Programming a DNA tetrahedral nanomachine as an integrative tool for intracellular microRNA biosensing and stimulus-unlocked target regulation

Abstract

The synchronous detection and regulation of microRNAs (miRNAs) are essential for the early tumor diagnosis and treatment but remains a challenge. An integrative DNA tetrahedral nanomachine was self-assembled for sensitive detection and negative feedback regulation of intracellular miRNAs. This nanomachine comprised a DNA tetrahedron nanostructure as the framework, and a miRNA inhibitor-controlled allosteric DNAzyme as the core. The DNA tetrahedron brought the DNAzyme and the substrate in spatial proximity and facilitated the cellular uptake of DNAzyme. In allosteric regulation of DNAzyme, the locked tetrahedral DNAzyme (L-tetra-D) and active tetrahedral DNAzyme (A-Tetra-D) were controlled by miRNA inhibitor. The combination of miRNA inhibitor and target could trigger the conformational change from L-tetra-D to A-Tetra-D. A-Tetra-D cleaved the substrate and released fluorescence for intracellular miRNA biosensing. The DNA tetrahedral nanomachine showed excellent sensitivity (with detection limit down to 0.77 pM), specificity (with one-base mismatch discrimination), biocompatibility and stability. Simultaneously, miRNA stimulus-unlocked inhibitor introduced by our nanomachine exhibited the synchronous regulation of target cells, of which regulatory performance has been verified by the upregulated levels of downstream genes/proteins and the increased cellular apoptosis. Our study demonstrated that the DNA tetrahedral nanomachine is a promising biosense-and-treat tool for the synchronous detection and regulation of intracellular miRNA, and is expected to be applied in the early diagnosis and tailored management of cancers.

Keywords: DNA nanomaterials; DNA tetrahedron; DNAzyme; Target regulation; microRNA detection.

Graphical abstract

Scheme 1

(A) Schematic illustration of the mechanism of DNA tetrahedral nanomachine for specific miRNA detection and regulation in living cells. (B) DNA tetrahedral nanomachine synthesis based on internal self-assembly of six oligonucleotides and amplified reaction process.

Fig. 1

Characterization and optimization of DNA tetrahedral nanomachine. (A) Experimental procedure of this DNA tetrahedral nanomachine. (B) Electrophoresis result to verify the construction of DNA tetrahedral nanomachine. Lane M: DNA ladder, Lane 1: P1, lane 2: P1+P2, lane 3: P1+P2+P4, lane4: P1+P2+P4+P5, lane 5: P1+P2+P4+P5+miRNA inhibitor strand I, lane 6: TD-DNAzyme (P1+P2+P3+P4+P5+I), lane 7: DNA tetrahedral nanomachine reacted with target miR-21,lane 8: DNA tetrahedral nanomachine without the inhibitor strand I, lane 9: the hybridization of target miR-21 and inhibitor I. (C) Characterization of DNA tetrahedral nanomachine via atomic force microscopy (AFM). Scale bars, 10 ​nm. (D) Optimum condition of toehold length at the 3′ end of the inhibitor. Optimum conditions of (E) reaction temperature and (F) reaction time between DNA tetrahedral nanomachine and target.

Fig. 2

Detection performance of DNA tetrahedral nanomachine in vitro and their feasibility in vivo. (A) Schematic illustration of the mechanism of DNA tetrahedral nanomachine for specific miRNA detection. (B) Fluorescence responses in the presence of different concentrations of miR-21. (C)The linear relationship between signals and miRNA concentrations. (D and E) Selectivity of DNA tetrahedral nanomachine toward Miss-1, Miss-2, Miss-3, different types of miRNAs (miR-155, miR-25, and let-7) and Mixture.

Fig. 3

(A) Schematic illustration of the mechanism of DNA tetrahedral nanomachine for intracellular miRNA detection. (B) Fluorescence characterization of DNA tetrahedral nanomachine degradation by incubating in 10% fetal bovine serum for the stated times. (C) Cell viability assay (CCK-8): HepG2 cells treated with DNA tetrahedral nanomachine of varied concentrations (0–200 ​nM) for 12 ​h at 37 ​°C. Error bars indicate means ​± ​SD (n ​= ​3). (D) Confocal fluorescence imaging of DNA tetrahedral nanomachine incubated with HepG2 cells for 1, 2, 4, 8 and 12 ​h. Scale bars are 20 ​μm.

Fig. 4

Study of DNA tetrahedral nanomachine for detecting miRNAs in vivo. (A) Confocal fluorescence imaging of DNA tetrahedral nanomachine for detection of miR-21 in L02 ​cells and HepG2 cells. (B) Fluorescence intensity and the relative expression levels of miR-21 in the HepG2 cells, and L02 ​cells. And the relative expression levels of miR-21 in corresponding cells. (C) Confocal fluorescence imaging of DNA tetrahedral nanomachine to detect miR-21 in HepG2 cells treated with miR-21 inhibitor, untreated HepG2 cells and HepG2 cells treated with mimic. (D) Fluorescence intensity of the HepG2 cells pretreated with miR-21 inhibitor, untreated HepG2 cells and HepG2 cells treated with mimic. And the relative expression levels of miR-21 in corresponding cells. Scale bars, 10 ​μm. Error bars indicate means ​± ​SD (n ​= ​3). (∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ∗∗∗∗P ​< ​0.0001).

Fig. 5

Study of intracellular regulatory function of DNA tetrahedral nanomachine. (A) Schematic illustration of the mechanism of DNA tetrahedral nanomachine for synchronous cell regulation. (B) The changes of downstream mRNA expression after the regulation of DNA tetrahedral nanomachine. (C) The changes of downstream protein expression after the regulation of DNA tetrahedral nanomachine. (D) Apoptosis of HepG2 cells after incubated with 100 ​nM DNA tetrahedral nanomachine for 48 ​h. 7-AAD: 7-Amino-Actinomycin; PE: Phycoerythrin. Error bars indicate means ​± ​SD (n ​= ​3). (∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001).