Biomedical subjects

E Luzi

Publications and source records attributed to E Luzi.

6 recordsLinked to original sources

PubMed2005

Aptamer-based biosensors for the detection of HIV-1 Tat protein.

Two biosensors have been constructed using an RNA aptamer as biorecognition element. The aptamer, specific for HIV-1 Tat protein, has been immobilised on the gold surface of piezoelectric quartz crystals or surface plasmon resonance (SPR) chips to develop a quartz crystal microbalance (QCM)-based and an SPR-based biosensor, respectively. Both the biosensors were modified with the same immobilisation chemistry based on the binding of a biotinylated aptamer on a layer of streptavidin. The binding between the immobilised aptamer and its specific protein has been evaluated with the two biosensors in terms of sensitivity, reproducibility and selectivity. A protein very similar to Tat, Rev protein, has been used as negative control. The two biosensors both were very reproducible in the immobilisation and the binding steps. The selectivity was high in both cases.

Biosensing Techniques↗

PubMed2004

Development of biosensors with aptamers as bio-recognition element: the case of HIV-1 Tat protein.

The in vitro selection of combinatorial libraries of RNA/DNA, has allowed the identification of specific nucleic acids (aptamers) which bind to a wide range of target molecules with high affinity and specificity. In this work, an RNA aptamer, specific for the protein trans-activator of transcription (Tat) of HIV-1, has been used as bio-recognition element to develop a biosensor (aptasensor). The biosensor was optimised using piezoelectric quartz-crystals as transducers and the aptamer was immobilised on the gold electrode of the crystal. The immobilisation procedure was based on the interaction between the biotinylated aptamer and streptavidin previously deposited on the electrode. The main analytical characteristics of the biosensor, such as sensitivity, selectivity and reproducibility, have been studied in details. An optimised regeneration procedure allowed the multiple use of the aptamer-coated crystal. The aptasensor has been compared with the corresponding immunosensor, based on the specific monoclonal anti-Tat antibody. The antibody was immobilised on a layer of carboxylated dextran previously deposited on the gold electrode. The results demonstrated that the use of a biosensor with a specific aptamer as bio-recognition element could be an interesting approach in the detection of proteins, which has been here examined considering a model system.

Biosensing Techniques↗

PubMed2000

HIV-1 LTR as a target for synthetic ribozyme-mediated inhibition of gene expression: site selection and inhibition in cell culture.

A library of three synthetic ribozymes with randomized arms, targeting NUX, GUX and NXG triplets, respectively, were used to identify ribozyme-accessible sites on the HIV-1 LTR transcript comprising positions -533 to 386. Three cleavable sites were identified at positions 109, 115 and 161. Ribozymes were designed against these sites, either unmodified or with 2'-modifications and phosphorothioate groups, and their cleavage activities of the transcript were determined. Their biological activities were assessed in cell culture, using a HIV-1 model assay system where the LTR is a promoter for the expression of the reporter gene luciferase in a transient expression system. Intracellular efficiency of the ribozymes were determined by cotransfection of ribozyme and plasmid DNA, expressing the target RNA. Modified ribozymes, directed against positions 115 and 161, lowered the level of LTR mRNA in the cell resulting in inhibition of expression of the LTR-driven reporter gene luciferase of 87 and 61%, respectively. In the presence of Tat the inhibitions were 43 and 25%. The inactive variants of these ribozymes exhibited a similar inhibitory effect. RNase protection revealed a reduction of RNA which was somewhat stronger for the active than the inactive ribozymes, particularly for ribozyme 115. Unmodified ribozymes showed no inhibition in the cell. The third ribozyme, targeting a GUG-triplet at position 109, possessed only low cleavage activity in vitro and no inhibitory effect in cell culture.

Base Sequence↗

PubMed1998

Designing ribozymes for the inhibition of gene expression.

Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. However, particular attention must be paid to the following points: the identification of regions on the mRNA accessible to the ribozyme; the delivery of ribozymes to cells by either exogenous or endogenous delivery; colocalization of the ribozyme with the target RNA in the cell; and differentiation between closely related sequences. This field is advancing rapidly, and results obtained with transgenic animals demonstrate the power of this strategy for the inhibition of gene expression.

Animals↗

PubMed1997

The newt ribozyme is part of a riboprotein complex.

Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a "hammerhead" domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

Animals↗

PubMed1997

Intermolecular cleavage by the newt ribozyme.

To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop II. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and III to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondary-structure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and III did. These results suggest that the sequence of the stems I and III impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.

Animals↗