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Herein, we report on the design of a programmable DNA ribbon using long-chain DNA molecules with a user-defined repetitive padlock sequence. The DNA ribbon can be further combined with gold nanoparticles (AuNPs) to create a composite nanomaterial that contains an AuNP core and a high-density DNA crown carrying a cancer-cell-targeting DNA aptamer, a fluorescent tag for location tracking, and a cell-killing drug. This composite material can be efficiently internalized by cancer cells and its cellular location can be tracked by fluorescence imaging. The system offers several attractive characteristics, including simple design, tunable DNA crown, high drug-loading capacity, selective cell targeting, and pH-sensitive drug release. These features make such a material a promising therapeutic agent., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It is crucial to deliver anticancer drugs to target cells with high precision and efficiency. While nanomaterials have been shown to enhance the delivery efficiency once they reach the target, it remains challenging for precise drug delivery to overcome the nonspecific adsorption and off-target effect. To meet this challenge, we report herein the design of a novel DNA nanostructure to act as a DNA nanoscale precision-guided missile (D-PGM) for highly efficient loading and precise delivery of chemotherapeutic agents to specific target cells. The D-PGM consists of two parts: a warhead (WH) and a guidance/control (GC). The WH is a rod-like DNA nanostructure as a drug carrier, whose trunk is a three-dimensionally self-assembled DNA nanoscale architecture from the programmed hybridization among two palindromic DNA sequences in the x - y dimension and two common DNA oligonucleotides in the z direction, making the WH possess a high payload capacity of drugs. The GC is an aptamer-based logic gate assembled in a highly organized fashion capable of performing cell-subtype-specific recognition via the sequential disassembly, mediated by cell-anchored aptamers. Because of the cooperative effects between the WH and the GC, the GC logic gates operate like the guidance and control system in a precision-guided missile to steer the doxorubicin (DOX)-loaded DNA WH toward target cancer cells, leading to selective and enhanced therapeutic efficacy. Moreover, fluorophores attached to different locations of D-PGM and DOX fluorescence dequenching upon release enable intracellular tracing of the DNA nanostructures and drugs. The results demonstrate that by mimicking the functionalities of a military precision-guided missile to design the sequential disassembly of the GC system in multistimuli-responsive fashion, our intrinsically biocompatible and degradable D-PGM can accurately identify target cancer cells in complex biological milieu and achieve active targeted drug delivery. The success of this strategy paves the way for specific cell identity and targeted cancer therapy.

MicroRNAs (miRNAs) play an important role in the regulation of various biological processes and have been used as potential biomarkers for biomedical research and clinical diagnosis. Here, nicking-mediated rolling circle amplification (N-RCA)/symmetric isothermal circular strand-displacement amplification (S-SDA)-integrated combined cascade amplification (rs-CCA) was proposed for let-7a miRNA detection. Via introducing a palindromic fragment-integrated recognition sites for nicking endonuclease Nt.AlWI into the padlock probe, the hybridization of target miRNA can induce N-RCA and continuously generate the nicked fragments (NFs). Because the released NFs each have a palindromic sequence and daughter nicking site at the 3' end, they hybridize with each other, followed by alternately extension by polymerase and nicking by endonuclease. This leads to S-SDA effect, and the released single-stranded products in turn hybridize with NFs, accomplishing the rs-CCA process. When the Sybr Green I dyes intercalate into double-stranded DNA products, the amplified fluorescence signal is achieved. Thus, the target miRNA can be detected down to 5 pM. Importantly, the rs-CCA system is capable of distinguishing the single base difference between target miRNAs, indicating the high sequence specificity. Moreover, its potential application in disease diagnosis was demonstrated via detecting target miRNA in complex biological matrix and analyzing the total RNAs extracted from HeLa cells. As a proof-of-concept building, the impressive rs-CCA scheme is expected to provide a valuable insight into constructing powerful signal amplification strategies via sophisticated combination of biotechnologies available for nucleic acid manipulation and significantly benefit biomedical research and disease diagnostics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A highly sensitive and selective colorimetric assay based on a multifunctional molecular beacon with palindromic tail (PMB) was proposed for the detection of target p53 gene. The PMB probe can serve as recognition element, primer, and polymerization template and contains a nicking site and a C-rich region complementary to a DNAzyme. In the presence of target DNA, the hairpin of PMB is opened, and the released palindromic tails intermolecularly hybridize with each other, triggering the autonomous polymerization/nicking/displacement cycles. Although only one type of probe is involved, the system can execute triple and continuous polymerization strand displacement amplifications, generating large amounts of G-quadruplex fragments. These G-rich fragments can bind to hemin and form the DNAzymes that possess the catalytic activity similar to horseradish peroxidase, catalyzing the oxidation of ABTS by H 2 O 2 and producing the colorimetric signal. Utilizing the newly proposed sensing system, target DNA can be detected down to 10 pM with a linear response range from 10 pM to 200 nM, and mutant target DNAs are able to be distinguished even by the naked eye. The desirable detection sensitivity, high specificity, and operation convenience without any separation step and chemical modification demonstrate that the palindromic molecular beacon holds the potential for detecting and monitoring a variety of nucleic acid-related biomarkers.

