Label-free Fluorescence Assay of Telomerase RNA Based on Strand Displacement Amplification†

doi:10.7503/cjcu20150524

Abstract

Abstract:

A novel fluorescence method was developed for detecting telomerase RNA(hTR) based on the fluorescence resonance energy transfer(FRET) and strand displacement amplification(SDA) technique. Graphene oxide(GO) served as DNA carrier and fluorescence quencher. SYBR Green Ⅰ(SGⅠ) and hairpin DNAs(hpDNA) are fluorescence probe and molecular recognition probes, respectively. The fluorescence of SGⅠ that intercalated into the stem of hairpin DNAs was quenched when hpDNA1 and hpDNA2 were adsorbed onto the surface of GO. In the presence of T1, the hybridization reaction between hpDNA1 and T1 opened the hairpin structure of hpDNA1 to trigger the SDA reaction between hpDNA2 and T1, leading to an accumulation of hpDNA1/hpDNA2 hybrids. The rigid dsDNA desorbed from GO surface to restore the fluorescence of SGⅠ. Based on the change in fluorescence intensity, T1 can be quantitatively detected from 0.2 nmol/L to 50 nmol/L, with a detection limit of 90 pmol/L. Therefore, it offers a label-free, highly sensitive and specific fluorescence strategy for detection of hTR.

Key words: Telomerase RNA, Strand displacement amplification, Fluorescence resonance energy transfer, Label-free

CLC Number:

O657.3

TrendMD:

ZHANG Xiafei, CHENG Rui, SHI Zhilu, JIN Yan. Label-free Fluorescence Assay of Telomerase RNA Based on Strand Displacement Amplification^†[J]. Chem. J. Chinese Universities, 2016, 37(1): 12.

Figures/Tables 9

Scheme 1 Schematic illustration of a label-free strand displacement amplification strategy for sensitive detection of hTR

Fig.1 Fluorescence emission spectra of SGⅠ^(A) a. SGⅠ; b. SGⅠ+hpDNA1+hpDNA2; c. SGⅠ+hpDNA1+hpDNA2+GO; d. SGⅠ+hpDNA1+hpDNA2+GO+T1. Inset of (A): photographs under UV light. a. SGⅠ+hpDNA1+hpDNA2; b. SGⅠ+htDNA1+hpDNA2+GO; c. SGⅠ+hpDNA1+T1; d. SGⅠ+hpDNA1+hpDNA2+GO+T1.

Fig.2 Gel electrophoretic analysis of strand displacement amplification strategy^Lane M: marker; lane 1: hpDNA1; lane 2: hpDNA2; lane 3: T1; lane 4: hpDNA1+T1; lane 5: hpDNA2+T1; lane 6: hpDNA1+hpDNA2; lane 6: hpDNA1+hpDNA2+T1.

Fig.3 Investigation of the selectivity of the proposed method^a. SGⅠ+hpDNA1+hpDNA2/GO; b. SGⅠ+HPDNA1+hpDNA2+T1; c. SGⅠ+hpDNA1+hpDNA2+single-base mismatched DNA; d. SGⅠ+hpDNA1+hpDNA2+two-base mismatched DNA; e. SGⅠ+hpDNA1+hpDNA2+completely mismatch DNA. Inset: photographs under UV light.

Fig.4 Fluorescence emission spectra of SGⅠ/hpDNA1/hpDNA2 in the presence of T1(A) and the linear relationship between the fluorescence intensity and the concentrations of T1(B)^ (A) c(T1)/(nmol·L-1), a—f: 0, 10, 20, 30, 40, 50.

Fig.5 Fluorescence emission spectra of GO/SGⅠ/hpDNA1/hpDNA2 in the presence of T1(A) and the linear relationship between the fluorescence intensity and concentrations of T1(B)^ (A) c(T1)/(nmol·L-1), a—m: 0, 0.2, 0.4, 0.6, 0.8, 10, 2, 5, 10, 20, 30, 40, 50. Inset of (B) is the linear relationship in T1 concentration range of 0.2—1.0 nmol/L.

Fig.6 Gel electrophoresis analysis of hpDNA1 treated with DNase 1 in the absence and presence of GO for 5, 10, 30 and 60 min

Fig.7 Cytotoxicity test of the GO in HeLa cell(A)and the color change of formazan crystals with different concentrations of GO(B)

Fig.8 Intracelluar images of HeLa cells incubated with GO/F-hpDNA1(A1—A3) and GO/F-hpDNA1/hpDNA2 for 8 h(B1—B3)^(A1), (B1): Bright-field images; (A2), (B2): fluorescence images; (A3), (B3): merge of fluorescence and bright-field images.

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