistribution and high tissue, organ or cell selectivity.communication we present novel chemical modifications in siRNAs to increase serum stability and a novel biocompatible gold nanoparticle (NP) as a carrier.5 The combination of these modified siRNAs and

ds a nanostructure that inhibits the gene expression of the luciferase gene in SH

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. Nature 1998, 391, 8062. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. Nature 2001, 411, 494

2009, 457, 426-433. 2009, 48, 1378-1398.

5. Giljohann, D. A., Seferos, D. S., Prigodich, A. E., Patel, P. C. & Mirkin, C. A. J. Am. Chem. Soc. 2009


, Ramón Eritja2

IX, 3ª Planta Avda Fco. Tomás y Valiente 7, Ciudad Universitaria de Cantoblanco

of genes in a very specific This process is triggered by double stranded RNAs known as small interfering RNAs (siRNAs).2

During the last years many efforts have been focused on the use of this technology as targeted therapy, expression of genes are involved, such

In order to have an effective treatment it would be desirable organ or cell selectivity.4 In this

communication we present novel chemical modifications in siRNAs to increase serum stability and a The combination of these modified siRNAs and

ds a nanostructure that inhibits the gene expression of the luciferase gene in SH-SY5Y

, 806-811. , 494-498.

2009, 131, 2072-2073.

Session VI – 1

26

pH-modulated Watson-Crick duplex - quadruplex equilibria of guanine-rich and

cytosine-rich DNA sequences upstream of the c-kit transcription initiation site

Pavel Bucek1, Joaquim Jaumot2, Anna Aviñó3, Ramon Eritja3, Raimundo Gargallo2

1Dept. of Chemistry, Faculty of Science, Masaryk University, Kotlárská 2, 61137, Brno, Czech Republic

2 Dept. of Analytical Chemistry, University of Barcelona, Martí i Franqués 1 - 11, E-08028 Barcelona,

Spain 3 Instituto de Biologia Molecular de Barcelona, CSIC, Jordi Girona 18-26, E-08034 Barcelona, Spain

Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures 1. Recently, it has been proposed the formation of a G-quadruplex structure near the c-kit transcription initiation site 2. In this work we have studied the acid-base equilibria and the thermal-induced unfolding of the structures formed by a guanine-rich and by its complementary cytosine-rich strands in c-kit by means of circular dichroism and molecular absorption spectroscopies. In addition, the competition between the Watson-Crick duplex and the isolated structures has been studied as a function of pH and temperature. Appropriate multivariate data analysis methods based on both hard- and soft-modeling analysis have been applied 3. The results have shown that G-quadruplex and i-motif coexist with the Watson-Crick duplex in the pH range 4 - 6. At pH 7.0, only a minor amount of isolated structures is present.

References 1. Huppert, J. L.. Biochimie 2008, 90, 1140-1148. 2. Fernando, H.; Reszka, A. P.; Huppert, J.; Ladame, S.; Rankin, S.; Venkitaraman, A. R.; Neidle S.; Balasubramanian, S. Biochemistry 2006, 45, 7854-7860 3. Jaumot, J.; Vives, M.; Gargallo, R. Analytical Biochemistry 2004, 327, 1-13.

Session VI – 2

27

A loop-loop RNA tertiary interaction motif shared by most natural hammerhead

ribozymes

David Dufour1, Marcos de la Peña2, Selma Gago2, Ricardo Flores2 and José Gallego1

1Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, 46012 Valencia, Spain

2Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Avda. de los Naranjos s/n, 46022

Valencia, Spain

Hammerhead ribozymes are small catalytic RNAs found in a variety of organisms that can also act, when conveniently manipulated, as antisense agents against specific RNA targets. Natural hammerheads are composed of a conserved catalytic core flanked by three helices usually capped by short loops, with loop-loop tertiary interactions playing a key role in catalytic activity. Using a combination of NMR spectroscopy, site-directed mutagenesis, kinetics analysis and infectivity bioassays, we have examined the structure and functional impact of loops 1 and 2 of the (+) and (-) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5’ side and an extrahelical U at its 3’ side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3’ side. These structural features promote a specific loop-loop interaction motif across the major groove. Unexpectedly, the essential features of this tertiary structure element, base pairing between the 5’ U of loop 1 and the 3’ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, were found to be likely shared by a significant fraction of natural hammerheads1. The discovery of this motif illustrates how conserved RNA conformations rather than sequence trigger a tertiary interaction leading to functional RNA structures.

