Ramon Eritja

Nanotechnology: Carbon nanotubes with DNA recognition

Since the discovery of their one-dimensional electronic band structure, the leading candidate that has emerged for nanodevice applications is single-walled carbon nanotubes (SWNTs) . Here we unite their unique properties with the specific molecular-recognition features of DNA by coupling SWNTs to peptide nucleic acid (PNA, an uncharged DNA analogue) and hybridizing these macromolecular wires with complementary DNA. Our findings provide a new, versatile means of incorporating SWNTs into larger electronic devices by recognition-based assembly, and of using SWNTs as probes in biological systems by sequence-specific attachment.

Crystal structure of a DNA Holliday junction

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 Å resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.

The structure of plasmid‐encoded transcriptional repressor CopG unliganded and bound to its operator

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon–helix–helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13‐bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19‐bp oligonucleotide, containing the pseudosymmetric element, with two β‐ribbons inserted into the major groove. The DNA is bent 60° by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix–turn–helix regions of unrelated DNA‐binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon–helix–helix and helix–turn–helix proteins.

Label-Free DNA Biosensors Based on Functionalized Carbon Nanotube Field Effect Transistors

A carbon nanotube transistor array was used to detect DNA hybridization. A new approach to ensure specific adsorption of DNA to the nanotubes was developed. The polymer poly (methylmethacrylate(0.6)-co-poly(ethyleneglycol)methacrylate(0.15)-co-N-succinimidyl methacrylate(0.25)) was synthesized and bonded noncovalently to the nanotube. Aminated single-strand DNA was then attached covalently to the polymer. After hybridization, statistically significant changes were observed in key transistor parameters. Hybridized DNA traps both electrons and holes, possibly caused by the charge-trapping nature of the base pairs.

Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea.

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice (Oryza sativa) constitutively expressing the afp gene was generated by Agrobacterium-mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp-expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T2 homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.

Diketopiperazine formation in solid phase peptide synthesis using p-alkoxybenzyl ester resins and Fmoc-amino acids

Summary Diketopiperazine formation rates under the usual conditions of a solid phase peptide synthesis cycle with Fmoc-amino acids have been studied on a p -alkoxybenzyl ester resin. Piperidine has been found to be an extremely efficient catalyst for the intramolecular aminolysis reaction.

Efficient Self-Assembly in Water of Long Noncovalent Polymers by Nucleobase Analogues

Molecular self-assembly is widely appreciated to result from a delicate balance between several noncovalent interactions and solvation effects. However, current design approaches for achieving self-assembly in water with small, synthetic molecules do not consider all aspects of the hydrophobic effect, in particular the requirement of surface areas greater than 1 nm(2) for an appreciable free energy of hydration. With the concept of a minimum hydrophobic surface area in mind, we designed a system that achieves highly cooperative self-assembly in water. Two weakly interacting low-molecular-weight monomers (cyanuric acid and a modified triaminopyrimidine) are shown to form extremely long supramolecular polymer assemblies that retain water solubility. The complete absence of intermediate assemblies means that the observed equilibrium is between free monomers and supramolecular assemblies. These observations are in excellent agreement with literature values for the free energy of nucleic acid base interactions as well as the calculated free energy penalty for the exposure of hydrophobic structures in water. The results of our study have implications for the design of new self-assembling structures and hydrogel-forming molecules and may provide insights into the origin of the first RNA-like polymers.

An aptamer-gated silica mesoporous material for thrombin detection

An aptamer-capped mesoporous material for the selective and sensitive detection of α-thrombin in human plasma and serum has been prepared and characterised.

α,γ-Peptide nanotube templating of one-dimensional parallel fullerene arrangements

The formation and full characterization of single self-assembling alpha,gamma-peptide nanotubes (alpha,gamma-SPNs) is described. The introduction of C(60) into cyclic peptides allows the preparation of supramolecular 1D fullerene arrangements induced by peptide nanotube formation under appropriate conditions.

Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence.

Abstract Oligonucleotide-peptide hybrid molecules containing a nuclear transport signal have been synthesized using two different approaches based on the specific reactivity of the thiol group.