RESUMO
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.
Assuntos
Ouro , Nanopartículas Metálicas , MicroRNAs , Humanos , MicroRNAs/análise , Ouro/química , Nanopartículas Metálicas/química , Animais , Invasividade Neoplásica , DNA/química , Camundongos , Neoplasias , Sondas de DNA/químicaRESUMO
While engineered DNA nanoframeworks have been extensively exploited for delivery of diagnostic and therapeutic regents, DNA tiling-based DNA frameworks amenable to applications in living systems lag much behind. In this contribution, by developing a Y-shaped backbone-based DNA tiling technique, we assemble Y-shaped backbone-rigidified supersized DNA tetrahedrons (RDT) with 100% efficiency for precisely targeted tumor therapy. RDT displays unparalleled rigidness and unmatched resistance to nuclease degradation so that it almost does not deform under the force exerted by the atomic force microscopy tip, and the residual amount is not less than 90% upon incubating in biological media for 24 h, displaying at least 11.6 times enhanced degradation resistance. Without any targeting ligand, RDT enters the cancer cell in a targeted manner, and internalization specificity is up to 15.8. Moreover, 77% of RDT objects remain intact within living cells for 14 h. The drug loading content of RDT is improved by 4-8 times, and RDT almost 100% eliminates the unintended drug leakage in a stimulated physiological medium. Once systemically administrated into HeLa tumor-bearing mouse models, doxorubicin-loaded RDTs preferentially accumulate in tumor sites and efficiently suppress tumor growth without detectable off-target toxicity. The Y-DNA tiling technique offers invaluable insights into the development of structural DNA nanotechnology for precise medicine.
Assuntos
DNA , Neoplasias , Humanos , Animais , Camundongos , Microscopia de Força Atômica , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Células HeLa , Neoplasias/tratamento farmacológicoRESUMO
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.
Assuntos
DNA , Humanos , DNA/química , DNA/metabolismo , DNA Catalítico/metabolismo , DNA Catalítico/química , Imagem Óptica/métodos , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Nanoestruturas/química , Neoplasias/metabolismo , Colesterol/química , Nanopartículas/químicaRESUMO
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.
Assuntos
Técnicas Biossensoriais , Nanoestruturas , Neoplasias , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Técnicas Eletroquímicas/métodos , Ouro/química , Humanos , Limite de Detecção , Mutação PuntualRESUMO
Nuclease-resistant assay probes are of significant importance for biochemical analysis and disease diagnosis. In this contribution, a reconfigurable lipidic moiety-attached DNA nanoparticle (LDN) is constructed from a cholesterol-conjugated multifunctional hairpin-type DNA probe (Chol-DP) by hydrophobicity-mediated self-assembly. The LDN holds high serum stability and displays a low false-positive signal even in a complex biological milieu. The hydrophobic cholesterol moiety enables the hydrophobicity-mediated assembly, while hydrophilic DNA sequence serves as a recognition element and a polymerization template. The initiator-activated strand displacement amplification (SDA) reaction can convert the hairpin-shaped probe into rigid double-stranded DNA (dsDNA), causing the conformational rearrangement-based LDN swelling that can be used to reliably and fluorescently signal the cancer-related p53 gene. The size increase and structural reconfiguration are confirmed by dynamic light scattering (DLS) analysis and confocal microscopy imaging, respectively. Target p53 is specifically detected down to 10 pM. The whole assay process involved only several simple mixing steps. Recovery test and blind test further confirm the feasibility of the use of the LDN for the detection of target DNA in a complex biological milieu, indicating a promising nanotool for biomedical applications.
