RESUMO
Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.
Assuntos
DNA Catalítico , Clivagem do RNA , Dobramento de RNA , RNA , DNA Catalítico/química , DNA Catalítico/metabolismo , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , DNA/química , DNA/metabolismo , Ribonuclease H/metabolismo , Sítios de Ligação , Especificidade por SubstratoRESUMO
Due to nucleic acid's programmability, it is possible to realize DNA structures with computing functions, and thus a new generation of molecular computers is evolving to solve biological and medical problems. Pioneered by Milan Stojanovic, Boolean DNA logic gates created the foundation for the development of DNA computers. Similar to electronic computers, the field is evolving towards integrating DNA logic gates and circuits by positioning them on substrates to increase circuit density and minimize gate distance and undesired crosstalk. In this minireview, we summarize recent developments in the integration of DNA logic gates into circuits localized on DNA substrates. This approach of all-DNA integrated circuits (DNA ICs) offers the advantages of biocompatibility, increased circuit response, increased circuit density, reduced unit concentration, facilitated circuit isolation, and facilitated cell uptake. DNA ICs can face similar challenges as their equivalent circuits operating in bulk solution (bulk circuits), and new physical challenges inherent in spatial localization. We discuss possible avenues to overcome these obstacles.
Assuntos
DNA , Lógica , DNA/química , Computadores MolecularesRESUMO
Cleavage of biological mRNA by DNAzymes (Dz) has been proposed as a variation of oligonucleotide gene therapy (OGT). The design of Dz-based OGT agents includes computational prediction of two RNA-binding arms with low affinity (melting temperatures (Tm ) close to the reaction temperature of 37 °C) to avoid product inhibition and maintain high specificity. However, RNA cleavage might be limited by the RNA binding step especially if the RNA is folded in secondary structures. This calls for the need for two high-affinity RNA-binding arms. In this study, we optimized 10-23 Dz-based OGT agents for cleavage of three RNA targets with different folding energies under multiple turnover conditions in 2â mM Mg2+ at 37 °C. Unexpectedly, one optimized Dz had each RNA-binding arm with a Tm ≥60 °C, without suffering from product inhibition or low selectivity. This phenomenon was explained by the folding of the RNA cleavage products into stable secondary structures. This result suggests that Dz with long (high affinity) RNA-binding arms should not be excluded from the candidate pool for OGT agents. Rather, analysis of the cleavage products' folding should be included in Dz selection algorithms. The Dz optimization workflow should include testing with folded rather than linear RNA substrates.
Assuntos
DNA Catalítico , RNA , RNA/química , DNA Catalítico/metabolismo , RNA Mensageiro , OligonucleotídeosRESUMO
Accessibility of synthetic oligonucleotides and the success of DNA nanotechnology open a possibility to use DNA nanostructures for building sophisticated enzyme-like catalytic centers. Here we used a double DNA crossover (DX) tile nanostructure to enhance the rate, the yield, and the specificity of 5'-5' ligation of two oligonucleotides with arbitrary sequences. The ligation product was isolated via a simple procedure. The same strategy was applied for the synthesis of 3'-3' linked oligonucleotides, thus introducing a synthetic route to DNA and RNA with a switched orientation that is affordable by a low-resource laboratory. To emphasize the utility of the ligation products, we synthesized a circular structure formed from intramolecular complementarity that we named "an impossible DNA wheel" since it cannot be built from regular DNA strands by enzymatic reactions. Therefore, DX-tile nanostructures can open a route to producing useful chemical products that are unattainable via enzymatic synthesis. This is the first example of the use of DNA nanostructures as a catalyst. This study advocates for further exploration of DNA nanotechnology for building enzyme-like reactive systems.
Assuntos
Nanoestruturas , Oligonucleotídeos , Oligonucleotídeos/química , DNA/química , Nanoestruturas/química , Nanotecnologia/métodos , RNA , Conformação de Ácido NucleicoRESUMO
Both tight and specific binding of folded biological mRNA is required for gene silencing by oligonucleotide gene therapy agents. However, this is fundamentally impossible using the conventional oligonucleotide probes according to the affinity/specificity dilemma. This study addresses this problem for cleaving folded RNA by using multicomponent agents (dubbed 'DNA nanomachine' or DNM). DNMs bind RNA by four short RNA binding arms, which ensure tight and highly selective RNA binding. Along with the improved affinity, DNM maintain the high sequence selectivity of the conventional DNAzymes. DNM enabled up to 3-fold improvement in DNAzymes catalytic efficiency (kcat/Km) by facilitating both RNA substrate binding and product release steps of the catalytic cycle. This study demonstrates that multicomponent probes organized in sophisticated structures can help to achieve the balance between affinity and selectivity in recognizing folded RNA and thus creates a foundation for applying complex DNA nanostructures derived by DNA nanotechnology in gene therapy.
