An RCA product is a promising scaffold for the construction of DNA nanostructures, but so far, there is no RCA scaffold-based dynamic reconfigurable nanorobot for biological applications. In this contribution, we develop an intracellular stimuli-responsive reconfigurable coiled DNA nanosnake (N-Snake) by using incomplete aptamer-functionalized (A) DNA tetrahedrons (T) to fold a long tandemly repetitive DNA strand synthesized by rolling circle amplification reaction (R) with the help of palindromic fragment (P). A DNA-assembled product, ARTP, including spiked aptamers, can retain the structural integrity even if exposed to fetal bovine serum (FBS) for 24 h and displays substantially enhanced target molecule-dependent cellular internalization efficiency. ARTP contains tetrahedral containers and linear containers, so that there are 500 doxorubicins (DOXs) and 12.5 siRNAs per ARTP. Moreover, ARTP can precisely transport anticancer drugs to cancerous sites and controllably release via the structural reconfiguration upon intracellular stimuli, almost 100% inhibiting tumor growth without detectable systemic toxicity owing to the synergistic RNAi-/Chemotherapy. Apparently, coiled N-snake, DOX/siPlk1-loaded ARTP, can specifically enter tumor cells, uncoil upon intracellular stimuli, and attack the cells from the inside, exerting precise cancer therapy.

Chemotherapy is commonly used to treat malignant tumors. However, conventional chemotherapeutic drugs often cannot distinguish between tumor and healthy cells, resulting in adverse effects and reduced therapeutic efficacy. Therefore, zigzag-shaped gear-occlude-guided cymbal-closing (ZGC) DNA nanotechnology was developed based on the mirror-symmetry principle to efficiently construct symmetric DNA polyhedra. This nanotechnology employed simple mixing steps for efficient sequence design and assembly. A targeting aptamer was installed at a user-defined position using an octahedron as a model structure. Chemotherapeutic drug-loaded polyhedral objects were subsequently delivered into tumor cells. Furthermore, anticancer drug-loaded DNA octahedra were intravenously injected into a HeLa tumor-bearing mouse model. Assembly efficiency was almost 100 %, with no residual building blocks identified. Moreover, this nanotechnology required a few DNA oligonucleotides, even for complex polyhedrons. Symmetric DNA polyhedrons retained their structural integrity for 24 h in complex biological environments, guaranteeing prolonged circulation without drug leakage in the bloodstream and promoting efficient accumulation in tumor tissues. In addition, DNA octahedra were cleared relatively slowly from tumor tissues. Similarly, tumor growth was significantly inhibited in vivo, and a therapeutic outcome comparable to that of conventional gene-chemo combination therapy was observed. Moreover, no systemic toxicity was detected. These findings indicate the potential application of ZGC DNA nanotechnology in precision medicine.

Although DNA probes have attracted increasing interest for precise tumor cell identification by imaging intracellular biomarkers, the requirement of commercial transfection reagents, limited targeting ligands, and/or non-biocompatible inorganic nanostructures has hampered the clinic translation. To circumvent these shortcomings, a reconfigurable ES-NC (Na+-dependent DNAzyme (E)-based substrate (S) cleavage core/shell DNA nanocluster (NC)) entirely from DNA strands is assembled for precise imaging of cancerous cells in a successive dual-stimuli-responsive manner. This nanoprobe is composed of a strung DNA tetrahedral satellites-based protective (DTP) shell, parallelly aligned target-responsive sensing (PTS) interlayer, and hydrophobic cholesterol-packed innermost layer (HCI core). Tetrahedral axial rotation-activated reconfiguration of DTP shell promotes the exposure of interior hydrophobic moieties, enabling cholesterol-mediated cellular internalization without auxiliary elements. Within cells, over-expressed glutathione triggers the disassembly of the DTP protective shell (first stimulus), facilitating target-stimulated signal transduction/amplification process (second stimuli). Target miRNA-21 is detected down to 10.6 fM without interference from coexisting miRNAs. Compared with transfection reagent-mediated counterpart, ES-NC displays a higher imaging ability, resists nuclease degradation, and has no detectable damage to healthy cells. The blind test demonstrates that the ES-NC is suitable for the identification of cancerous cells from healthy cells, indicating a promising tool for early diagnosis and prediction of cancer. A reconfigurable core/shell DNAzyme-based ES-NC nanoprobe is demonstrated for the specific identification of cancerous cells. Axial rotation of the tetrahedral shell turns interior cholester moieties inside out and makes ES-NC grow a wiggling tentacle, contributing to cellular internalization. The disassembly of the protective shell by endogenous stimuli exposes the target miRNA binding site and catalytic core, activating the signal transduction/amplification process for cell imaging. image

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

Developing DNA nanostructures as delivery vectors into organelles is a key technical barrier because of their innate poor pharmacokinetic properties. Herein, we propose a unique concept for high precision cancer therapy by using small gold nanoparticles (AuNPs) as adhesive nano-tapes to fold a long string of aptamerattached tetrahedral DNA pearls (Apt-Nano-Tetra) into a spherical multicenter DNA nanostructure that can serve as a nucleus-targeted drug delivery carrier (Apt-ADMC) with a 75 similar to 85 times enhanced doxorubicin (Dox)-loading capacity. After specifically entering target tumor cells, Dox-Apt-ADMC is disassembled into individual AuNPs surrounded by short Apt-Nano-Tetra, finally reaching cell nucleus and causing cell apoptosis. Even if Dox-Apt-ADMC is treated with fetal bovine serum for 12 h, the drug delivery ability is not compromised, contributing to high functional stability in circulation system. As such, the systemically-administrated Dox-Apt-ADMC shows 7 times enhanced tumor accumulation and almost 100% inhibits tumor growth without detectable systemic toxicity. (c) 2022 Elsevier Ltd. All rights reserved.

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.

Nucleic acid probes have the advantages of excellent biocompatibility, biodegradability, versatile functionalities and remarkable programmability. However, the low biostability of nucleic acid probes under complex physiological conditions limits their in vivo application. Despite impressive progress in the development of inorganic material-mediated biostable nucleic acid nanostructures, uncertain systemic toxicity of composite nanocarriers has hindered their application in living organisms. In the field of biomedicine, as a promising alternative capable of avoiding potential cytotoxicity, biologically stable nanostructures composed entirely of DNA oligonucleotides have been rapidly developed in recent years, offering an exciting in vivo tool for cancer diagnosis and clinical treatment. In this review, we summarize the recent advances in the development of nuclease-resistant DNA nanostructures with different geometrical shapes, such as tetrahedron, octahedron, DNA triangular prism (DTP), DNA nanotubes and DNA origami, introduce innovative assembly strategies, and discuss unique structural advantages and especially biological applications in cellular imaging and targeted drug delivery in an organism. Finally, we conclude with the challenges in the clinical development of DNA nanostructures and present an outlook of the future of this rapidly expanding field.