Abnormal expression of miRNAs is often detected in various human cancers. DNAzyme machines combined with gold nanoparticles (AuNPs) hold promise for detecting specific miRNAs in living cells but show short circulation time due to the fragility of catalytic core. Using miRNA-21 as the model target, by introducing a circular bulging DNA shield into the middle of the catalytic core, we report herein a self-protected DNAzyme (E) walker capable of fully stepping on the substrate (S)-modified AuNP for imaging intracellular miRNAs. The DNAzyme walker exhibits 5-fold enhanced serum resistance and more than 8-fold enhanced catalytic activity, contributing to the capability to image miRNAs much higher than commercial transfection reagent and well-known FISH technique. Diseased cells can accurately be distinguished from healthy cells. Due to its universality, DNAzyme walker can be extended for imaging other miRNAs only by changing target binding domain, indicating a promising tool for cancer diagnosis and prognosis.
Due to high programmability and good biocompatibility, DNA has been recognized as a powerful building block for engineering of sophisticated nanostructures for different purposes. Herein, we present the first example of a bead-string-shaped DNA nanowire (BS-nanow) with long-range structural order for in vivo bioimaging and targeted drug delivery. BS-nanow is assembled from DNA tetrahedron units with precise nanometer-scale spatial control, capable of accommodating chemotherapeutic agents with high payload capacity (1204 binding sites) as well as possessing a 60-fold enhanced binding affinity for target cells. Confocal fluorescence imaging and in vivo experiments on CEM subcutaneous tumor-bearing mice show that specific bioimaging of living cells and even systemic delivery of drugs into internal tumor organs and tissues were accomplished, thereby achieving an efficient inhibition of tumor growth in the xenograft model without systemic toxicity. BS-nanow’s show potential in vivo applications in accurate diagnosis and targeted therapy for human cancer.
The abnormal expression of miRNA-21 is often found in tumor specimens and cell lines, and thus, its specific detection is an urgent need for the diagnosis and effective therapy of cancers. In this contribution, we demonstrate a palindrome-based hybridization chain reaction (PHCR) upon the stimuli of a short oligonucleotide trigger to perform the autonomous assembly of cross-linked network structures (CNSs) for the amplification detection of miRNA-21 and sensitive fluorescence imaging of cancerous cells. The building blocks are only two palindromic hairpin-type DNA strands that are separately modified with different fluorophores (Cy3 and Cy5), which is easily combined with the catalytic hairpin assembly (CHA) technique that can further amplify the signal output. Utilizing the CHA−PHCR assay system, a small amount of miRNA-21 can activate many triggers via CHA and in turn induce the PHCR-based CNS assembly from more DNA building blocks, bringing Cy3 and Cy5 into close proximity to each other and generating ultrasensitive fluorescence resonance energy transfer signals. As a result, target miRNA can be quantitatively detected down to as low as 10 pM with high assay specificity. The coexisting nontarget miRNAs and other biomacromolecules do not interfere with signal transduction. The developed assay system is suitable for screening different expression levels of miRNA-21 in living cells by fluorescence imaging. The palindrome-based cross-linking assembly can enhance the intracellular stability of assembled nanostructures by at least fivefold and exhibit the good universality for the detection of other miRNAs. Moreover, cancerous cells can be distinguished from healthy cells, and the CHA−PHCR assay is in good accordance with the gold standard PCR method, indicating a promising platform for the diagnosis of human cancers and other genetic diseases.
Sensitive and selective detection of proto-oncogenes, especially recognition of point mutation, is of great importance in cancer diagnosis. Here, a ligation-mediated technique is demonstrated for the construction of an intertwined three-dimensional DNA nanosheet (3D SDN) on an electrode surface from only two palindromic hairpin probes (HP1 and HP2), creating a powerful electrochemical biosensor (E-biosensor) for the detection of the p53 gene. First, a capturing probe (CP) is immobilized on an electrode surface via Au−S chemistry, forming an electrochemical sensing interface. In the presence of the target p53 (T), the triggering probe is covalently linked to CP by a ligase. Moreover, target hybridization/ligation/dehybridization process is repeated, amplifying the target hybridization event and increasing the content of surface-confined triggering fragments. As a result, HP1 is opened and in turn interacts with HP2, forming intertwined 3D SDN where HP1 and HP2 are alternately arranged in parallel. Common hybridization and interaction between palindromic fragments are responsible for the assembly in the horizontal and vertical directions, respectively. An electrochemical indicator, methylene blue (MB), can be inserted into 3D SDN, generating a strong electrochemical signal. Utilizing the 3D SDN-based E-biosensor, the target DNA is detected down to 3 fM with a linear response range from 10 fM to 10 nM. Single point mutations are reliably identified even in fetal bovine serum and cellular homogenate. Because of the several advantages of simple design, good universality, inexpensive instrumentation, high assay specificity, and sensitivity, the 3D SDN-based E-biosensor is expected to provide a potential platform for screening point mutation required by early clinical diagnostics and medical research.
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