Assuntos
DNA Catalítico , Nanoestruturas , RNA , DNA Catalítico/química , DNA Catalítico/metabolismo , RNA/química , RNA/metabolismo , Nanoestruturas/química , Conformação de Ácido Nucleico , Nanotecnologia/métodos , Dobramento de RNA , DNA/química , DNA/metabolismo , CatáliseRESUMO
We have developed a hook-equipped DNA nanomachine (HDNM) for the rapid detection of specific nucleic acid sequences without a preamplification step. HDNM efficiently unwinds RNA structures and improves the detection sensitivity. Compared to the hookless system, HDNM offers an 80-fold and 13-fold enhancement in DNA and RNA detection, respectively, reducing incubation time from 3 to 1 h.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , DNA Catalítico/química , Corantes Fluorescentes/química , DNA/química , RNA , Sequência de BasesRESUMO
Aptamers that bind non-fluorescent dyes and increase their fluorescence can be converted to fluorescent sensors. Here, we discuss and provide guidance for the design of split (binary) light up aptameric sensors (SLAS) for nucleic acid analysis. SLAS consist of two RNA or DNA strands and a fluorogenic organic dye added as a buffer component. The two strands hybridize to the analyzed DNA or RNA sequence and form a dye-binding pocket, followed by dye binding, and increase in its fluorescence. SLAS can detect nucleic acids in a cost-efficient label-free format since it does not require conjugation of organic dyes with nucleic acids. SLAS design is preferable over monolith fluorescent sensors due to simpler assay optimization and improved selectivity. RNA-based SLAS can be expressed in cells and used for intracellular monitoring and imaging biological molecules.
Assuntos
Aptâmeros de Nucleotídeos , Ácidos Nucleicos , Aptâmeros de Nucleotídeos/genética , DNA/análise , Corantes Fluorescentes/química , RNA/químicaRESUMO
Conventional methods for the detection and differentiation of Bacillus cereus group species have drawbacks mostly due to the complexity of genetic discrimination between the Bacillus cereus species. Here, we describe a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay uses a universal fluorescent reporter and four all-DNA binding fragments, three of which are responsible for "opening up" the folded rRNA while the fourth stand is responsible for detecting single nucleotide variation (SNV) with high selectivity. Binding of the DNM to 16S rRNA results in the formation of the 10-23 deoxyribozyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. This developed biplex assay enables the detection of B. thuringiensis 16S rRNA at fluorescein and B. mycoides at Cy5 channels with a limit of detection of 30 × 103 and 35 × 103 CFU/mL, respectively, after 1.5 h with a hands-on time of ~10 min. The new assay may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis. The DNM proposed here may become an advantageous tool for detecting SNV in clinically significant DNA or RNA samples and can easily differentiate SNV under broadly variable experimental conditions and without prior amplification.
Assuntos
Bacillus , Bacillus/genética , Bacillus cereus/genética , RNA Ribossômico 16S/genética , DNA Ribossômico/genética , DNA BacterianoRESUMO
Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population's health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.
Assuntos
Ácidos Nucleicos , Peroxidase , DNA , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Peroxidases , Sensibilidade e EspecificidadeRESUMO
Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA-based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA-cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation-inspired molecular device for controlling gene expression and cancer treatment.
Assuntos
Biomarcadores Tumorais/metabolismo , DNA Catalítico/metabolismo , MicroRNAs/metabolismo , Neoplasias/diagnóstico , RNA/metabolismo , Biomarcadores Tumorais/genética , DNA Catalítico/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oligonucleotídeos/uso terapêutico , RNA Interferente Pequeno/uso terapêuticoRESUMO
DNA-based computers can potentially analyze complex sets of biological markers, thereby advancing diagnostics and the treatment of diseases. Despite extensive efforts, DNA processors have not yet been developed due, in part, to limitations in the ability to integrate available logic gates into circuits. We have designed a NAND gate, which is one of the functionally complete set of logic connectives. The gate's design avoids stem-loop-folded DNA fragments, and is capable of reusable operations in RNaseâ H-containing buffer. The output of the gate can be translated into RNA-cleaving activity or a fluorescent signal produced either by a deoxyribozyme or a molecular beacon probe. Furthermore, three NAND-gate-forming DNA strands were crosslinked by click chemistry and purified in a simple procedure that allowed ≈1013 gates to be manufactured in 16â h, with a hands-on time of about 30â min. Two NAND gates can be joined into one association that performs a new logic function simply by adding a DNA linker strand. Approaches developed in this work could contribute to the development of biocompatible DNA logic circuits for biotechnological and medical applications.