Assuntos
Técnicas Biossensoriais , Nanopartículas , Neoplasias , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Humanos , Neoplasias/genética , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
The detection of disease-related biomarkers, including microRNA (miRNA), is of crucial importance in reducing the morbidity and mortality of cancer. Thus, there is a great desire to develop an efficient and simple sensing method to fulfill the detection of miRNAs. In this study, a novel amplification assay strategy is demonstrated for the highly sensitive detection of miRNA-21 by combining a structure-switchable molecular beacon with nicking-enhanced rolling circle amplification (SMB-NRCA). A circular padlock probe (CPP) contains a target recognition sequence, two binding sites for nicking endonuclease and three hybridization sites for SMBs. miRNA-21 can hybridize with the CPP and act as polymerization primer that initiates the rolling circle amplification (RCA) reaction and two different nicking-mediated RCA processes, releasing a large amount of SMBs and leading to a significantly amplified fluorescence signal originating from the restoration of pre-quenched fluorescence via their structural switching. Via the signal amplification based on the combination of RCA, nicking and SDA, this assay system can quantitatively detect miRNA-21 in a linear change of three orders of magnitude with a detection limit of 1 pM. The assay specificity is very high so that there is no interference from coexisting miRNAs. Moreover, the sensing system possesses ideal anti-interference ability in complicated milieux such as human serum. The novel sensing strategy shows tremendous prospects for application in tumor diagnosis and clinical therapy guidance.
Assuntos
MicroRNAs , Bioensaio , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodosRESUMO
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 8â¯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.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , DNA/química , Doxorrubicina , Sistemas de Liberação de Medicamentos/métodos , Neoplasias/tratamento farmacológicoRESUMO
DNAzymes with enzymatic activity identified from random DNA pools by in vitro selection have recently attracted considerable attention. In this work, a DNAzyme-based autonomous-motion (AM) molecular machine is demonstrated for sensitive simultaneous imaging of different intracellular microRNAs (miRNAs). The AM molecular machine consists of two basic elements, one of which is a target-analogue-embedded double-stem hairpin substrate (TDHS) and the other is a locking-strand-silenced DNAzyme (LSDz). LSDz can be activated by target miRNA and catalytically cleave TDHS, generating Clv-TDHS and releasing free target analogue capable of triggering the next round of cleavage reaction. As such, the molecular machine can exert sustainable autonomous operation, producing an enhanced signal. Because the active target analogue comes from the machine itself and offers cyclical stimulation in a feedback manner, this target-induced autonomous cleavage circuit is termed a self-feedback circuit (SFC). The SFC-based molecular machine can be used to quantify miRNA-21 down to 10 pM without interference from nontarget miRNAs, indicating a substantial improvement in assay performance compared with its counterpart system without an SFC effect. Moreover, due to the enzyme-free process, the AM molecular machine is suitable for miRNA imaging in living cells, and the quantitative results are consistent with the gold standard PCR assay. More interestingly, the AM molecular machine can be used for the simultaneous fluorescence imaging of several intracellular miRNAs, enabling the accurate discrimination of cancerous cells (e.g., HeLa and MCF-7) from healthy cells. The SFC-based autonomous-motion machine is expected to be a promising tool for the research of molecular biology and early diagnosis of human diseases.
Assuntos
DNA Catalítico , MicroRNAs , DNA , Células HeLa , Humanos , MicroRNAs/genética , Imagem ÓpticaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Sistemas de Liberação de Medicamentos , Nanoestruturas/química , Neoplasias/patologia , Humanos , Técnicas In VitroRESUMO
DNA nanostructures have shown potential in cancer therapy. However, their clinical application is hampered by the difficulty to deliver them into cancer cells and susceptibility to nuclease degradation. To overcome these limitations, we report herein a periodically ordered nick-hidden DNA nanowire (NW) with high serum stability and active targeting functionality. The inner core is made of multiple connected DNA double helices, and the outer shell is composed of regularly arranged standing-up hairpin aptamers. All termini of the components are hidden from nuclease attack, whereas the target-binding sites are exposed to allow delivery to the cancer target. The DNA NW remained intact during incubation for 24â h in serum solution. Animal imaging and cell apoptosis showed that NWs loaded with an anticancer drug displayed long blood-circulation time and high specificity in inducing cancer-cell apoptosis, thus validating this approach for the targeted imaging and therapy of cancers.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Nanofios/química , Nanomedicina Teranóstica , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/metabolismo , DNA/metabolismo , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Portadores de Fármacos/química , Endocitose , Corantes Fluorescentes/química , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Transplante HeterólogoRESUMO
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.