References 1. Dufour, D.; de la Peña, M.; Gago, S.; Flores, R.; Gallego, J. Nucleic Acids Research 2009, 37, 368-381.

Session VI – 3

28

Carbohydrate-oligonucleotide conjugates: how sugars other than (deoxy-)ribose affect DNA stability and allow to study intermolecular forces

José J. Reina1, Ricardo Lucas1, Anna Aviñó2, Irene Gómez-Pinto3, Irene Díaz1, Pedro M. Nieto1, Carlos

Gonzalez3, Ramón Eritja2, Juan C. Morales1*

1Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC - Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla

2 Instituto de Investigación Biomédica, Barcelona, Parc Cientific, IBMB - CSIC, CIBER - BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Josep Samitier 1, E-08028 Barcelona

3 Instituto de Química Física 'Rocasolano', CSIC, C/. Serrano 119, 28006 Madrid

Carbohydrate-oligonucleotide conjugates are being used to facilitate the internalization of the so

promising oligonucleotide-based drugs.1 Recently, these type of conjugates are also being applied in

nanomaterials.2 Our research group is currently studying how different saccharide units may affect DNA stability in these carbohydrate-oligonucleotide conjugates. We have observed that mono- and disaccharides, such as glucose or lactose, attached to the 5’-end of short DNA sequences are capable

of stabilizing a duplex up to 1.3 kcal.mol-1 respect to the DNA control. These results are quite surprising since these carbohydrates are highly hydrophilic molecules.

Secondly, we have used carbohydrate-oligonucleotide conjugates to study carbohydrate molecular interactions. Protein-carbohydrate recognition is of fundamental importance for a large number of biological processes, where carbohydrate-aromatic stacking is a widespread structural motif found

but poorly understood.3 We describe, for the first time, the measurement of carbohydrate-aromatic interactions by their contribution to the stability of a dangling-ended carbohydrate-DNA model system

that functions as an energetic probe.4 We observe clear differences in the energetic of the interaction of several monosaccharides with a benzene moiety depending on the number of hydroxyl groups, the stereochemistry and the presence of a methyl group in the pyranose ring. A fucose-

benzene pair is the most stabilizing of the studied series (-0.4 Kcal.mol-1), placing this interaction in the same range of other more studied interactions with aromatic residues of proteins, such as Phe-Phe, Phe-Met or Phe-His.

References 1. H. Lönnberg, Bioconjugate Chem., 2009, DOI: 10.1021/bc800406a. 2. G. A. Burley et al. J. Am. Chem. Soc. 2006, 128,1398-1399. 3. a) A. B. Boraston, D. N. Bolam, H. J. Gilbert, G. J. Davies, Biochem. J. 2004, 382, 769; b) H. Lis, H. Sharon, Chem. Rev.

1998, 98, 637. 4. J. C. Morales, J. J. Reina, I. Díaz, A. Aviñó, P. M. Nieto, R. Eritja, Chem. Eur. J. 2008, 14, 7828-7835. (Commented in Nature

Chem. Biol., 2008, 4(10), 586-587).