Assuntos
Computadores Moleculares , DNA , Lógica , Nanoestruturas , Nanotecnologia , Sondas Moleculares , NanomedicinaRESUMO
This Minireview discusses the design and applications of binary (also known as split) light-up aptameric sensors (BLAS). BLAS consist of two RNA or DNA strands and a fluorogenic organic dye added as a buffer component. When associated, the two strands form a dye-binding site, followed by an increase in fluorescence of the aptamer-bound dye. The design is cost-efficient because it uses short oligonucleotides and does not require conjugation of organic dyes with nucleic acids. In some applications, BLAS design is preferable over monolithic sensors because of simpler assay optimization and improved selectivity. RNA-based BLAS can be expressed in cells and used for the intracellular monitoring of biological molecules. BLAS have been used as reporters of nucleic acid association events in RNA nanotechnology and nucleic-acid-based molecular computation. Other applications of BLAS include the detection of nucleic acids, proteins, and cancer cells, and potentially they can be tailored to report a broad range of biological analytes.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Bactérias/química , Computadores Moleculares , DNA/análise , DNA/química , Humanos , Lógica , Compostos Orgânicos/análise , Proteínas/análise , RNA/análise , RNA/químicaRESUMO
Soon after their discovery, RNA-cleaving deoxyribozymes (RCDZ) were explored as anticancer gene therapy agents. Despite low toxicity found in clinical trials, there is no clinically significant anticancer RCDZ-based therapy. Some of the reported disadvantages of RCDZ agents include poor accessibility to folded nucleic acids, low catalytic efficiency inside cells, and problems of intracellular delivery. On the other hand, structural DNA nanotechnology provides an opportunity to build multifunctional nano-associations that can address some of these problems. Herein we discuss the possibility of building RCDZ-based multifunctional DNA nanomachines equipped with RNA unwinding, cancer marker recognition, and RCDZ-based RNA-cleavage functions. An important advantage of such "nanomachines" is the possibility to cleave a housekeeping gene mRNA in a cancer-cell-specific manner. The proposed design could become a starting point for building sophisticated DNA-based nanodevices for cancer treatment.
Assuntos
DNA Catalítico , Nanotecnologia , Neoplasias/tratamento farmacológico , RNA/metabolismo , DNA Catalítico/química , DNA Catalítico/uso terapêutico , Humanos , Clivagem do RNARESUMO
Hybridization probes are RNA or DNA oligonucleotides or their analogs that bind to specific nucleotide sequences in targeted nucleic acids (analytes) via Watson-Crick base pairs to form probe-analyte hybrids. Formation of a stable hybrid would indicate the presence of a DNA or RNA fragment complementary to the known probe sequence. Some of the well-known technologies that rely on nucleic acid hybridization are TaqMan and molecular beacon (MB) probes, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), antisense, siRNA, and CRISPR/cas9, among others. Although invaluable tools for DNA and RNA recognition, hybridization probes suffer from several common disadvantages including low selectivity under physiological conditions, low affinity to folded single-stranded RNA and double-stranded DNA, and high cost of dye-labeled and chemically modified probes. Hybridization probes are evolving into multifunctional molecular devices (dubbed here "multicomponent probes", "DNA machines", and "DNA robots") to satisfy complex and often contradictory requirements of modern biomedical applications. In the definition used here, "multicomponent probes" are DNA probes that use more than one oligonucleotide complementary to an analyzed sequence. A "DNA machine" is an association of a discrete number of DNA strands that undergoes structural rearrangements in response to the presence of a specific analyte. Unlike multicomponent probes, DNA machines unify several functional components in a single association even in the absence of a target. DNA robots are DNA machines equipped with computational (analytic) capabilities. This Account is devoted to an overview of the ongoing evolution of hybridization probes to DNA machines and robots. The Account starts with a brief excursion to historically significant and currently used instantaneous probes. The majority of the text is devoted to the design of (i) multicomponent probes and (ii) DNA machines for nucleic acid recognition and analysis. The fundamental advantage of both designs is their ability to simultaneously address multiple problems of RNA/DNA analysis. This is achieved by modular design, in which several specialized functional components are used simultaneously for recognition of RNA or DNA analytes. The Account is concluded with the analysis of perspectives for further evolution of DNA machines into DNA robots.
Assuntos
Computadores Moleculares , Sondas de DNA/química , DNA/química , RNA/química , DNA/análise , DNA Catalítico/química , Limite de Detecção , Nanotecnologia/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA/análiseRESUMO
Theranostic approaches rely on simultaneous diagnostic of a disease and its therapy. Here, we designed a DNA nanodevice, which can simultaneously report the presence of a specific RNA target through an increase in fluorescence and cleave it. High selectivity of RNA target recognition under near physiological conditions was achieved. The proposed approach can become a basis for the design of DNA nanomachines and robots for diagnostics and therapy of viral infections, cancer, and genetic disorders.