Assuntos
Antineoplásicos/administração & dosagem , DNA/química , Sistemas de Liberação de Medicamentos , Ouro/química , Nanopartículas Metálicas/química , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Humanos , Microscopia de Força AtômicaRESUMO
We report a tetrahedron-based DNAzyme probe (Tetra-ES) for intracellular miRNA detection. Two DNA tetrahedra (Tetra) were arranged at the different positions of the enzyme (E)/substrate (S) complex in a unique direction. A Na+-dependent DNAzme was designed to be initially locked to inhibit the activity of the DNAzyme. Fluorescence imaging and gel electrophoresis analyses demonstrated that the silenced DNAzyme could be specifically initiated by intracellular target miRNA. The activated DNAzyme repeatedly cleaved the substrates, allowing a controllable signal transduction and amplification effect. The combination of spatially controlled arrangement of DNA tetrahedra with the stimuli-responsive behavior of the locked DNAzyme improved cell permeability and desirable nuclease resistance. The Tetra-ES detector exhibited at least 10 times higher detection sensitivity (LOD of 16 pM) than that of the nonamplification molecular beacon counterpart and was capable of discriminating the miRNA target from the corresponding family members. The expression levels of target miRNA inside the cells of interest as well as different miRNAs inside the same type of cell lines were reliably screened utilizing the Tetra-ES detector. As an intracellular probe, Tetra-ES may provide valuable insight into developing a homogeneous DNA nanostructure-based controllable signal transduction strategy suitable for detection of miRNA and potential application to cancer diagnosis, prognosis, and therapeutics.
Assuntos
Sondas de DNA/química , DNA Catalítico/química , MicroRNAs/análise , Linhagem Celular Tumoral , Sondas de DNA/genética , Sondas de DNA/metabolismo , DNA Catalítico/genética , DNA Catalítico/metabolismo , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/química , MicroRNAs/genética , Microscopia de Fluorescência , Nanoestruturas/química , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Sódio/metabolismo , p-Dimetilaminoazobenzeno/análogos & derivados , p-Dimetilaminoazobenzeno/químicaRESUMO
DNA as a programmable molecule shows great potential in a wide variety of applications, with the dynamic DNA nanodevices such as DNA motors and walkers holding the most promise in controlled functions for biosensing and nanomedicine. However, a motor or walker that consists of DNA exclusively has not been shown to function within cells because of its susceptibility to endogenous nuclease-mediated degradation. In this contribution, we demonstrate a Y-shaped backbone-rigidified triangular DNA scaffold (YTDS)-directed DNAzyme walker that functions inside living cells to detect microRNAs (miRNAs) with high sensitivity. A novel Y-shaped backbone offers access to geometrically well-defined configurations and increases the rigidity of DNA assemblies, providing a unique, circular, and rigid DNA track within living cells without non-nucleic acid auxiliary materials and enabling the stepwise movement of DNAzyme in an inchworm fashion. This strategy is extended to the construction of larger rigid planar geometric polygon-based DNA walkers, demonstrating unprecedented opportunities to build dynamic DNA nanostructures with precise geometry and versatile functionality.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , DNA/química , MicroRNAs/análise , Imagem Molecular/métodos , Células HeLa , Humanos , MicroRNAs/genéticaRESUMO
The microRNA profiles within living cells are informative for diagnosis and prognosis of human cancers. In the present work, we developed a new sensing strategy based on branched DNA junction-enhanced isothermal circular strand displacement polymerization (B-ICSDP) for the detection and intracellular imaging of microRNAs in living cells of interest. A circular DNA template consisting of three repetitive fragments serves as the scaffold for the self-assembly of sophisticated signaling probes, resulting a shrunk branched DNA junction. Target microRNA triggers the opening of molecular beacon, not only restoring the quenched fluorescence but also activating a circular polymerization-based strand displacement reaction. Thus, patulous branched DNA junction is abundantly formed, generating the amplified signal. It is noteworthy that great heaps of branched product assemblies can be also achieved in living cells, and the intracellular enzymatic assembly based strategy is able to be used to recognize specific microRNA-expressed cancer cells. Moreover, different microRNAs coexisting in the same living cells can be simultaneously screened without any interference from each other by confocal laser scanning microscopy. The measured data from confocal fluorescence imaging of different cancer cells demonstrates that the B-ICSDP-based system is a promising alternative for in vivo analysis of microRNAs in complicated biological samples.
Assuntos
DNA Circular/química , MicroRNAs/análise , Células Cultivadas , DNA Circular/genética , Células HEK293 , Humanos , Células MCF-7 , Sondas de Oligonucleotídeos/química , Imagem Óptica , PolimerizaçãoRESUMO
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 H2O2 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.
