Real-time determination of telomerase activity in cell extracts using an optical biosensor

Item Type:	Article
Title:	Real-time determination of telomerase activity in cell extracts using an optical biosensor
Creators Name:	Schmidt, P.M. and Matthes, E. and Scheller, F.W. and Bienert, M. and Lehmann, C. and Ehrlich, A. and Bier, F.F.
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.
Keywords:	Biosensor, Grating Coupler, Phosphorothioate-Modified Oligonucleotides, Telomerase, TRAP Assay
Source:	Biological Chemistry
ISSN:	1431-6730
Publisher:	de Gruyter
Volume:	383
Number:	10
Page Range:	1659-1666
Date:	October 2002
Official Publication:	https://doi.org/10.1515/BC.2002.186
PubMed:	View item in PubMed

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