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. 2018 Nov 19;10(5):1442-1449.
doi: 10.1039/c8sc04887h. eCollection 2019 Feb 7.

Dual-microRNA-controlled double-amplified cascaded logic DNA circuits for accurate discrimination of cell subtypes

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Dual-microRNA-controlled double-amplified cascaded logic DNA circuits for accurate discrimination of cell subtypes

Ke Quan et al. Chem Sci. .

Abstract

Accurate discrimination between different cells at the molecular level is particularly important for disease diagnosis. Endogenous RNAs are such molecular candidates for cancer cell subtype identification. But the key is that there is often low abundance of RNAs in live cells, or some RNAs are often shared by multiple types of cells. Thus, we have designed dual-microRNA-controlled double-amplified cascaded logic DNA circuits for cancer cell subtype identification. The basic idea is to improve sensitivity by cascading DNAzyme and hybridization chain reaction (HCR), and improve accuracy by simultaneous detection of miR-122 and miR-21. The in-tube and in-cell experimental results show that the cascaded logic DNA circuits can work and serve to differentiate the liver cancer cells Huh7 from other normal cells and cancer cells. We anticipate that this design can be widely applied in facilitating basic biomedical research and accurate disease diagnosis.

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Figures

Scheme 1
Scheme 1. Schematic illustration of the dual-microRNA-controlled double-amplified cascaded logic DNA circuits. The green and red dots represent FAM and TAMRA, respectively. The triangle denotes a cleavage site (rA).
Fig. 1
Fig. 1. (a) The scheme of the signal-activable DNAzyme amplified circuit. The green dot denotes FAM and the black dot denotes BHQ. The triangle denotes a cleavage site. (b) Electrophoresis analysis of the DNAzyme amplified circuit: lane 1 (5); lane 2 (1) + (4) + Zn2+; lane 3 (1) + (4); lane 4 (1); lane 5 (4). (c) Electrophoresis analysis of the input strand (3) triggered DNAzyme amplified circuit: lane 1 (1) + (2) + (3) + (4) + Zn2+; lane 2 (1) + (2) + (4) + Zn2+; lane 3 (5); lane 4 (4); lane 5. (1) + (2) + (3); lane 6 (2) + (3); lane 7 (3); lane 8 (1) + (2); lane 9 (2); lane 10. (1). (d) Response of the signal-activable DNAzyme amplified circuit to different concentrations of target miR-122.
Fig. 2
Fig. 2. (a) The scheme of the FRET-based HCR amplified circuit. The red dot denotes TAMRA and the green dot denotes FAM. (b) Electrophoresis analysis of the HCR amplified circuit: lane 1 (5); lane 2 (7); lane 3 (5) + (7); lane 4 (5) + (7) + 0.5 (6); lane 5 (5) + (7) + 0.1 (6); lane 6 (4); lane 7 (4) + (7) + (6). (c) Electrophoresis analysis of the DNAzyme-HCR cascaded circuits: lane 1 (5); lane 2 (7); lane 3 (5) + (7) +0.1 (6); lane 4 (1) + (4) + (7); lane 5 (1) + (4) + (7) + (6). (d) Response of the FRET-based HCR amplified circuit to different concentrations of target miR-21 (6).
Fig. 3
Fig. 3. (a) Schematic diagram of the cascade AND logic circuitry. (b) The histograms show the FA/FD under different situations. Error bars represent the standard deviation from three independent measurements. The inset shows the truth table based on the input–output signal correlation pattern. “1” and “0” represent the presence and absence of miR-122 or miR-21, respectively.
Fig. 4
Fig. 4. The fluorescence images of the cascaded logic DNA circuits with three cell lines: Huh 7, Hela and HEK 293 T. The imaging was performed with a 60× oil immersion objective. Green field = FAM fluorescence and red field = TAMRA fluorescence. The scale bar is 10 μm.
Fig. 5
Fig. 5. (a) Flow cytometric assay of the cascaded circuits with the three cell lines. (b) Flow cytometric assay of the cascaded circuits with the three different cell lines by artificially changing the intracellular expression level of miR-122 and miR-21. Each histogram displays the count of cells versus fluorescence of TAMRA. “1” and “0” represent the presence and absence of miR-122 or miR-21, respectively.

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