Novel Label-free Assay of Telomerase Based on DNAzyme†

doi:10.7503/cjcu20141048

Abstract

Abstract:

A novel method for telomerase detection based on DNAzyme and toehold-mediated strand displacement was developed. Optimal designing of a hairpin probe ensured efficient inhibition of DNAzyme activity by preventing it from forming the G-quadruplex structure. After the substrate TS was elongated by telomerase, the original hairpin structure could be destroyed through toehold-mediated strand displacement, so that the free DNAzyme was released, which catalyzed the oxidation of ABTS^2- to generate an absorbance increase at 415 nm. The results indicated that the proposed method could detect the telomerase as low as 500 Hela cells equi-valent. Besides, this method is simple and requires no fluorescence labeling or complicated surface modification, and thus is expected to have a wide use in routine telomerase analysis of tumor cells.

Key words: Telomerase, DNAzyme, Horseradish peroxidase, Signal amplification, Toehold-mediated strand displacement

CLC Number:

O657

TrendMD:

FAN Hongliang, ZHANG Tao, JIN Wei, JIN Qinhan. Novel Label-free Assay of Telomerase Based on DNAzyme^†[J]. Chem. J. Chinese Universities, 2015, 36(4): 625.

Figures/Tables 7

Table 1 DNA sequences used in this experiment

DNA	Sequence
DZ	5'-TGGGTAGGGCGGGTTGGGA-3'(Free DNAzyme)
TM-1	5'-CTAACCCTAA CTCTGCT TGGGTAGGGCGGGTTGGGA GCAGAG-3'
TM-2	5'-CTAACCCTAA CTCTGCTC TGGGTAGGGCGGGTTGGGA GCAGAG-3'
TM-3	5'-CTAACCCTAA CTCTGCTCC TGGGTAGGGCGGGTTGGGA GCAGAG-3'
TM-4	5'-CTAACCCTAA CTCTGCTCCC TGGGTAGGGCGGGTTGGGA GCAGAG-3'
TM-31	5'-AACCCTAA CTCTGCTCC TGGGTAGGGCGGGTTGGGA GCAGAG-3'
TM-32	5'-CCCTAA CTCTGCTCC TGGGTAGGGCGGGTTGGGA GCAGAG-3'
TS	5'-AATCCGTCGAGCAGAGTT
TS-02	5'-AATCCGTCGAGCAGAGTT AG-3'
TS-04	5'-AATCCGTCGAGCAGAGTT AGGG-3'
TS-06	5'-AATCCGTCGAGCAGAGTT AGGGTT-3'
TS-08	5'-AATCCGTCGAGCAGAGTT AGGGTTAG-3'
TS-R2	5'-AATCCGTCGAGCAGAGTT AGGGTTAGGGTT-3'

Scheme 1 Illustration of the proposed telomerase assay

Fig.1 Hairpin structures of TM1—TM4

Fig.2 Time-dependent absorbance changes of ABTS2-(5 mmol/L)/H2O2(5 mmol/L)/Hemin(300 nmol/L) in the presence of different probes(100 nmol/L)(A), absorbance changes of Hemin(5 mmol/L)/ABTS2-(5 mmol/L)/H2O2(300 nmol/L)/TM-x(100 nmol/L) after adding TS or TS-R2 for 120 s(B) and ratios of absorbance changes caused by TS-R2(200 nmol/L) and TS(200 nmol/L)(C)

Fig.3 Absorbance changes of TM-3(100 nmol/L)/Hemin(300 nmol/L)/ABTS2-(5 mmol/L)/H2O2(5 mmol/L) in the presence of various telomerase products(200 nmol/L)(A) and effects of overhang length on the absorbance change of TM-x(100 nmol/L)/Hemin(300 nmol/L)/ABTS2-(5 mmol/L)/H2O2(5 mmol/L) in the presence of TS or TS-R2(B)

Fig.4 Effects of Hemin concentration on absorbance change of TM-31(100 nmol/L)/ABTS(5 mmol/L)/H2O2(5 mmol/L) in the presence of TS or TS-R2(A) and effects of Hemin and ABTS2- concentrations on RTS-R2/TS value(B)

Fig.5 Time-dependent absorbance changes of TM-31(100 nmol/L)/Hemin(300 nmol/L)/ABTS(7.5 mmol/L)/H2O2(5 mmol/L) caused by TS-R2(A) and Hela cell extracts(B) (A) a. [TS]=200 nmol/L, TS-R2/(nmol·L-1): b. 20, c. 40, d. 60, e. 80, f. 100, g. 150, h. 200; (B) a. heat deactivated cells, b. 500 cells, c. 1000 cells, d. 2000 cells, e. 5000 cells.

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