Session VII – 1

29

Antiviral and biological properties of glucose-conjugated antisense

oligodeoxynucleotides that inhibit HIV-1 replication in cell culture

Jóse A. Reyes-Darias1, Francisco J. Sánchez-Luque1, Sonia Perez2, Ramón Eritja2, Juan Carlos

Morales3, Cristina Romero-López1, Soledad Marton-García1, Vicente Augustin Vacas1, Alfredo Berzal-Herranz1

1Instituto de Parasitología y Biomedicina “López-Neyra”, Parque tecnológico de Ciencias de la Salud, CSIC. Avda. del Conocimiento s/n, Armilla, 18100, Granada, Spain 2Instituto de química avanzada de Cataluña-CSIC. Grupo de Química de Ácidos Nucleicos. Jordi Girona 18-26, 08034 Barcelona, Spain. 3Instituto de Investigaciones Químicas-CSIC. Grupo de Carbohidratos. Américo Vespucio, 49 Isla de la Cartuja 41092 Sevilla, Spain

Antisense oligodeoxynucleotides (ODNs) are short synthetic strands of DNA, which are complementary to a target sequence of RNA designed to halt a biological event, such as transcription, translation or splicing. Unfortunately the therapeutic potential of unmodified ODNs is dramatically limited by their poor cellular uptake and lack of stability against enzymatic degradation. Under in vivo conditions they are rapidly hydrolyzed by nucleases and then cleared from the body through renal excretion. Therefore intense research activity in this field has produced a vast number of ODNs bearing chemical modifications in the backbone, mainly at the sugar moiety or at the phosphodiester linkages, in order to assure a prolonged in vivo half- life, still preserving if not improving the hybridization capabilities of the oligomers versus the target complementary sequences. Phosphorothioate-modified (PS) oligonucleotides, in which an oxygen atom of the phosphodiester linkage has been replaced by a sulphur atom, are the most widely used molecules in cell cultures, animals and humans. In addition to backbone and sugar modifications, various ligands have been attached or conjugated to ODNs such as cholesterol, folic acid, fatty acids, etc. to improved uptake, biodistribution, target delivery and mechanism of antisense action. To evaluated antiviral and biological properties of glucose-conjugated antisense oligodeoxynucleotides the 25-mer phosphorothioate oligodeoxynucleotide GEM911, which is complementary to the initiation codon of gag gene of HIV-1 RNA, was selected as a model in this study. We had synthesized unmodified (GEM91) and modified (GEM91-PS) oligonucleotides conjugated at the 5’ end with a glucose residue. Our results show that GEM91 and Glucose-GEM91 have antiviral activity only when they are delivered by transfect agents to culture cells. However, GEM91-PS and Gluc-GEM91-PS display antiviral activity in cell culture in the presence or absence of transfection agent. When the GEM91-PS and Gluc-GEM91-PS were added free to infected Jurkat cells the formation of syncytia was blocked. GEM91-PS and Gluc-GEM91-PS have a half-life approximately 40-50-fold higher than GEM91 and Glucose-GEM91. Taken together these results suggest that the conjugation of a glucose residue at the 5’ end of ODNs do not negatively interfere with their biological properties.

References

1. Lisziewicz J, Sun D, Weichold FF, Thierry AR, Lusso P, Tang J, Gallo RC, Agrawal S. PNAS 1994 16;91(17):7942-6.

Session VII – 2

30

Structural basis for the binding of the new antitumor drug Zalypsis to selected DNA triplets using molecular dynamics

Juan Antonio Bueren-Calabuig1, Ana Negri1, Carmen Cuevas2, Carlos María Galmarini2, Federico Gago1

1Departamento de Farmacología, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain

2Departamento de Biología Celular, Avda. de los Reyes, 1, 28770 Colmenar Viejo, PharmaMar, Madrid, Spain