The detection of biomarkers is of crucial importance in reducing the morbidity and mortality of complex diseases. Thus, there is a great desire to develop highly efficient and simple sensing methods to fulfill the different diagnostic and therapeutic purposes. Herein, using tumor suppressor p53 gene as model target DNA, we developed a novel palindromic fragment-incorporated molecular beacon (P-MB) that can perform multiple functions, including recognition element, signal reporter, polymerization template and primer. Upon specific hybridization with target DNA, P-MBs can interact with each other and are extended by polymerase without any additional probes. As a result, hybridized targets are peeled off from P-MBs and initiate the next round of reactions, leading to the unique strand displacement amplification (SDA). The newly-proposed enzymatic amplification displays the detection limit as low as 100pM and excellent selectivity in distinguishing single-base mutation with the linear response range from 100pM to 75nM. This is the simplest SDA sensing system so far because of only involving one type of DNA probe. This impressive sensing paradigm is expected to provide new insight into developing new-type of DNA probes that hold tremendous potential with important applications in molecular biology research and clinical diagnosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

The accurate detection of cancer-related genes is of great significance for early diagnosis and targeted therapy of cancer. In this contribution, an automatically cycling operation of a functional overhang-containing molecular beacon (OMB)-based sensing system was proposed to perform amplification detection of the p53 gene. Contrary to the common molecular beacon (MB), a target DNA is designated to hybridize with a label-free recognition probe (RP) with a hairpin structure rather than OMB. In the presence of a target DNA of interest, the locked primer in RP opens and triggers the subsequent amplification procedures. The newly-developed OMB is not only capable of accomplishing cyclical nucleic acid strand-displacement polymerization (CNDP) with the help of polymerase and nicking endonuclease, but is also cleaved by restriction endonucleases, removing the quencher away from the fluorophore. Thus, the target DNA at an extremely low concentration is expected to generate a considerable amount of double-stranded and cleaved OMBs, and the quenched fluorescence is completely restored, leading to a dramatic increase in fluorescence intensity. Utilizing this sensing platform, the target gene can be detected down to 8.2 pM in a homogeneous way, and a linear response range of 0.01 to 150 nM could be obtained. More strikingly, the mutant genes can be easily distinguished from the wild-type ones. The proof-of-concept demonstrations reported herein are expected to promote the development of DNA biosensing systems, showing great potential in basic research and clinical diagnosis.

DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Herein, we report on the design of a programmable DNA ribbon using long‐chain DNA molecules with a user‐defined repetitive padlock sequence. The DNA ribbon can be further combined with gold nanoparticles (AuNPs) to create a composite nanomaterial that contains an AuNP core and a high‐density DNA crown carrying a cancer‐cell‐targeting DNA aptamer, a fluorescent tag for location tracking, and a cell‐killing drug. This composite material can be efficiently internalized by cancer cells and its cellular location can be tracked by fluorescence imaging. The system offers several attractive characteristics, including simple design, tunable DNA crown, high drug‐loading capacity, selective cell targeting, and pH‐sensitive drug release. These features make such a material a promising therapeutic agent. [ABSTRACT FROM AUTHOR]

