Biomedical subjects

Jan Weiler

Publications and source records attributed to Jan Weiler.

6 recordsLinked to original sources

PubMed2005

Design of a genome-wide siRNA library using an artificial neural network.

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

Algorithms↗

PubMed2005

The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27Kip1 protein levels.

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27Kip1 was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCFSkp2 ubiquitin ligase has been reported to mediate p27Kip1 degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27Kip1, and prevent cellular proliferation. Elevation of p27Kip1 protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27Kip1 with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCFSkp2) ubiquitin ligase substrate p27Kip1, but has no concomitant effect on the level of IkBalpha and beta-catenin, which are known substrates of a closely related SCF ligase.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity.

A cellular assay has been developed to allow measurement of the inhibitory activity of large numbers of oligonucleotides at the protein level. The assay is centred on an mRNA fusion transcript construct comprising of a full-length reporter gene with a target region of interest inserted into the 3'-untranslated region. Luciferase and fluorescent reporter genes were used in the constructs. The insert can be from multiple sources (uncharacterised ESTs, partial or full-length genes, genes from alternate species, etc.). Large numbers of oligonucleotides were screened for antisense activity against a number of such constructs bearing different reporters, in different cell lines and the inhibitory profiles obtained were compared with those observed through screening the oligonucleotides against the corresponding endogenous genes assayed at the mRNA level. A high degree of similarity in the profiles was obtained indicating that the fusion constructs are suitable surrogates for the endogenous messages for characterisation of antisense oligonucleotides (ASOs). Furthermore, the results support the hypothesis that the secondary structure of mRNAs are divided into domains, the nature of which is determined by primary nucleotide sequence. Oligonucleotides whose activity is dependent on the local structure of their target mRNAs (e.g. ASOs, short interfering RNAs) can be characterised via such fusion RNA constructs.

Animals↗

PubMed2002

Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin.

BACKGROUND: In Schizosaccharomyces pombe, Hus1 is a component of the radiation sensitive (Rad) machinery that has been identified as playing a role in DNA repair and cell cycle G2/M checkpoint control pathways. Hus1 has been shown to exist in a discrete complex with at least two Rad family members, Rad1 and Rad9. Furthermore, Hus1 is essential for checkpoint activation, since Hus1 mutants fail to arrest the cell cycle in response to DNA damage or unreplicated DNA. To establish the role and relevance of human Hus1 in cell cycle regulation, the authors applied antisense technology to selectively downregulate the expression of Hus1 mRNA. METHODS: Transfection of 2'-O-methoxyethyl-modified Hus1 antisense oligoribonucleotides into human H1299 nonsmall lung carcinoma cells was performed using Lipofectin as the carrier. The authors prepared RNA from transfected cells, and levels of Hus1 expression were analyzed by real time polymerase chain reaction. The growth and viability of cells treated with Hus1 antisense oligonucleotides in the presence or absence of cisplatin were analyzed and compared to controls. RESULTS: Transfection of selected Hus1 antisense oligonucleotides into p53 deficient H1299 cells resulted in significant downregulation of Hus1 mRNA, up to 80%; RNA analyses reveal a maximal Hus1 antisense activity at a concentration of 200 nM with an IC50 determined to be 90 nM. The design and transfection of oligonucleotides containing three mismatches to their corresponding antisense counterparts had no or only minor effects on Hus1 mRNA levels, showing the specificity of Hus1 mRNA downregulation. The cisplatin IC50 in untransfected H1299 cells was found to be 20 microM and could be reduced significantly to only 7 microM after transfection of a Hus1 antisense oligonucleotide. CONCLUSIONS: Experiments addressing the proliferation and viability of transfected H1299 cells suggest that downregulation of Hus1 by specific antisense oligonucleotides sensitizes human cells to treatment with the DNA damaging agent cisplatin.

Antineoplastic Agents↗

PubMedDate not supplied

A role for oligonucleotide-based RNA-knock down technologies in functional genomics.

Functional genomics is inundating the pharmaceutical industry with large numbers of potential gene targets from several sources such as gene expression profiling experiments (DNA microchips, proteomics) or database mining. Oligonucleotide-based RNA-knock down technologies such as antisense or RNA interference can aid in the filtering and prioritization of target candidates in the drug discovery process.

Drug Industry↗

PubMedDate not supplied

Linear polyethylenimine as a tool for comparative studies of antisense and short double-stranded RNA oligonucleotides.

Despite the recently enlarged field of available RNA knock-down technologies, e.g., antisense oligonucleotides (ASOs) and duplexes of synthetic 21 nucleotides RNAs (siRNAs), no versatile transfection reagent has been reported to deliver different nucleic acids formats at high rates of efficiency. We have evaluated the versatility and efficacy of linear PEI in transfecting and properly delivering a broad panel of nucleic acids such as short oligonucleotides and double-stranded RNA into cells in culture.

Animals↗