Zalypsis is a new synthetic dimeric isoquinoline alkaloid (Fig.1) developed by PharmaMar S.A.U. (Colmenar Viejo, Madrid) which is presently in phase I clinical trials for the treatment of solid tumours and hematological malignancies. This molecule is structurally related to jorumycin, a natural compound isolated from the skin and mucus of the Pacific nudibranch Jorunna funebris, and also to renieramycins from sponges and tunicates, safracins and saframycins from bacteria and marine sponges, and ecteinascidins from marine tunicates.1 These carbinolamine-containing compounds react, through an iminium intermediate that is generated by dehydration, with the exocyclic amino group of selected guanines in the minor groove of DNA. The resulting adduct, which is additionally stabilized through van der Waals interactions and one or more hydrogen bonds with neighbouring nucleotides in the opposite strand of the DNA double helix, creates the equivalent to a functional interstrand crosslink that can lead to strong inhibition of the early phases of transcription.2 The most favourable DNA triplets for adduct formation, as assessed from experiments measuring increases in the melting temperatures of fluorescently labelled oligodeoxynucleotides, are AGG, GGC, AGC, CGG and TGG.1 To gain insight into the structural basis for these findings, we studied in atomic detail the feasibility of achieving the geometries required to activate dehydration of the carbinolamine prior to the nucleophilic attack that leads to covalent bond formation in a series of oligodeoxynucleotides of general sequence 5’-d(ATAATAXYZATAATA)/5’-d(TATTATZ’Y’X’TATTAT), where XYZ/Z’Y’X’ stands for AGA/TCT, AGG/CCT and AGC/GCT. The initial complexes were built as previously reported for trabectedin,3 refined using energy minimization techniques in the AMBER force field4 and simulated in an explicit water box using unrestrained molecular dynamics simulations for 10 ns. Analysis of hydrogen-bonding patterns (Fig.2) in the molecular models of the resulting Zalypsis®-DNA precovalent complexes provided a rationale that accounts for the distinct binding of the drug to three types of central triplets representative of good (AGC), intermediate (AGG) or poor (AGA) binding sites for this drug.

Figure 1. Structure of Zalypsis ® Figure 2. View of the central part of a

DNA−Zalypsis precovalent complex

References 1. Leal, J.F.M. Biochem Pharmacol 2009, doi:10.1016/j.bcp.2009.04.003. 2. Casado J.A. Mol Cancer Ther 2008, 7, 1309–1318. 3. Marco E. J Med Chem 2006, 49, 6925–6929. 4. Case D.A. J Comput Chem 2005, 26, 1668–1688.

Session VII – 3

31

Structure and function of the leader transcription-regulating sequence of

Transmissible Gastroenteritis Virus

David Dufour1*, Pedro A. Mateos2*, Isabel Sola2, Luis Enjuanes2 and José Gallego1

1Centro de Investigación Príncipe Felipe. Avda. Autopista del Saler 16, 46012 Valencia, Spain

2Centro Nacional de Biotecnología. C/ Darwin 3, Cantoblanco, 28049 Madrid, Spain

Transcription of the Transmissible Gastroenteritis Virus (TGEV) genome generates a nested set of 5’ and 3’ coterminal subgenomic mRNAs. This discontinuous transcription process depends on transcription-regulating sequences located at the 3’-end of the leader region (TRS-L) and preceding each viral gene (TRS-B). Discontinuous transcription implies the template-switch of the nascent negative strand from the TRS-B region to the leader TRS, that would act as the acceptor RNA. All TRS-L and TRS-Bs contain a highly conserved CUAAAC core sequence (CS) critical for the template switch process1. To study the functional impact of TRS-L structure on TGEV transcription, we are using a combination of site-directed mutagenesis, NMR spectroscopy and UV thermal experiments together with in vivo transcriptional analyses. The results indicate that a 35-nucleotide oligomer representing the wild-type TRS-L forms a hairpin in which the CS and its neighbouring residues occupy the apical loop and are not involved in stable intramolecular interactions. TRS-L mutations stabilizing the hairpin structure and/or hiding the CS in a double-stranded region have a detrimental effect on viral transcription, indicating that the CS structure needs to remain labile so that this sequence can hybridize with the nascent negative RNA strand during the template switch process.

* Both authors have equally contributed to this work.

References 1. Zúñiga, S.; Sola, I.; Alonso, S.; Enjuanes, L. Journal of Virology 2004, 78, 980-994.

Session VIII – 1

Screening for small molecules interacting with RNA by NMR

I. Gómez

1Instituto de Química

2Dept. of Pharmaceutical3Departament de Química Orgànica. Universidad de Barcelona.

