Related Experiment Video
Updated: Aug 15, 2026
Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction
Published on: May 3, 2019
Optimization of the TRAP assay to evaluate specificity of telomerase inhibitors
Kamilla Piotrowska1, Elke Kleideiter, Thomas E Mürdter
1Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany. kamilla.piotrowska@ikp-stuttgart.de
Abstract:
Telomerase inhibition represents a promising approach to anticancer treatment. In order to clarify the therapeutic potential of telomerase inhibitors we examined different substances (small molecule compounds BIBR1532 and BRACO19, as well as hTR antisense oligonucleotides 2'-O-methyl RNA and PNA) in A-549, MCF-7, and Calu-3 cell lines in a cell-free TRAP assay. We demonstrated that each of the tested agents inhibited telomerase in all used cell lines and that the antisense oligonucleotides represent the most potent inhibitors. Interestingly, upon evaluating the specificity of telomerase inhibitors we found out that not all agents acted specifically against telomerase. We observed that BRACO19 and PNA had an inhibitory effect also on PCR amplification of the TSR8 oligonucleotide which is provided in the TRAP(EZE) kit as a PCR control. By modifying the experimental protocol and using a different reverse primer we were able to enhance PNA selectivity, although the PCR inhibition of the TSR8 control template by BRACO19 could not be prevented. We propose an explanation for the lack of target specificity and suggest caution when testing putative telomerase inhibitors, as it appears that some of those substances may not affect specifically telomerase or telomeric G-rich sequences and thus can lead to the misinterpretation of experimental results.
Insights
Telomerase inhibitors show promise for cancer treatment. However, some compounds, like BRACO19 and PNA, may non-specifically inhibit PCR assays, potentially leading to inaccurate results.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Telomerase inhibition is a key strategy in anticancer drug development.
- Evaluating the specificity of telomerase inhibitors is crucial for accurate therapeutic assessment.
Purpose of the Study:
- To assess the efficacy and specificity of various telomerase inhibitors.
- To investigate potential off-target effects of small molecule compounds and oligonucleotides.
Main Methods:
- Utilized a cell-free telomere repeat amplification protocol (TRAP) assay.
- Tested small molecule compounds (BIBR1532, BRACO19) and oligonucleotide inhibitors (2'-O-methyl RNA, PNA) in A-549, MCF-7, and Calu-3 cell lines.
- Evaluated inhibition of PCR amplification using a control oligonucleotide (TSR8).
Main Results:
- All tested agents inhibited telomerase activity across all cell lines.
- Antisense oligonucleotides demonstrated the most potent telomerase inhibition.
- BRACO19 and PNA exhibited non-specific inhibition of PCR amplification of the TSR8 control.
- Modified protocols improved PNA selectivity but did not prevent BRACO19's PCR inhibition.
Conclusions:
- While antisense oligonucleotides are potent telomerase inhibitors, some agents like BRACO19 and PNA lack target specificity.
- Non-specific inhibition of PCR controls can lead to misinterpretation of telomerase inhibitor efficacy.
- Caution is advised when evaluating potential telomerase inhibitors to ensure specific targeting of telomerase or telomeric sequences.
