Real-time determination of telomerase activity in cell extracts using an optical biosensor.
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.
Schmidt
Peter M
PM
Fraunhofer-Institut für Biomedizinische Technik, Abt. Molekulare Bioanalytik und Bioelektronik, Arthur-Scheunert-Allee 114-116, D-14558 Potsdam-Rehbrucke, Germany.
Matthes
Eckart
E
Scheller
Frieder W
FW
Bienert
Michael
M
Lehmann
Christine
C
Ehrlich
Angelika
A
Bier
Frank F
FF
D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002021:Buffers; D002457:Cell Extracts / Q000032:analysis*; D005347:Fibroblasts / Q000201:enzymology; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D055095:Optics and Photonics / Q000295:instrumentation; D020135:Peptide Nucleic Acids / Q000494:pharmacology; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013499:Surface Properties; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002021:Buffers; D002457:Cell Extracts; D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D019098:Telomerase