Ramon Güimil García

ESPRESSO: the Echelle spectrograph for rocky exoplanets and stable spectroscopic observations

ESPRESSO, the Echelle SPectrograph for Rocky Exoplanets and Stable Spectroscopic Observations, will combine the efficiency of modern echelle spectrograph design with extreme radial-velocity precision. It will be installed on ESOs VLT in order to achieve a gain of two magnitudes with respect to its predecessor HARPS, and the instrumental radialvelocity precision will be improved to reach cm/s level. Thanks to its characteristics and the ability of combining incoherently the light of 4 large telescopes, ESPRESSO will offer new possibilities in various fields of astronomy. The main scientific objectives will be the search and characterization of rocky exoplanets in the habitable zone of quiet, nearby G to M-dwarfs, and the analysis of the variability of fundamental physical constants. We will present the ambitious scientific objectives, the capabilities of ESPRESSO, and the technical solutions of this challenging project.

The structural protein p54 is essential for African swine fever virus viability

Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and beta-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.

A COMPARATIVE STUDY OF SUPPORTS FOR THE SYNTHESIS OF OLIGONUCLEOTIDES WITHOUT USING AMMONIA

Abstract A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2-(2-nitrophenyl)ethyl, 9-fluorenylmethyl, and 2-nitrobenzyl supports in 0.5M DBU solutions is described. A decrease in cleavage efficiency is observed when small oligonucleotides containing thymidine are linked to the supports. In these conditions oxalyl supports gave the best yields followed by 2-(2-nitrophenyl)ethyl and 9-fluorenylmethyl supports.

Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-2'-deoxycytidine: examination of the intertwined roles of co-factor, target, transition state structure and enzyme conformation.

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyts inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.

Parallel-Stranded Hairpins Containing 8-Aminopurines. Novel Efficient Probes for Triple-Helix Formation

We describe novel oligomers with a greater propensity to form triplexes than oligomers containing only natural bases. They consist of a polypyrimidine sequence linked head-to-head with a polypurine sequence carrying one or several 8-aminoadenine or 8-aminoguanines. The presence of 8-aminopurines also stabilised the parallel-stranded duplex structure.

Synthesis of Oligodeoxynucleotides Containing 2-Substituted Guanine Derivatives Using 2-Fluoro-2′-Deoxyinosine as Common Nucleoside Precursor

Abstract Oligonucleotides containing 2-substituted guanine derivatives with double-helix stabilizing molecules such as spermine, spermidine and propylimidazole have been prepared using protected 2-fluoro-2′-deoxyinosine phosphoramidite and two different protective strategies: the p-nitrophenylethyl (NPE) and the t-butylphenoxyacetyl groups. Melting studies show a large increase on the melting temperatures of duplexes containing these 2-substituted guanine derivatives.

Preparation and properties of oligodeoxynucleotides containing 4-O-butylthymine, 2-fluorohypoxanthine and 5-azacytosine1

Abstract Oligonucleotides carrying the ammonia sensitive bases 4-O-butylthymine, 2-fluorohypoxanthine and 5-azacytosine have been prepared for the first time using a special protocol that avoids the use of nucleophiles during the final deprotection.

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

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

Protection of the guanine residue during synthesis of2′-O-alkylguanosine derivatives

Highly selective 2′-O-alkylation of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl) guanosine has been achieved by using an alkyl halide and a sterically hindered strong organic base, when the 6-O atom is protected with either a 2-nitrophenyl or a tert-butyldiphenylsilyl group prior to the alkylation. A minimum of chromatography is required, the yields are high and none of the unwanted isomer is produced. Moreover, the highly versatile intermediates enable the synthesis of several new 2′-O-alkylguanosine derivatives as well as base-modified analogues.

Synthesis of Oligoribonucleotides Containing 4‐Thiouridine Using the Convertible Nucleoside Approach and the 1‐(2‐Fluorophenyl)‐4‐Methoxypiperidin‐4‐yl Group

Oligoribonucleotides containing 4‐thiouridine were prepared using the Fpmp group for protection of the 2′‐OH. Two uridine derivatives with the 1,2,4‐triazolyl and the 2‐nitrophenyl groups at position 4 were used to obtain 4‐thiouridine by postsynthetic substitution with sodium hydrogen sulfide. Both uridine derivatives allow the preparation of the desired oligonucleotides in good yields.