Real-Time Determination of Telomerase Activity in Cell Extracts Using an Optical Biosensor

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 3'-end 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 signal-to-noise 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.

Assays for Detection of Telomerase Activity

“…Then, an oligonucleotide containing a short noncomplementary region on its 3’-end complementarily binds to the immobilized DNA. The prominent 3’-end of the DNA is modified with phosphorothioate, which enhances the affinity of telomerase-primer binding by a factor of 10 [50]. After treatment with a telomerase-containing extract, the enzyme is removed from the sensor’s surface by proteinase K in the presence of dNTP ( Fig.…”

DNA Microarrays

“…Since its activity is regulated down in differentiated cells, the amount of activity has been established as a significant tumour marker in recent years [25]. Schmidt et al succeeded to demonstrate that telomerase acts also on artificial telomeres immobilised on a sensor surface [26,27].…”

Distinguishing Tumor Cells via Analyzing Intracellular Telomerase Activity

“…Among them, telomere extension assay (TEA) 7 and polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) 6 have been widely employed in the quantitative assay of telomerase activity, in the process of which some harmful radiation or expensive fluorescent labels have to be used. To eliminate these drawbacks, researchers have proposed several techniques, such as incorporating silver staining techniques with TRAP, [8][9][10][11] and some other optical 12,13 or electrochemical 14,15 techniques.…”