Oligonucleotides

A clear difference in the enthalpy changes derived from spectroscopic and calorimetric measurements has recently been shown. The exact interpretation of this deviation varied from study to study, but it was generally attributed to the non-two-state transition and heat capacity change. Although the temperature-dependent thermodynamics of the duplex formation was often implied, systemic and extensive studies have been lacking in universally assigning the appropriate thermodynamic parameter sets.

10p system191 01-06-2013 36 4   Download

Laboratory for Lipoprotein Chemistry and 4Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research, Faculty of Medicine, Department of Biochemistry, Ghent University, Belgium; 2Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Belgium; 3Ecole Normale Supe´rieure, Paris, France Penetratin is a 16-amino-acid peptide, derived from the homeodomain of antennapedia, a Drosophila transcription factor, which can be used as a vector for the intracellular delivery of peptides or oligonucleotides....

9p system191 01-06-2013 33 4   Download

The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino-acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full-length cDNA encoding a protein of 330 amino-acid residues. N-terminal amino-acid sequencing of derived peptides and the purified protein confirmed 76% of the sequence and demonstrated presence of a cleavable signal peptide of 23 residues, leaving a mature protein of 307 amino acids.

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The decoy approach against nuclear factor kB (NF-kB) is a useful tool to alter NF-kB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF-kB specific cis-elements. Unfortunately, ODNs are not stable and need to be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N-(2-aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases.

10p system191 01-06-2013 42 4   Download

Wilson disease (WD), an inherited disorder affecting copper metabolism, is characterized by hepatic cirrhosis and neuronal degeneration, which result from toxic levels of copper that accumulate in the liver and brain, respectively. We reported previously that the  1.3-kb promoter of the WD gene contains four metal response elements (MREs). Among the four MREs, MREa plays the most important role in the transcriptional activation of the WD promoter.

11p research12 01-06-2013 53 3   Download

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin.

5p zingzing09 24-04-2013 60 5   Download

The uptake by mammalian cells of phosphorothioate oli-gonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10– 100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumula-tion in all cases. ...

8p research12 23-04-2013 46 2   Download

We have analyzed the response of a number of human cell lines to treatment with antisense oligodeoxynucleotides (ODNs) directed against RNA polymerase II, replication protein A, and Ha-ras. ODN-delivery to the cells was liposome-mediated or via electroporation, which resulted in di€erent intracellular locations of the ODNs. The ODN-mediated target mRNA reduction varied consider-ably between the cell lines.

10p research12 23-04-2013 38 3   Download

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. A promising strategy to overcome this problem consists in the chemical conjugation (or fusion) to cell penetrating peptides (CPP) derived from proteins able to cross the plasma membrane. A large number of di€erent cargo mol-ecules such as oligonucleotides, peptides, peptide nucleic acids, proteins or even nanoparticles have been internalized in cells by this strategy.

8p research12 23-04-2013 31 4   Download

Special oligonucleotides for targeted gene correction have attracted increasing attention recently, one of which is the chimericRNAÆDNAoligonucleotide (RDO) system. RDOs for targeted gene correction were first designed in 1996, and are typically 68 nucleotides in length including continuous RNA andDNA sequences (RNA is 2¢-O-methyl-modified).

6p tumor12 22-04-2013 34 2   Download

GToligomers, showingadose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins.By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides.Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC)....

12p tumor12 20-04-2013 34 1   Download

Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTRcells that constitutively over-expressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTRcells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3.

10p fptmusic 16-04-2013 36 4   Download

Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence-specific manner,and may be used for functional genomics,target validation and thera-peuticpurposes.Three typesof anti-mRNAstrategies canbe distinguished. Firstly,the use of single stranded antisense-oligonucleotides; secondly,the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly,RNA interference induced by small interfering RNA molecules.

17p fptmusic 16-04-2013 40 2   Download

We report on a sequence-specific double-stranded DNA labelling strategy in which a stem–loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the for-mation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investi-gated. ...

10p fptmusic 12-04-2013 29 2   Download

Embryos and larvae of the brine shrimp,Artemia francis-cana, were shown previously to possess a protein, now termed p49, which cross-links microtubules in vitro. Molecular characteristics of p49 were described, but the protein’s identity and its role in the cell were not determined. Degenerate oligonucleotide primers designed on the basis of peptide sequence obtained by Edman degradation during this studywereusedtogeneratep49cDNAsbyRT-PCRand thesewere clonedand sequenced.

11p fptmusic 12-04-2013 51 2   Download

Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle-specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core-SRF (the 124–245 fragment of serum response fac-tor) with specific oligonucleotides from c-fos anddesminpromoters, were determined by spectroscopy.

15p fptmusic 11-04-2013 43 4   Download

RecA protein plays a pivotal role in homologous recombination inEscheri-chia coli. RecA polymerizes on single-stranded (ss) DNA forming a nucleo-protein filament. Then double-stranded (ds) DNA is bound and searched for segments homologous to the ssDNA. Finally, homologous strands are exchanged, a new DNA duplex is formed, and ssDNA is displaced. We report a quantitative analysis of RecA interactions with ss d(pN)n of var-ious structures and lengths using these oligonucleotides as inhibitors of RecA filamentation on d(pT)20....

0p awards 06-04-2013 34 3   Download

Base excision repair (BER), a major pathway for the removal of simple lesions in DNA, requires the co-ordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. We here address the role of poly(ADP-ribose) polymerase-1 (PARP-1) in BER. Using anin vitro cross-linking assay, we reveal that PARP-1 is always involved in repair of a uracil-containing oligonucleotide and that it binds to the damaged DNA during the early stages of repair.

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The ability ofEntamoeba histolyticaTATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box for E. histolytica, was demonstrated by gel-shift assays. ...

13p awards 06-04-2013 45 1   Download

The interaction of the homeodomain of the sunflower KNOX protein HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl radical footprinting and missing nucleoside experiments. Binding of HAKN1 to different oligonucleotides indicated that HAKN1 prefers the sequence TGACA (TGTCA), with changes within the GAC core more pro-foundly affecting the interaction. Footprinting and missing nucleoside experiments using hydroxyl radical cleavage of DNA showed that HAKN1 interacts with a 6-bp region of the strand carrying the GAC core, covering the core and nucleotides towards the 3¢ end....

13p awards 05-04-2013 40 4   Download