The general goal of this research is to obtain ligands that selectively interact with target RNA

structures involved in human diseases. Moreover, the connection between the target structures and disease opens up the possibility of findings compounds with therapeutic application.target is telomerase, since telomerase activity has been shown in several studcell immortality, and chromosome stability in cancer cells.telomerase as a potential target for cancer diagnosis and therapy.ribonucleoprotein enzyme essential for telomere length maintenance by adding telomeric sequence onto chromosome ends. The core enzyme contains the protein TERT and the RNA component TER. The RNA moiety contains one highly conserved putative pseudoknot which is required for telomeraseassembly RNP (Fig. 1). Our goal is to develop a method to discover novel noncompounds that interact with high affinity and specificity with a new target: the structured P2b hairpin in telomerase RNA in order to inhibi

flexible docking program called MORDOR (MOlecular Recognition with a Driven dynamics OptimazeR). MORDOR allows flexibility not only in the ligand but also in the receptor. 200 compounds witexperimentally for binding using saturation transfer difference NMR. We experimentally found 54% binders with the new program MORDOR. Most of the compounds bind to the U bulge in the P2b hairpin. Specifity of the binders to hTR RNA was also tested by STD NMR using aminoacylspecific to hTR RNA. This preliminary data shows that the new program MORDOR obtained scaffolds that bind to a unique tthe required specificity; binding affinity of telomerase RNA is responsive to ligand characteristics and can be modulated by addiction or deletion of certain chemical groups.

The second RNA target is a pre-such as Pick’s disease, corticobasal degeneration, Alzheimer disease and frontoparkinsonism linked to chromosome 17 (FTDPintron junction forms a stable stemsplice site and regulates the splicing of exon 10identify several ligands that bind with high affinity aa promising start in the search for new therapeutic agents.wild type Tau RNA fragment and two mutants have been explored by NMR methods. Preliminary data reveals a strong interaction in all cases which occurs preferentially with the nucleotides in a bulged region of the stem-loop structure.

Fig.1. Vertebrate telomerase RNA secondary structure.

32


I. Gómez-Pinto1,2, P. López-Senín3, V. Marchán3

C. Guilbert2, T.L. James2

Instituto de Química-Física Rocasolano. CSIC. Madrid Dept. of Pharmaceutical-Chemistry, University of California, San Francisco, USA

Departament de Química Orgànica. Universidad de Barcelona.

The general goal of this research is to obtain ligands that selectively interact with target RNA human diseases. Moreover, the connection between the target structures and

disease opens up the possibility of findings compounds with therapeutic application.target is telomerase, since telomerase activity has been shown in several studies to be correlated with cell immortality, and chromosome stability in cancer cells. These facts lead to the general supposition of telomerase as a potential target for cancer diagnosis and therapy. Telomerase is a eukaryotic

ential for telomere length maintenance by adding telomeric sequence onto chromosome ends. The core enzyme contains the protein TERT and the RNA component TER. The RNA moiety contains one highly conserved putative pseudoknot which is required for telomeraseassembly RNP (Fig. 1). Our goal is to develop a method to discover novel noncompounds that interact with high affinity and specificity with a new target: the structured P2b hairpin in telomerase RNA in order to inhibit telomerase activity. To find such lead compounds we tested a new

flexible docking program called MORDOR (MOlecular Recognition with a Driven dynamics OptimazeR). MORDOR allows flexibility not only in the ligand but also in the receptor. 200 compounds with the highest scores were assayed experimentally for binding using saturation transfer difference NMR. We experimentally found 54% binders with the new program MORDOR. Most of the compounds bind to the U bulge in the P2b hairpin. Specifity of the binders o hTR RNA was also tested by STD NMR using HIVaminoacyl-tRNA site (A-SITE) of ribosomal RNA. Results show 14 binders specific to hTR RNA. This preliminary data shows that the new program MORDOR obtained scaffolds that bind to a unique tertiary RNA structure with the required specificity; binding affinity of telomerase RNA is responsive to ligand characteristics and can be modulated by addiction or deletion of certain chemical groups.