Assuntos
Colorimetria/métodos , DNA Catalítico/química , DNA/análise , Genes p53/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , Benzotiazóis/química , Técnicas Biossensoriais/métodos , Compostos Cromogênicos/química , DNA/genética , DNA Catalítico/genética , Neoplasias/diagnóstico , Neoplasias/genética , Hibridização de Ácido Nucleico , Ácidos Sulfônicos/químicaRESUMO
DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver inâ situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for inâ situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence inâ situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , MicroRNAs/análise , Biocatálise , DNA Polimerase Dirigida por DNA/química , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Células MCF-7 , MicroRNAs/metabolismo , Imagem ÓpticaRESUMO
In this work, we have proposed a chain anadiplosis-structured DNA nanowire by using two well-defined assembly strands (AS1 and AS2). The presence of a target analyte would drive the single-stranded AS1 dissociate from the pre-formatted nanowire, converting into a fully double-stranded form responsible for extensive accumulation of G-rich cleavage fragment1 (GCF1) because of an autonomously performed polymerization/nicking/displacement process. In turn, the produced GCF1 is able to hybridize with the un-peeled AS2, allowing the replication over AS2 to occur and generate large amounts of G-rich cleavage fragment2 (GCF2) with the ability to hybridize with the un-peeled AS1, thereafter initiating new enzymatic reactions for further collection of GCF1. Because the reactions occur repeatedly, the assembled nanowires gradually dissociated and completely collapsed in the end, achieving the goal of substantial signal amplification for the colorimetric readout of the target analytes. The sensing feasibility is firstly verified by one trigger primer (TP), and then exemplified with the detection of the target, the kras oncogene, with high sensitivity and specificity. As a proof-of-concept strategy, the intelligent signal readout pathway and desired assay ability provide unique insights into the materials research and biological studies.
Assuntos
Técnicas Biossensoriais , Colorimetria , DNA/química , Nanofios , Humanos , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos , Proteínas Proto-Oncogênicas p21(ras)/análiseRESUMO
DNA catenanes are assemblies made up of two or more DNA rings linked together through mechanical bonds, and they are desirable for engineering unique nanoscale devices. However, current methods of synthesizing DNA catenanes rely on the formation of strong linking duplexes between component units to enable interlocking and thus do not permit the synthesis of complex single-stranded DNA structures with freely functioning units. We have recently reported DNA sequences that can thread through a DNA circle without the formation of a linking duplex. Here we show that these unique DNA molecules can be further used to make intricate symmetric or asymmetric DNA [3]catenanes, single-stranded DNA assemblies made up of a central mother ring interlocked to two identical or fraternal twin daughter rings, which have never been reported before. These addressable freely functioning interlocked DNA rings should facilitate the design of elaborate nanoscale machines based on DNA.
Assuntos
DNA Catenado/química , Enzimas de Restrição do DNA , DNA Catenado/síntese química , DNA Catenado/metabolismo , Eletroforese em Gel de Poliacrilamida , Nanoestruturas/química , Técnicas de Amplificação de Ácido NucleicoRESUMO
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.
Assuntos
Técnicas Biossensoriais , DNA/química , Sondas Moleculares , Técnicas de Amplificação de Ácido Nucleico , Oncogenes , Endonucleases , HumanosRESUMO
Construction of a simple, reconfigurable, and stimuli-responsive DNA nanocarrier remains a technical challenge. In this contribution, by designing three palindromic fragments, a simplest four-sticky end-contained 3D structural unit (PS-unit) made of two same DNA components is proposed. Via regulating the rotation angle of central longitudinal axis of PS-unit, the oriented assembly of one-component spherical architecture is accomplished with high efficiency. Introduction of an aptamer and sticky tail warehouse into one component creates a size-change-reversible targeted siRNA delivery nanovehicle. Volume swelling of 20 nm allows one carrier to load 1987 siPLK1s. Once entering cancer cells and responding to glutathione (GSH) stimuli, siPLK1s are almost 100% released and original size of nanovehicle is restored, inhibiting the expression of PLK1 protein and substantially suppressing tumor growth (superior to commercial transfection agents) in tumor-bearing mice without systemic toxicity.