Assuntos
DNA Catalítico/genética , Neoplasias/genética , RNA/química , RNA/metabolismo , Viroses/diagnóstico , DNA Catalítico/química , DNA Catalítico/metabolismo , Fluorescência , Humanos , Neoplasias/química , Nanomedicina TeranósticaRESUMO
DNA nanotechnology uses oligonucleotide strands to assemble molecular structures capable of performing useful operations. Here, we assembled a multifunctional prototype DNA nanodevice, DOCTR, that recognizes a single nucleotide mutation in a cancer marker RNA. The nanodevice then cuts out a signature sequence and uses it as an activator for a "therapeutic" function, namely, the cleavage of another RNA sequence. The proposed design is a prototype for a gene therapy DNA machine that cleaves a housekeeping gene only in the presence of a cancer-causing point mutation and suppresses cancer cells exclusively with minimal side effects to normal cells.
Assuntos
Biomarcadores Tumorais/genética , DNA/química , Terapia Genética , Nanotecnologia , Neoplasias/genética , RNA Neoplásico/genética , Sequência de Bases , Humanos , MutaçãoRESUMO
Hybridization probes have been used for the detection of single nucleotide variations (SNV) in DNA and RNA sequences in the mix-and-read formats. Among the most conventional are Taqman probes, which require expensive quantitative polymerase chain reaction (qPCR) instruments with melting capabilities. More affordable isothermal amplification format requires hybridization probes that can selectively detect SNVs isothermally. Here we designed a split DNA aptamer (SDA) hybridization probe based on a recently reported DNA sequence that binds a dapoxyl dye and increases its fluorescence ( Kato, T.; Shimada, I.; Kimura, R.; Hyuga, M., Light-up fluorophore-DNA aptamer pair for label-free turn-on aptamer sensors. Chem. Commun. 2016 , 52 , 4041 - 4044 ). SDA uses two DNA strands that have low affinity to the dapoxyl dye unless hybridized to abutting positions at a specific analyte and form a dye-binding site, which is accompanied by up to a 120-fold increase in fluorescence. SDA differentiates SNV in the inhA gene of Mycobacterium tuberculosis at ambient temperatures and detects a conserved region of the Zika virus after isothermal nucleic acid sequence based amplification (NASBA) reaction. The approach reported here can be used for detection of isothermal amplification products in the mix-and-read format as an alternative to qPCR.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA Bacteriano/análise , DNA Viral/análise , Corantes Fluorescentes/química , Aptâmeros de Nucleotídeos/genética , Proteínas de Bactérias/genética , Sondas de DNA/química , Sondas de DNA/genética , DNA Bacteriano/genética , DNA Viral/genética , Limite de Detecção , Mycobacterium tuberculosis/química , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oxirredutases/genética , Mutação Puntual , Espectrometria de Fluorescência/métodos , Sulfonas/química , Zika virus/químicaRESUMO
Synthetic molecular machines have been explored to manipulate matter at the molecular level. Here, we designed a multifunctional DNA nano-construct, dubbed a 'DNA minimachine' (DMM), which (i) tightly binds complementary DNA; (ii) recognizes specific fragments with high selectivity and (iii) amplifies output signals. DMM1 detects lower concentrations of both single-stranded DNA and double-stranded DNA compared to a conventional probe. This study sets a direction towards the development of molecular machines for selective, sensitive and cost-efficient DNA analysis.
Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , DNA/química , Limite de Detecção , Nanoestruturas/química , Sequência de Bases , DNA/genética , NanotecnologiaRESUMO
Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme-based DNA machine that can i)â recognize the sequence of a cancer biomarker with high selectivity, ii)â tightly bind a structured fragment of a housekeeping gene mRNA, and iii)â cleave it with efficiency greater than that of a traditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.
Assuntos
Biomarcadores Tumorais/metabolismo , DNA de Neoplasias/metabolismo , Terapia Genética , Nanoestruturas/química , Neoplasias/terapia , RNA Mensageiro/genética , Biomarcadores Tumorais/química , DNA Catalítico/química , DNA Catalítico/metabolismo , DNA de Neoplasias/química , Humanos , Nanotecnologia , Neoplasias/metabolismo , Dobramento de RNA , RNA Mensageiro/metabolismoRESUMO
Aptameric sensors can bind molecular targets and produce output signals, a phenomenon that is used in bioassays. In some cases, it is important to distinguish between monomeric and oligomeric forms of a target. Here, we propose a strategy to convert a monomer/oligomer-nonselective sensor into an oligomer-selective sensor. We designed an aptazyme that produced a high fluorescent output in the presence of oligomeric α-synuclein (a molecular marker of Parkinson's disease) but not its monomeric form. The strategy is potentially useful in the design of point-of-care tests for the diagnosis of neurodegenerative diseases.