-mRNA related with Tau gene associated to neurodegenerative diseases,

such as Pick’s disease, corticobasal degeneration, Alzheimer disease and frontoparkinsonism linked to chromosome 17 (FTDP-17). A 25-nucleotide-long RNA covering the exon 10

nction forms a stable stem-loop structure that limits the access of the splicing machinery to the 5’splice site and regulates the splicing of exon 10. Dynamic combinatorial chemistry

bind with high affinity and stabilize this secondary structure which represents a promising start in the search for new therapeutic agents. The interaction of these compounds with the wild type Tau RNA fragment and two mutants have been explored by NMR methods. Preliminary data

veals a strong interaction in all cases which occurs preferentially with the nucleotides in a bulged region


Francisco, USA

Departament de Química Orgànica. Universidad de Barcelona.

The general goal of this research is to obtain ligands that selectively interact with target RNA human diseases. Moreover, the connection between the target structures and

disease opens up the possibility of findings compounds with therapeutic application. The first studied ies to be correlated with

These facts lead to the general supposition of Telomerase is a eukaryotic

ential for telomere length maintenance by adding telomeric sequence onto chromosome ends. The core enzyme contains the protein TERT and the RNA component TER. The RNA moiety contains one highly conserved putative pseudoknot which is required for telomerase activity and

-peptide, non-nucleotide compounds that interact with high affinity and specificity with a new target: the structured P2b hairpin in

t telomerase activity. To find such lead compounds we tested a new flexible docking program called MORDOR (MOlecular Recognition with a Driven dynamics OptimazeR). MORDOR allows flexibility not only in the ligand

h the highest scores were assayed experimentally for binding using saturation transfer difference NMR. We experimentally found 54% binders with the new program MORDOR. Most of the compounds bind to the U bulge in the P2b hairpin. Specifity of the binders

HIV-1 TAR and the 16 S SITE) of ribosomal RNA. Results show 14 binders

specific to hTR RNA. This preliminary data shows that the new program ertiary RNA structure with

the required specificity; binding affinity of telomerase RNA is responsive to ligand characteristics and can be modulated by addiction or deletion of certain

gene associated to neurodegenerative diseases, such as Pick’s disease, corticobasal degeneration, Alzheimer disease and fronto-temporal dementia with

long RNA covering the exon 10–5’-access of the splicing machinery to the 5’-

ynamic combinatorial chemistry has been used to nd stabilize this secondary structure which represents

The interaction of these compounds with the wild type Tau RNA fragment and two mutants have been explored by NMR methods. Preliminary data

veals a strong interaction in all cases which occurs preferentially with the nucleotides in a bulged region

Session VIII – 2

33

Effects of Major Groove Substitution on RNA Interference

Montserrat Terrazas,1 Eric T. Kool2

1Departamento de Nanotecnología Química y Biomolecular (Grupo de Química de Ácidos Nucleicos), Instituto de Química Avanzada de Cataluña (IQAC)-Consejo Superior de Investigaciones Científicas

(CSIC), Jordi Girona 18-26, 08034 Barcelona

2 Department of Chemistry, Stanford University

RNA interference (RNAi) represents a powerful molecular strategy for the inhibition of gene

expression.1 This process can be induced by 21-23 nt double stranded RNAs, known as short interfering RNAs (siRNAs).2 These oligoribonucleotides are recognized by the RNA-induced silencing complex (RISC)3 –a protein located in the cytoplasm– which induces endonucleolytic cleavage of the target mRNA, preventing its translation into protein. Despite the significance of RNAi both as a biological tool and as a potential therapeutic strategy, some important challenges remain, including development of strategies for effective siRNA delivery, improving stability against serum nuclease degradation and addressing low sequence specificity that results in undesired "off tagert" activities.4 To address these limitations and to further understand the mechanism of silencing, several research groups have been actively studying siRNAs with various chemical modifications.5 However, most of the focus to date has been on modifying the RNA backbone, and few laboratories have modified the bases of siRNAs, despite their central involvement in target recognition.

