Reliable Förster Resonance Energy Transfer Probe Based on Structure-Switching DNA for Ratiometric Sensing of Telomerase in Living Cells

Abstract

In situ detection and monitoring of telomerase is of great importance as it is a relatively specific cancer target. However, the complexity of the biological system makes it difficult for the nanoprobe to keep absolutely stable and have a low background in living cells. This study designs a probe termed Förster resonance energy transfer (FRET) nanoflare to achieve ratiometric fluorescent detection of intracellular telomerase with higher specificity, which can effectively resist the disturbance from DNase I and GSH, etc. The probe is composed of a gold nanoparticle (AuNP) which is functioned with telomerase primer sequences (TS) and flares fluorescently labeled donors and acceptors at two terminals. In the presence of telomerase, flares are displaced from the primer sequences and form hairpin structures, so that the donors and acceptors are brought into close proximity, resulting in high FRET efficiency. The probe exhibits good performance for efficiently distinguishing tumor cells from normal cells and monitoring the change of intracellular telomerase activity during treatment with telomerase-related drugs, showing great potential for cancer diagnosis and estimating therapeutic effect.

Advances in optical assays for detecting telomerase activity

“…The nicked molecular beacon could be unfolded by the hybridization of TEP to produce a telomerase‐dependent signal probe. Yang et al developed a ratio FRET to detect intracellular telomerase activity (Figure ) . The TS primer was immobilized onto the surface of AuNPs and predesigned to hybridize with a reporter probe.…”

“…Schematic illustration of the fluorescence resonance energy transfer (FRET) probe for ratiometric detection of intracellular telomerase. Reprinted with permission from ref …”

Fluorescent Platforms Based on Organic Molecules for Chemical and Biological Detection

“…Two fluorophore‐labeled DNA sequences were immobilized on gold nanoparticles to construct FRET‐based fluorescent platforms. Analytes such as H + , potassium ions, mRNA, and proteins interact with the fluorophore‐labeled DNA and change its conformation. This structural change alters the distance between the donor and acceptor, resulting in a change in ratiometric fluorescence response.…”

Telomerase and poly(ADP-ribose) polymerase-1 activity sensing based on the high fluorescence selectivity and sensitivity of TOTO-1 towards G bases in single-stranded DNA and poly(ADP-ribose)

“…These methods do not have good selectivity because of the high background signals. Because PAR lacks unique properties as the extended telomerase primer, very few methods have been developed for its detection even though its activity is very important for assessing cancer development 16,17…”