A smart ZnO@polydopamine-nucleic acid nanosystem for ultrasensitive live cell mRNA imaging by the target-triggered intracellular self-assembly of active DNAzyme nanostructures

Abstract

Efficient strategies for the ultrasensitive imaging of gene expression in living cells are essential in chemistry and cell biology. Here, we report a novel and efficient enzyme-free dual signal amplification strategy for live cell mRNA imaging by using a smart nucleic acid hairpin-based nanosystem. This nanosystem consists of a ZnO nanoparticle core, an interlayer of polydopamine and an outer layer of four hairpin DNA (hpDNA) probes. Such a core-shell nanosystem facilitates the cellular uptake of molecular hairpin payloads, protects them from nuclease digestion, and delivers them into the cytoplasm by the acid-triggered dissolution of the ZnO core. In the presence of target mRNA, the released hpDNA probes self-assemble via HCR into wire-shaped active DNAzymes that catalyze the generation of a fluorescence signal. The target-initiated HCR events and DNAzyme cascades offer efficient dual amplification and enable the ultrasensitive detection of mRNA with a femtomolar detection limit. Live cell assays show an intense fluorescence response from a tumor-related biomarker survivin mRNA only in tumor cells untreated with a survivin expression repressor YM155, but not in normal cells. The developed nanosystem provides a potential platform for the amplified imaging of low-abundance disease-related biomarkers in live cells.

Scheme 1. (A) Preparation of the polydopamine-coated ZnO nanoparticles (ZnO@PDA NPs) and functional hairpin DNA strands (hpDNAs)-immobilized ZnO@PDA (ZnO@PDA-hpDNAs) nanosystem. (B) Illustration of the ZnO@PDA-hpDNAs nanosystem for live cell mRNA imaging via the target-triggered HCR-mediated intracellular self-assembly of wire-shaped active DNAzyme nanostructures.

Fig. 1. (A) Polyacrylamide gel electrophoresis: H0, H1 and H2 with the target sequence (lane 1); H0, H1 and H2 mixture (lane 2); H1 and H2 with the target sequence (lane 3); H1 (lane 4); H2 (lane 5); H0 (lane 6); target sequence (lane 7); H0 with the target sequence (lane 8); H1 and H2 mixture (lane 9). (B) AFM image and cross-section analysis of wire-shaped DNA nanostructures formed by the target-triggered HCR event.

Fig. 2. (A) Fluorescence spectra obtained by incubating the nanosystem carrying H0, H1, H2 and H3 with the (a) target sequence, (b) nontarget sequence, and (c) assay buffer as well as the target sequence. (B) Fluorescence spectral responses to the target sequence with different concentrations: (a) 0 M, (b) 1 × 10^–16 M, (c) 1 × 10^–15 M, (d) 1 × 10^–14 M, (e) 1 × 10^–13 M, (f) 1 × 10^–12 M, (g) 1 × 10^–11 M, (h) 1 × 10^–10 M, (i) 1 × 10^–9 M, (j) 1 × 10^–8 M, and (k) 1 × 10^–7 M target sequence. (C) Plot of fluorescence peak intensities (at 526 nm) versus target concentrations. (D) Fluorescence peak intensities versus target concentrations on a logarithmic scale.

Fig. 3. CLSM images of HeLa cells after being incubated with different nanosystems: (A) ZnO@PDA-hpDNAs nanosystem (10 μg mL^–1), (B) ZnO@PDA-hpDNAs nanosystem (5 μg mL^–1), (C) ZnO@PDA-H6 nanosystem (10 μg mL^–1), (D) ZnO@PDA-H3 nanosystem (10 μg mL^–1), and (E) ZnO@PDA-hpDNAs nanosystem (10 μg mL^–1) without any additional Mg²⁺ ions. Scale bar = 25 μm.

Fig. 4. (A) CLSM images for HeLa cells treated with varying concentrations of YM155 followed by incubation with the ZnO@PDA-hpDNAs nanosystem (10 μg mL^–1). (a) Green fluorescence images and (b) the merged images of fluorescence and bright field. Scale bar = 25 μm. (B) Fluorescence intensity comparison of the cells by flow cytometry. MFI represents the mean fluorescence intensity of intracellular FAM. (C) Relative expression levels of survivin mRNA in HeLa cells treated with varying concentrations of YM155.

Fig. 5. (A) CLSM images of different cells incubated with the ZnO@PDA-hpDNAs nanosystem (10 μg mL^–1). (a) Green fluorescence images and (b) the merged images of fluorescence and bright field. Scale bar = 25 μm. (B) Fluorescence intensity comparison of the different cells by flow cytometry. (C) Relative expression levels of survivin mRNA in the L02, C166, MCF-10A, HepG2, SMCC-7721, and MCF-7 cells.