A Robust Probe for Lighting Up Intracellular Telomerase via Primer Extension To Open a Nicked Molecular Beacon

Abstract

A nicked molecular beacon (MB)-functionalized probe has been designed for in situ imaging and detection of intracellular telomerase activity. The nick separates the MB into two segments: a shorter telomerase primer (TSP) sequence as a part of the 5'-end stem and a longer sequence to form a loop with one thiol-labeled 3'-end stem. The MB can be opened by substitutional hybridization of the telomerase-triggered stem elongation product, which leads to separation of the Cy5 at the 5'-end nick from the gold nanoparticle (AuNP) as the nanocarrier and thus inhibits the energy transfer from Cy5 to AuNP. Upon endocytosis of the probe, the TSP can be extended by intracellular telomerase at its 3' end to produce the telomeric repeated sequence, which leads to the inner chain substitution and thus turns on the fluorescence of Cy5. The probe provides a one-step incubation technique for quantification and monitoring of the telomerase activity in living cells. The practicality of the proposed approach for distinguishing tumor cells from normal cells and monitoring the decrease of telomerase activity during treatment with antitumor drugs demonstrates its potential in clinical diagnostic and therapeutic monitoring.

Sensing telomerase: From in vitro detection to in vivo imaging

“…To simplify the procedures, Ju further demonstrated the use of gold nanoparticles and the nicked molecular beacon for one-step imaging of intracellular telomerase activity. 53,54 They designed a nicked molecular beacon with a nick at the 5′-end stem which separates the beacon into two parts: the telomerase primer sequence and a loop structure (Fig. 10).…”

“…10). 53 The loop structure is labeled with Cy5 at 5′-end and modified by thiol at the 3′-end. The MB is conjugated to an AuNP via the thiol-labeled 3′-end, and the Cy5 is quenched by AuNP via FRET between Cy5 and AuNP.…”

“…In addition to the fluorescent imaging methods, 49,52,53 Kuang demonstrated the use of in situ Raman scattering spectroscopy for the intracellular telomerase assay (Fig. 11).…”

Quencher Group Induced High Specificity Detection of Telomerase in Clear and Bloody Urines by AIEgens

“…For example, a fluorescence strategy based on a molecular beacon functionalized gold nanoparticle (AuNP) probe has been developed to detect activity of telomerase. 17 However, conventional fluorophores used in these fluorescence strategies are aggregation-caused quenching (ACQ) molecules. They often give bright emission in dilute solution, whereas the emission is weakened or quenched in concentrated solution or in the aggregated state.…”

Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in < 25 min

“…Therefore, microfluidic PCR systems are often coupled with dedicated cell lysis and nucleic acid purification steps to remove cell debris and compounds that could inhibit DNA amplification (Witek et al, 2006; Park et al, 2011). Another promising approach in this regard could be the use of intracellular enzymes to perform amplification (Qian et al, 2014) or translocation of the enzymes inside the cells (Patel et al, 2014; Wang et al, 2014). …”