Label-free molecular beacons-based cascade amplification DNA machine for sensitive detection of telomerase activity.
Sensitive detection of telomerase activity is critical to cancer diagnosis, screening of anticancer drugs and evaluation of cancer therapy. Herein, a label-free molecular beacons-based DNA machine was developed for sensitive detection of telomerase activity. The DNA machine consisted of T7 exonuclease (T7 Exo), label-free recognition molecular beacon (RMB) and signal molecular beacon (SMB) with projecting 5'-terminuses, which can protect RMB and SMB from being digested by T7 Exo. Firstly, telomerase elongated telomerase substrate (TS) primer, generating a telomerase elongation production (TEP) with tandem repeats (TTAGGG)n. Next, TEP activated the DNA machine by hybridizing with RMB, unfolding RMB with a recessed 5'-terminus, making RMB deprotection from T7 Exo. Then T7 Exo-assisted cycling cleavage was incurred, releasing intact TEP and numerous DNA fragments (trigger DNA), which got recycling I. Subsequently, trigger DNA specifically opened SMB and was recycled by T7 Exo, liberating multiple G-quadruplex (G4) structures, which got recycling II. Finally, TEP and the liberative G4 structures strongly interacted with N-methyl-mesoporphyrin IX (NMM), yielding a significantly enhanced fluorescence together. In this way, per telomerase-mediated elongation event was efficiently converted into the greatly amplified fluorescence signals. Telomerase activity in crude HeLa cells extracts equivalent to 50 cells/mL was successfully measured with a linear range from 50 cells/mL to 2000 cells/mL. Besides, the strategy was also successfully used to assay the inhibition effect of a telomerase-inhibiting drug, demonstrating the strategy holds the potential to screen telomerase inhibitors.
Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
Kan
K
Li
School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China.
Lei
L
Wang
Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China. Electronic address: xuxw@sdu.edu.cn.
Xiaowen
X
Xu
Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China. Electronic address: wjiang@sdu.edu.cn.
Wei
W
Jiang
D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013050:Spectrometry, Fluorescence; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D015335:Molecular Probes; D004247:DNA; C509186:TERT protein, human; D019098:Telomerase
Cascade amplification; DNA machine; Fluorescent strategy; Label-free molecular beacon; Telomerase activity detection