The development of robust biosensing platforms for accurate detection of cancer-related gene is of great significance for early diagnosis and treatment of cancer. In this paper, we proposed a new concept, exponential rolling circle amplification (E-RCA), for the circular oligonucleotide-mediated tumor suppressor p53 gene detection. Specifically, PMB (primer-containing molecular beacon), PPT (primer-containing polymerization template) and padlock probe are involved. PMB is capable of recognizing target DNA and serves as signal reporter, while PPT is designed to harbor the half binding site of nicking endonuclease and is used as the primary polymerization primer. After the target-activated hybridization between PMB and PPT, besides the as-designed PPT can promote the cyclical target strand-displacement polymerization (CTDP) and nicking-mediated strand-displacement polymerization (NSDP), the nicked primer-induced RCA can make subsequent NSDPs and RCAs proceed repeatedly and exponentially, increasing dramatically the optical signal due to the opening of a large amount of PMBs. The p53 gene can be detected down to 1 pM, and point mutation can be easily discriminated. The exciting breakthrough in the genetic diagnosis concept, desirable analytical characteristics and measured data for real sample assay indicate the potential application in biomedical research and early clinical diagnostics. [ABSTRACT FROM AUTHOR]

The microRNAs (miRNAs) play a critical role in many biological processes and are essential biomarkers for diagnosing disease. However, the sensitive and specific quantification of microRNAs (miRNAs) expression in living cells still faces a huge challenge. Our study designed a multifunctional linear DNA nanostructure (MLN) as a carrier of molecular beacons (MB-21) for detecting and intracellular imaging miRNA-21. The MLN-MB consists of three parts: aptamer, MLN, and MB-21. The aptamer (AS1411) could media MLN-MB enter live cells without additional transfection reagents. Once inside the cells, the intracellular miRNA-21 could hybridize the MB-21s, resulting in significantly enhanced fluorescence signals. The whole process was enzyme-free, autonomous, and continuous, which avoided the necessity of adding external fuel strands or enzymes. We demonstrated that the MLN-MB could be used to screen the miRNA-21 with a detection limit of 320 pM in a short time (10 min) and show high specificity toward miRNA-21 against other miRNAs. Moreover, the proposed MLN-MB could detect the miRNA-21 in complex matrixes stably. With its outstanding stability, dual recognition, and biocompatibility, MLN-MB is capable of delivering into living cells to identify specific cancer cells. Therefore, our sensing approach, with high sensitivity, specificity, and simplicity advantages, holds great potential for early cancer diagnosis. [Display omitted] • The well-design MLN-MB can enter live cells without additional transfection reagents. • The MLN-MB system exhibits high sensitivity, excellent specificity, and a wide linear detection range in vitro. • It can improve cancer detection accuracy by offering a double-check platform to identify specific cancer cells. • The MLN-MB system can be used to image different levels of intracellular miRNA in cancerous cells and normal cells. • The unique method holds potential for biomedical research and molecular diagnostic applications. [ABSTRACT FROM AUTHOR]

The structural DNA nanotechnology holds great potential application in bioimaging, drug delivery and cancer therapy. Herein, an intelligent aptamer-incorporated DNA nanonetwork (Apt-Nnes) is demonstrated for cancer cell imaging and targeted drug delivery, which essentially is a micron-scale pattern with the thickness of double-stranded monolayer. Cancer cell-surface receptors can make it perform magical transformation into small size of nanosheet intermediates and specifically enter target cells. The binding affinity of Apt-Nnes is increased by 3-fold due to multivalent binding effect of aptamers and it can maintain the structural integrity in fetal bovine serum (FBS) for 8 h. More interestingly, target cancer cells can cause the structural disassembly, and each resulting unit transports 4963 doxorubicin (Dox) into target cells, causing the specific cellular cytotoxicity. The cell surface receptor-mediated disassembly of large size of DNA nanostructures into small size of fractions provides a valuable insight into developing intelligent DNA nanostructure suitable for biomedical applications. An aptamer-incorporated DNA nanonetwork is designed to be disassembled by target cancer cells, and each resulting unit transports drugs into the cells for intelligent targeted drug delivery. [Display omitted] • A micron-scale DNA pattern is presented for cell imaging and targeted drug delivery. • Disassembly of large size of DNA nanostructures into the small size. • Multivalent binding effect of aptamers provides a 3-fold higher binding affinity. • DNA nanonetworks retain the structural integrity in fetal bovine serum (FBS) over 8 h. • Each nanosheet intermediate can transport 4963 doxorubicin (Dox) into target cells. [ABSTRACT FROM AUTHOR]

