DNAzyme‐Based Probes for Telomerase Detection in Early‐Stage Cancer Diagnosis

Abstract

Human telomerase is a polymerase enzyme that adds tandem repeats of DNA (TTAGGG) in the telomere regions. Since telomerase can be detected in immortalized, but not normal, somatic cells, it has been considered a selective target for cancer chemotherapy. In this paper, we describe a DNAzyme-based probe method to determine telomerase in cell lysates. Telomerase elongates the primer site on the probe. Subsequent addition of the PbII cofactor activates the DNAzyme, which cleaves the elongated fragment at the RNA site, releasing the probe for repetitive cycling and signal amplification. The cleaved fragment is detected by a reporter molecular beacon. Enzymatic amplification with rapid turnover allows detection of telomerase in the range of 0.1 μg to 1 μg cell lysate, with a 5-fold increase in signal level for cancer cells over normal cells. This probe design can provide a simple, yet rapid and sensitive, measurement of telomerase activity.

Advances in the detection of telomerase activity using isothermal amplification

“…Among them, 8-17 DNAzyme which could catalyze the cleavage of its complementary substrate with the aid of its corresponding metal ion cofactor is attracting growing interest as a high selectivity and sensitivity biosensor 67 . Tan et al described a Pb II -dependent 8-17 DNAzyme-based probe to detect the telomerase activity in cell lysates 68 . This single-molecule probe could detect total protein in cell lysate range from 0.1 to 20 μg and discriminate tumor cells from normal cells with a 5-fold signal enhancement.…”

Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification

“…It is known that the sensitivity of EXPAR for nucleic acid detection is mainly limited by nonspecific background amplification. 22 Given these results, EXPAR itself is not suitable for Cas9 cleaved blunt-ended dsDNA detection and a new strategy is needed to dramatically reduce nonspecific amplification.…”

“…The point of inflection (POI), which is defined as the time at which the fluorescence curve rises significantly above the background, was used for quantification. 22 The POI values linearly correspond to the logarithms of dsDNA concentration with the correlation equation of POI = –14.54 – 2.69 lg[dsDNA] ( R 2 = 0.981). To further characterize the NTEXPAR reaction, the amplified products were analyzed by gel electrophoresis (Fig.…”

“… 18 – 25 For instance, the exponential isothermal amplification of telomere repeat (EXPIATR) assay has been demonstrated for telomerase activity analysis and achieved single cell level sensitivity. 20 , 26 – 28 Most conventional isothermal amplifications, such as exponential amplification reaction (EXPAR), 22 loop-mediated isothermal amplification (LAMP), 29 strand displacement amplification (SDA) 30 and rolling circle amplification (RCA), 31 require a single strand DNA (ssDNA) to trigger a downstream reaction. However, Cas9 cleaved dsDNA only provides a newly formed blunt end at the cleavage site which could not be directly used for isothermal amplification.…”

An AIEgens and exonuclease III aided quadratic amplification assay for detecting and cellular imaging of telomerase activity

“…This process will lead to cancer cells immortality and has been considered to be involved in carcinogenesis. Telomerase is therefore a common biomarker for the early diagnosis of cancers and screening anticancer drug [6][7][8][9][10][11][12][13]. Nonetheless, telomerase levels are still very low even in cancer cells.…”