Technique / Molecular Biology / Telomere and telomerase assay

Interaction of ferrocenylnaphthalene diimide 1 with the tetraplex oligonucleotide, AGGG(TTAGGG)3, which is a part of human telomere sequence, was studied in 0.1 M AcONa-AcOH (pH 5.5) containing 0.1 M NaCl coupled with 22-meric single and double stranded oligonucleotides using spectrophotometric titration experiment to search for the more suitable tetraplex DNA-binding ligand to achieve the electrochemical telomerase assay. CD spectra and polyacrylamide gel electrophoresis revealed that this tetraplex oligonucleotide kept to the single conformational structure of basket type G-quadruplex under these conditions. Scatchard analysis showed that 1 can bind to the tetraplex oligonucleotide with the binding constant of 10(5) M(-1) order, which was the highest value among the other oligonucleotides. Binding number of 1 for these oligonucleotides were ca. 2, 11, and 3 for tetraplex, double stranded, and single stranded oligonucleotides, respectively. These values were reasonable when considering with the binding mode of 1 as a threading intercalator.

Genomic and proteomic efforts have discovered a complex list of biomarkers that identify human disease, stratify risk of disease within populations, and monitor drug or therapy responses for treatment. Attention is needed to characterize these biomarkers and to develop high-throughput technologies to evaluate their accuracy and precision. Telomerase activity is correlated with tumor progression, indicating cells that express telomerase possess aggressive clinical behavior and that telomerase activity could be a clinically important cancer biomarker. Traditionally, the detection of cancer has involved invasive procedures to procure samples. There is a need for less invasive approaches suitable for population- and clinic-based assays for cancer early detection. Esophageal balloon cytology (EBC) is a low-invasive screening technique, which samples superficial epithelial cells from the esophagus. Since telomerase activity is absent in superficial cells of normal esophageal squamous epithelium but is often present in superficial cells from dysplastic lesions and ESCCs, measuring telomerase activity in EBC samples may be a good way to screen for these lesions. The development of rapid real-time telomerase activity assays raises the possibility of extending such screening to high-risk populations. In this study, we evaluate the feasibility of using rapid Real-Time Telomerase Repeat Amplification Protocol (RTTRAP) for the analysis of NIST telomerase candidate reference material and esophageal clinical samples. The telomerase activity of eight EBC samples was also measured by capillary electrophoresis of RTTRAP products, RApidTRAP, and hTERT mRNA RT-PCR assays. These findings demonstrate the feasibility of using the RTTRAP assay in EBC samples and suggest that individuals from high-risk populations can be screened for telomerase activity.

Spectroscopic studies revealed that ferrocenylnaphthalene diimide (1) can bind to tetraplex DNA at high potassium ion concentration. The tetraplex DNA was stabilized by the binding of 1, and this effect was larger than that of any other tetraplex stabilizers, which are known as a telomerase inhibitor. Quantitative analysis with circular dichroism and a quartz crystal microbalance strongly suggested a 3:1 binding stoichiometry of 1 to the tetraplex DNA. The telomere sequence could be extended by telomerase with the telomerase substrate primer on the surface of an electrode as proven by an increased current signal of 1 bound to the tetraplex DNA formed on the electrode. This is the first example of electrochemical detection of telomerase activity without relying on PCR.