• Innovates miRNA detection with cholesterol-enhanced 3D DNA Walker, enhancing precision. • Uses micelle structure for nuclease protection, cutting false positives in samples. • Allows cancer cell autonomous miRNA imaging, no need for external cofactors. • Enables precise cancer subtype identification via multiple miRNAs, boosting accuracy. • Advances molecular diagnostics, key for early detection and personalized medicine. Achieving reliable miRNA detection and imaging remains a critical and formidable challenge. To address this challenge, we developed a three-dimensional (3D) DNA walker nanoprobe (DWN), derived from a multifunctional molecular beacon with a lipidic moiety. This innovative structure employs a cholesterol-conjugated multifunctional molecular beacon (Chol-MMB) assembled through hydrophobic interactions, thereby bypassing the cumbersome and inefficient process of immobilizing DNA strands onto rigid surfaces. Once the walker nanoprobe enters cells, the binding between the target miRNA and the nanoprobe triggers an intermolecular polymerization process, leading to a conformational change in DWN. This change liberates a walking strand, initiating the walking reaction. As a result, the 3D DNA walker nanoprobe illuminates the fluorophore, enabling precise quantitative analysis. The efficacy of the DWN was demonstrated by the limit of detection of 0.326 fmol for miR-21 with a reaction duration of 1 h. In addition, this strategy effectively differentiates various cells based on the specific miRNA. Further refinement of this method will lead to an efficacious miRNA-based platform for cell typing and liquid biopsy. [ABSTRACT FROM AUTHOR]

The practical application of DNA biosensors is impeded by numerous limitations in complicated physiological environments, particularly the susceptibility of common DNA components to nuclease degradation, which has been recognized as a major barrier in DNA nanotechnology. In contrast, the present study presents an anti-interference and reinforced biosensing strategy based on a 3D DNA-rigidified nanodevice (3D RND) by converting a nuclease into a catalyst. 3D RND is a well-known tetrahedral DNA scaffold containing four faces, four vertices, and six double-stranded edges. The scaffold was rebuilt to serve as a biosensor by embedding a recognition region and two palindromic tails on one edge. In the absence of a target, the rigidified nanodevice exhibited enhanced nuclease resistance, resulting in a low false-positive signal. 3D RNDs have been proven to be compatible with 10% serum for at least 8 h. Once exposed to the target miRNA, the system can be unlocked and converted into common DNAs from a high-defense state, followed by polymerase- and nuclease-co-driven conformational downgrading to achieve amplified and reinforced biosensing. The signal response can be improved by approximately 700% within 2 h at room temperature, and the limit of detection (LOD) is approximately 10-fold lower under biomimetic conditions. The final application to serum miRNA-mediated clinical diagnosis of colorectal cancer (CRC) patients revealed that 3D RND is a reliable approach to collecting clinical information for differentiating patients from healthy individuals. This study provides novel insights into the development of anti-interference and reinforced DNA biosensors. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have been identified as important biomarkers with great significance for diagnosis and treatment of various diseases. However, their unique properties, such as small size, high sequence homology, and low abundance, make quantitative analysis of miRNAs extremely challenging. Herein, we reported a cascade catalytic hairpin assembly (CCHA) for sensitive and selective detection of miRNA with three kinds of hairpin probes (HP1, HP2, and HP3). In the presence of target miRNA, a series of toehold-mediated intermolecular DNA strand displacement and hybridization was activated among HP1, HP2, and HP3 to assembly numbers of DNA nanoobjects. During this period, the fluorescence response was greatly intensified to indicate the presence and expression level of interested target miRNA. We have demonstrated that the proposed method exhibits a high assay sensitivity to detect low concentration target and an excellent sequence specificity to distinguish even a single-nucleotide difference in vitro. Moreover, we also demonstrated that our design enables the intracellular imaging of miRNA in live cancer and normal cells. These results showing the promising potential of our CCHA for powerful biosensing, clinic diagnosis, or prognosis. [Display omitted] • We have developed a target-triggered cascade catalytic hairpin assembly (CCHA) using three hairpin probes. • The CCHA system can be used for robustly detection of miRNA-21. • The CCHA system can be applied to image intracellular miRNA in various types of cell lines. • This unique method holds promising potential for biomedical study and clinical molecular diagnostics. [ABSTRACT FROM AUTHOR]