M & L Jaargang 3/1

RedactioneelH. Kennes, L. Mondelaers m.m.v. S. Van Aerschot Mechelen: het verhaal van een binnenstad. [Mechelen: the tale of an inner-city.]J. Grootaers De Vijgenboom (1500-1510) en De Duivels (1545-1550), twee pronkstukken van het unieke laat-middeleeuwse houtbouwpatrimonium te Mechelen. [The Vijgenboom (Figtree) and the Duivels (Devils), two fine specimen of the unique late-medieval timber Construction in Mechelen.]A. Bergmans Verdwenen muurschilderingen in de Sint-Aldegondiskerk te Alken. [The vanished mural paintings in Saint-Aldegondis church in Alken.]SummaryM&L Binnenkran

Biological effects of hexitol and altritol-modified siRNAs targeting B-Raf

Increasing the effectiveness of siRNAs through chemical modification is an important task. Here we describe altritol and hexitol modified oligonucleotides targeting the B-Raf oncogene that is critical for the growth and survival of melanoma cells. Using assays for apoptosis, DNA synthesis, colony formation and B-Raf protein and message levels, we demonstrate that certain hexitol modifications can improve the effectiveness of B-Raf siRNAs and also increase duration of action. Altritol modified siRNAs were similar to or slightly less effective than unmodified B-Raf siRNA. Modifications at the 3′ or 5′ end of the sense strand, at the 3′ end of the antisense strand, or within either strand were well tolerated. The basis for the increased effectiveness of the hexitol-modified siRNAs is not fully understood but may be partly due to increased stability to nucleases

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

AbstractOligonucleotides of a novel type containing 2′-O-β-ribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein-nucleic acid interactions

2′-O-HYDROXYALKOXYMETHYLRIBIBONUCLEOSIDE AND THEIR INCORPORATION INTO OLIGORIBONUCLEOTIDES

A simple and efficient method for the preparation of pyrimidine 2′-O-hydroxyethoxymethylribonucleosides and 2′-O-hydmxypropoxymethylribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were shown to form stable RNA/RNA duplexes. The effect of 2′-O-modification in the antisense and sense strands of small interference RNA was evaluated in multi-drug resistant NIH 3T3 cells

Inhibition of MDR1 expression with altritol-modified siRNAs

Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3′ end of the sense or antisense strands, while modification at the 5′ end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5′-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness

Synthesis of nucleosides fluorinated in the sugar moiety - the application of diethylaminosulfur trifluoride to the synthesis of fluorinated nucleosides

. A survey is given of the different methods that have been usea for the synthesis of nucleosides fluorinated in the carbohydrate moiety. In this article we describe the use of diethylaminosulfur trifluoride (DAST) as a fluorinating agent in the nucleoside field. © 1989, Taylor & Francis Group, LLC. All rights reserved.status: publishe

Synthesis of 8-mercapto-2',3'-dideoxyadenosine and 8-mercapto-2',3'-dideoxyinosine

status: publishe

Synthesis of thymidine from 5-iodo-2'-deoxyuridine

A simple, high yield synthesis of thymidine from 5-iodo-2'-deoxyuridine is described, using tetramethyltin and tetrakis(triphenylphosphine)palladium(0) in hexamethylphoshoric triamide. Likewise, 5-phenyl- and 5-vinyl-2'-deoxyuridine can be obtained, and through reduction of the latter the 5-ethyl analogue is also at hand.status: publishe

Identification of a peptide inhibitor against glycosomal phosphoglycerate kinase of Trypanosom brucei by a synthetic peptide library approach

A synthetic peptide library, composed of 2.5 million L-amino acid pentapeptides anchored on polystyrene beads was prepared with each bead bearing a single pentapeptide sequence. This library was screened for interaction with glycosomal phosphoglycerate kinase (gPGK) of Trypanosoma brucei labelled with fluorescein or with biotin. Affinity beads that bound the enzyme were selected with a pipette or with streptavidin coated magnetic beads. The beads that bound to the enzyme were individually subjected to Edman microsequence analysis to determine the sequence of the corresponding peptide ligands. The corresponding peptide-sequences were synthesised as free peptide acids and evaluated for enzyme activity inhibition. The pentapeptide NWMMF was able to selectively inhibit gPGK with an IC50 of approximate to 80 mu M

Inhibition of gene expression with altritol- and hexitol-modified siRNAs

status: publishe