A new class of intracellular nanoprobe, termed AuNP-based hairpin-locked-DNAzyme probe, was developed to sense miRNA in living cells. Briefly, it consists of an AuNP and hairpin-locked-DNAzyme strands. In the absence of target miRNA, the hairpin-locked-DNAzyme strand forms a hairpin structure by intramolecular hybridization, which could inhibit the catalytic activity of DNAzyme strand and the fluorescence is quenched by the AuNP. However, in the presence of target, the target-probe hybridization can open the hairpin and form the active secondary structure in the catalytic cores to yield an “active” DNAzyme, which then cleaves the self-strand with the assist of Mg2+. The cleaved two shorter DNA fragments are separated with the target. As a result, the fluorophores are released from the AuNP and the fluorescence is enhanced. Meanwhile, the target is also released and binds to another hairpin-locked-DNAzyme strand to drive another cycle of activation. In such a way, the target-recycling amplification leads to significant signal enhancement and thus offers high detection sensitivity.

With the rapid development of DNA nanotechnology, various DNA nanostructures with different shapes and sizes have been self-assembled using “bottom-up” fabrication strategies and applied to a wide range of fields such as biosensors, drug delivery and tools for molecular biology. As a classical and simple polyhedron, DNA tetrahedron can be easily synthesised by a one-step assembly. Due to the excellent biocompatibility and cellular permeability, it provides a universal and promising platform to construct a series of biosensors and drug delivery systems for living cells studies. Moreover, the high programmability of DNA tetrahedron determines its capability to perform artful design and combine with other materials. Herein, we review and summarise the development and applications of DNA tetrahedron in living cell studies. We mainly focus on two parts, cellular biosensors for the detection of nucleic acids, proteins, small molecules and cancer cells and drug delivery systems for chemotherapy, immunotherapy, photodynamic therapy and gene silencing. With the rapid progress in DNA tetrahedron as well as DNA nanotechnology, new avenues and opportunities have opened up in analytical chemistry, molecular biology and medicine.

The donor donor–acceptor (DD–A) FRET model has proven to have a higher FRET efficiency than donor–acceptor acceptor (D–AA), donor–acceptor (D–A), and donor donor–acceptor acceptor (DD–AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the “DD–A” FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary “D–A” model. Furthermore, MnO2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn2+ ions by intracellular GSH. The results indicated that the MnO2 nanosheet mediated “DD–A” FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO2 nanosheet mediated “DD–A” FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

Due to its low cytotoxicity, high resistance to enzymatic degradation, and cellular permeability, a DNA tetrahedron-based molecular beacon (DTMB) is designed for tumor-related TK1 mRNA detection in living cells, where the target sequence can induce the tetrahedron from contraction to extension, resulting in fluorescence restoration.

A FRET-based sensor is anchored on the cell surface through streptavidin–biotin interactions. Due to the excellent properties of the pH-sensitive i-motif structure, the sensor can detect extracellular pH with high sensitivity and excellent reversibility.

Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers’ attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity.

To date, a few of DNAzyme-based sensors have been successfully developed in living cells; however, the intracellular aptazyme sensor has remained underdeveloped. Here, the first aptazyme sensor for amplified molecular probing in living cells is developed. A gold nanoparticle (AuNP) is modified with substrate strands hybridized to aptazyme strands. Only the target molecule can activate the aptazyme and then cleave and release the fluorophore-labeled substrate strands from the AuNP, resulting in fluorescence enhancement. The process is repeated so that each copy of target can cleave multiplex fluorophore-labeled substrate strands, amplifying the fluorescence signal. Results show that the detection limit is about 200 nM, which is 2 or 3 orders of magnitude lower than that of the reported aptamer-based adenosine triphosphate (ATP) sensors used in living cells. Furthermore, it is demonstrated that the aptazyme sensor can readily enter living cells and realize intracellular target detection.

Recently, DNA tetrahedron-based sensors for intracellular detection have attracted more attention due to many interesting properties, including good structural rigidity, excellent biocompatibility, high resistance to enzymatic degradation, and the ability to enter cells without the use of transfection agents. However, the previous designs are still restricted by their lack of accuracy, reliability, and generality. Herein, to solve these limitations, we describe self-assembly of the competition-mediated FRET-switching DNA tetrahedron molecular beacon (CF-DTMB), and its applications for intracellular tumor-related mRNA detection. In brief, the recognition strand is partially complementary to its competitor, which is a hairpin stem-loop structure inserted in one edge of the DNA tetrahedron. In the absence of a target, a long domain of the recognition strand hybridizes with the competitor and makes the hairpin structure open, inducing the two labeled dyes to be spatially separated (FRET off). However, in the presence of the target, the competitor is substituted for the target to bind with the recognition strand, subsequently leading the formation of a stem-loop structure, which draws two dyes together (FRET on). The results demonstrate that the current strategy possesses the merits of the previous DNA tetrahedron-based sensors, but also improves the accuracy and reliability. Furthermore, we have also demonstrated that our design can become a general strategy for detecting and imaging a variety of other molecules, such as adenosine triphosphate (ATP) in living cells. Therefore, the CF-DTMB can serve as an excellent intracellular molecular detection tool, which is promising for biological and disease studies.Keywords: DNA nanotechnology; DNA tetrahedron; FRET; intracellular detection; molecular beacon;

The donor donor–acceptor (DD–A) FRET model has proven to have a higher FRET efficiency than donor–acceptor acceptor (D–AA), donor–acceptor (D–A), and donor donor–acceptor acceptor (DD–AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the “DD–A” FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary “D–A” model. Furthermore, MnO2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn2+ ions by intracellular GSH. The results indicated that the MnO2 nanosheet mediated “DD–A” FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO2 nanosheet mediated “DD–A” FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

Due to its low cytotoxicity, high resistance to enzymatic degradation, and cellular permeability, a DNA tetrahedron-based molecular beacon (DTMB) is designed for tumor-related TK1 mRNA detection in living cells, where the target sequence can induce the tetrahedron from contraction to extension, resulting in fluorescence restoration.

A FRET-based sensor is anchored on the cell surface through streptavidin–biotin interactions. Due to the excellent properties of the pH-sensitive i-motif structure, the sensor can detect extracellular pH with high sensitivity and excellent reversibility.