A Smart DNA Tweezer for Detection of Human Telomerase Activity

Abstract

Reliable and accurate detection of telomerase activity is crucial to better understand its role in cancer cells and to further explore its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially designed single-stranded oligonucleotides: a central strand dually labeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs' complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG) . TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/μL for HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.

Advances in optical assays for detecting telomerase activity

“…Under the elongation of telomerase, the TEP strand hybridized with c‐DNA and released t‐DNA which can trigger cyclic amplification process under the aid of Nt.BbvCI, resulting in the specific cleavage of the molecular beacon and fluorescence enhancement. A smart DNA tweezer (DT) based on FRET was constructed to detect telomerase activity (Figure ) . The fluorescence intensity ratio between the TAMRA acceptor and the FAM donor was employed to reveal the telomerase activity.…”

“…Schematic illustration of the smart DNA tweezer for detection of telomerase activity. Reprinted with permission from ref …”

Applications of Catalytic Hairpin Assembly Reaction in Biosensing

“…In the past few decades, nucleic acids have been investigated in “dynamic DNA nanotechnology” instead of depending on the simple phosphate‐sugar backbone. Dynamic DNA nanotechnology includes the dynamic displacement and movement of nanostructures stimulated with the transitions of nucleic acids, such as DNA tweezers, DNA walkers, DNA dendrimers, and DNA circuits . Among them, toehold‐mediated strand displacement (TMSD), which is powered by the free energy of strand displacement, is a typical example.…”

Nonradioactive direct telomerase activity detection using biotin‐labeled primers

“…Therefore, many research teams continue to explore alternative methods for detecting telomerase, most of which are based on nanoparticle materials. These include colorimetry based on gold nanoparticles, 5–7 exponential isothermal amplification of telomere repeat (EXPIATR), 8,9 electrochemistry, 10 fluorescence, 11–17 surface‐enhanced Raman scattering (SERS), 18–22 surface plasmon resonance (SPR), 23,24 and the intracellular telomerase assay 25–28 . Although these methods do not incorporate PCR, and are sensitive, they still have their shortcomings including complicated manipulation, time‐consuming procedures, or the need for elaborate instruments and expensive fluorescent labels 5 .…”