Real-Time Determination of Telomerase Activity in Cell Extracts Using an Optical Biosensor
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P. M. Schmidt
Abstract
A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS) modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signaltonoise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.
Copyright © 2002 by Walter de Gruyter GmbH & Co. KG
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Artikel in diesem Heft
- Biological Chemistry 125th Anniversary
- Kai Simons Felix Hoppe-Seyler Lecturer 2002
- Lipid Rafts in Protein Sorting and Cell Polarity in Budding Yeast Saccharomyces cerevisiae
- Paper of the Year 2001
- RNA-Mediated RNA Degradation in Transgene- and Virus-Induced Gene Silencing
- Immunopharmacology of CpG DNA
- Induction and Regulation of Endogenous Granulocyte Colony-Stimulating Factor Formation
- In the Cellular Garden of Forking Paths: How p38 MAPKs Signal for Downstream Assistance
- The Human Steroid Hydroxylases CYP11B1 and CYP11B2
- Rate-Limiting Steps in Cytochrome P450 Catalysis
- YidC, a Newly Defined Evolutionarily Conserved Protein, Mediates Membrane Protein Assembly in Bacteria
- Vascular Endothelial Growth Factor Receptor Family Genes: When Did the Three Genes Phylogenetically Segregate?
- The TNFTNF Receptor System
- Fast Regulation of Cytochrome P450 Activities by Phosphorylation and Consequences for Drug Metabolism and Toxicity
- Protein Transduction: A Novel Tool for Tissue Regeneration
- NF-κB A Potential Therapeutic Target for Inhibition of Human Cytomegalovirus (Re)activation?
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