In this work, we have evaluated steric and stability effects on RNAi activity by use of siRNAs modified with propynyl and methyl functionalities at the C-5 position of pyrimidine nucleobases (siRNAs containing mU, mC and pU substitutions).6 Cellular gene suppression experiments showed that, at the 5' half of the guide strand, the bulky 5-propynyl modification was detrimental to RNA interference activity, probably due to disruption of interactions in the major groove of the active RISC complex. However, the smaller methyl group was found to have positive effects on gene suppression activity of the RNAs. The major groove modifications also increased the serum stability of siRNAs.

1. Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D.W.Y. Nat. Chem. Biol. 2006, 2, 711-719. 2. Elbashir, S.M.; Lendeckel, W.; Tuschl, T. Genes Dev. 2001, 15, 188-200. 3. Valencia-Sanchez, M.A.; Liu, J.; Hannon, G.J.; Parker, R. Genes Dev. 2006, 20, 515-524. 4. Alemán, L.M.; Doench, J.; Sharp, P.A. RNA 2007, 13, 385-395. 5. Manoharan, M. Curr. Opin. Chem. Biol. 2004, 8, 570-579. 6. Terrazas, M.; Kool, E.T. Nucleic Acids Res. 2009, 37, 346-353.

35

List of Participants

Javier Alguacil Blanco Universitat de Barcelona

[email protected]

Margarita Alvira Torre Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

Annalisa Arcella Institut de Recerca Biomèdica

[email protected]

Gerard Artigas Solé Universitat de Barcelona

[email protected]

Anna Aviñó Andrés Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

Flavia Barragán Clavero Universitat de Barcelona

[email protected]

María Cruz Bonache de Marcos Instituto de Química Médica, CSIC

[email protected]

Juan Antonio Bueren Calabuig Universidad de Alcalá

[email protected]

Marta Camacho Artacho Universidad de Alcalá

[email protected]

María José Camarasa Rius Instituto de Química Médica, CSIC

[email protected]

Elena Casanova Malpica Instituto de Química Médica, CSIC

[email protected]

Chiara Lara Castellazi Istitut de Recerca Biomedica

[email protected]

Ana María Claudio Montero Universitat de Barcelona

[email protected]

Claire Coderch Bou Universidad de Alcalá

[email protected]

Maria Cristina de Almagro García Facultad de Farmacia, Universidad de Barcelona

[email protected]

Fernando Díez García Instituto de Química Física Rocasolano, CSIC

[email protected]

David Dufour Rausell Centro de Investigación Príncipe Felipe

[email protected]

Ramon Eritja Casadellà Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

Núria Escaja Sánchez Universitat de Barcelona

[email protected]

Carme Fàbrega Claveria Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

36

Ignacio Faustino Plo Universitat de Barcelona

[email protected]

Carles Fenollosa Bielsa Parc Científic de Barcelona

[email protected]

Oscar Flores Guri Istitut de Recerca Biomedica

[email protected]

Federico Gago Badenas Universidad de Alcalá

[email protected]

José Gallego Sala Centro de Investigación Príncipe Felipe

[email protected]

Raimundo Gargallo Gómez Universitat de Barcelona

[email protected]

Irene Gómez Pinto Instituto de Química Física Rocasolano, CSIC

[email protected]

Luis González Bulnes Centro de Investigación Príncipe Felipe

[email protected]

Carlos González Ibáñez Instituto de Química Física Rocasolano, CSIC

[email protected]

Anna Grandas Sagarra Universitat de Barcelona

[email protected]

Thomas L. James University of California San Francisco

[email protected]

Douglas V. Laurents Instituto de Química Física Rocasolano, CSIC

[email protected]

Jorge Pedro López Alonso Instituto de Química Física Rocasolano, CSIC

[email protected]

Paula López Senín Universitat de Barcelona

[email protected]

Ricardo Lucas Rodríguez Instituto de Investigaciones Químicas, CSIC-US

[email protected]

Brendan J. Manning Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

Vicente Marchán Sancho Universitat de Barcelona

[email protected]

Saúl Martínez Montero Universidad de Oviedo

[email protected]

Nerea Martín-Pintado Zugasti Instituto de Química Física Rocasolano, CSIC

[email protected]

Juan Carlos Morales Sánchez Instituto de Investigaciones Químicas, CSIC-US

[email protected]

Sandra Milena Ocampo Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

Modesto Orozco López IRB-UB

[email protected]

Samuel Ortega Carrillo Universitat de Barcelona

[email protected]

Miguel Ángel Pardo Cea Instituto de Química Física Rocasolano, CSIC

[email protected]

37

Enrique Pedroso Muller Universitat de Barcelona

[email protected]

Alberto Pérez Antoñanzas Barcelona Supercomputing Center

[email protected]

Sónia Pérez Rentero

Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

Jose Antonio Reyes Darias

Instituto de Parasitología y Biomedicina López-Neyra

[email protected]

Albert Sánchez González Universitat de Barcelona

[email protected]

Ana San-Félix García Instituto de Química Médica, CSIC

[email protected]

Álvaro Somoza Calatrava IMDEA-Nanociencia

[email protected]

Montserrat Terrazas Martínez Instituto de Química Avanzada de Cataluña, CSIC

[email protected]

Roger Tresanchez Carrera Universitat de Barcelona

[email protected]

39

List of Authors

Alguacil, J. II-3 Almagro, M. C. I-2 Arcella, A. III-1 Aviñó, A. II-1, VI-1, VI-3 Berzal-Herranz, A. VII-1 Bonache, M. C. IV-2 Bucek, P. VI-1 Bueren-Calabuig, J. A. VII-2 Camarasa, M. J. I-3, IV-2 Casanova, E. I-3 Castellazzi, C. I-1 Chakrabartty, A. IV-3 Ciudad, C. J. I-2 Claudio, A. II-3 Coderch, C. III-2 Cordeiro, A. IV-2 Cuevas, C. VII-2 Damha, M. I-4 Defaus, S. II-3 Díaz, I. VI-3 Dufour, D. VI-2, VII-3 Enjuanes, L. VII-3 Eritja, R. II-1, V-2, VI-1, VI-3, VII-1 Faustino, I. V-1 Fenollosa, C. IV-1 Fernández, S. III-3 Ferrero, M. III-3 Flores, R. VI-2 Gago, F. III-2, VII-2 Gago, S. VI-2 Gallego, J. II-2, VI-2, VII-3 Galmarini, C. M. VII-2 Gargallo, R. VI-1 Gómez-Pinto, I. I-4, IV-3, VI-3, VIII-1 González, C. I-4, IV-3, VI-3 González, L. II-2 Gotor, V. III-3 Grandas, A. III-4 Guilbert, C. VIII-1 James, T. L. KC, VIII-1 Jaumot, J. VI-1 Jimeno, M. L. I-3, IV-2

Kool, E. T. VIII-2 Laurents, D. V. IV-3 Leigh, S. II-1 López-Alonso, J. P. IV-3 López-Senín, P. VIII-1 Lucas, R. VI-3 Manning, B. J. II-1, V-2 Marchán, V. III-4, VIII-1 Martínez-Montero, S. III-3 Martín-Pintado, N. I-4 Marton-García, S. VII-1 Mateos, P. A. VII-3 Morales, J. C. VI-3, VII-1 Negri, A. VII-2 Nieto, P. M. VI-3 Noé, V. I-2 Orozco, M. I-1, I-4, III-1, V-1 Ortega, S. III-4 Pardo-Cea, M. A. IV-3 Peña, M. VI-2 Pérez, A. I-1, III-1, V-1 Pérez, S. II-1, VII-1 Pérez-Pérez, M. J. I-3 Portela, G. I-4 Preece, J. A. II-1 Priego, E. M. I-3 Quesada, E. IV-2 Ramos, R. II-1 Reina, J. J. VI-3 Reyes-Darias, J. A. VII-1 Robles, J. II-3 Romero-López, C. VII-1 San Félix, A. IV-2 Sánchez-Luque, F. J. VII-1 Sanghvi, Y. S. III-3 Sola, I. VII-3 Soler, M. I-1 Somoza, A. V-2 Terrazas, M. V-2, VIII-2 Vacas, V. A. VII-1 Watts, J. I-4

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