ABSTRACT
Nucleic acid amplification techniques have been among the most powerful tools for biological and biomedical research, and the vast majority of the bioassays rely on thermocycling that uses time-consuming and expensive Peltier-block heating. Here, we introduce a plasmonic photothermal method for quantitative real-time PCR, using gold bipyramids and light to achieve ultrafast thermocycling. Moreover, we successfully extend our photothermal system to other biological assays, such as isothermal nucleic acid amplification and restriction enzyme digestion.
ABSTRACT
Recent advances in RNA research have posed new directives in biology and chemistry to uncover the complex roles of ribonucleic acids in cellular processes. Innovative techniques to visualize native RNAs, particularly, short, low-abundance RNAs in live cells, can dramatically impact current research on the roles of RNAs in biology. Herein, we report a novel method for real-time, microRNA imaging inside live cells based on programmable oligonucleotide probes, which self-assemble through the Cascade Hybridization Reaction (CHR).
Subject(s)
Fluorescence Resonance Energy Transfer/methods , MicroRNAs/analysis , Nucleic Acid Hybridization/methods , Base Sequence , Cell Survival , HeLa Cells , Humans , Oligonucleotide Probes/chemistry , Optical ImagingABSTRACT
A method for the amplified analysis of a gene sequence by a Fok I/DNA machine is described. A tailored nucleic acid that includes a recognition sequence for the analyzed gene and a hybridized sequence of the gene itself acting as the "fuel" substrate of the "machine". Upon recognition of the gene by the "fuel" substrate, the autonomous Fok I-induced cleavage of the resulting duplex proceeds, resulting in the duplication of the gene sequence and the horseradish peroxidase mimicking hemin/G-quadruplex DNAzyme as a chemiluminescence reporter. The autonomous duplication of the gene sequence and the catalytic activity of the DNAzyme provide a double amplification path that enables the analysis of target DNA with a detection limit of 1 x 10(-14) M.
Subject(s)
DNA/chemistry , Deoxyribonucleases, Type II Site-Specific/chemistry , Catalysis , DNA, Catalytic/chemistry , G-Quadruplexes , Genes, Reporter , Hemin/chemistry , Hydrogen Peroxide/chemistry , Luminol/chemistry , Models, Chemical , Models, Genetic , Nucleic Acid Conformation , Oxygen/chemistry , Sequence Analysis, DNA , Time FactorsABSTRACT
Alpha and beta conjugated bis-aptamers against thrombin act as bidentate "glue" for the self-assembly of thrombin nanowires; mixing the bidentate aptamer with a tripodal tridentate alpha aptamer construct yields branched thrombin nanowire structures.
ABSTRACT
The amplified detection of DNA or of single-base mismatches in DNA is achieved by the use of nucleic acid-functionalized magnetic particles that separate the recognition duplexes and, upon amplification, yield chemiluminescence-generating DNAzymes as reporter units. The analysis of M13 phage ssDNA is achieved by the hybridization of the analyte to capture nucleic acid-functionalized magnetic particles followed by the binding of a DNA machine unit to the analyte domain. The magnetic separation of the multi-component-functionalized magnetic particles, followed by their reaction with polymerase, dNTPs, and the nicking enzyme (Nb.BbvCI) activate the autonomous synthesis of the horseradish peroxidase-mimicking DNAzyme that acts as chemiluminescent reporter. The single-base mutation in DNA is achieved by coupling of the DNA machine to the mutant DNA/capture nucleic acid-functionalized magnetic particles hybrid structure. The activation of the polymerization/nicking cycles yield the chemiluminescent reporting DNAzyme. The magnetic separation of the DNA recognition hybrids improves the signal-to-noise ratio of the analytical protocol as compared to related DNAzyme synthesizing schemes.
Subject(s)
DNA, Catalytic/analysis , DNA/analysis , Point Mutation , Animals , Humans , Luminescent Measurements , MagneticsABSTRACT
A unique DNA scaffold was prepared for the one-step self-assembly of hierarchical nanostructures onto which multiple proteins or nanoparticles are positioned on a single template with precise relative spatial orientation. The architecture is a topologically complex ladder-shaped polycatenane in which the "rungs" of the ladder are used to bring together the individual rings of the mechanically interlocked structure, and the "rails" are available for hierarchical assembly, whose effectiveness has been demonstrated with proteins, complementary DNA, and gold nanoparticles. The ability of this template to form from linear monomers and simultaneously bind two proteins was demonstrated by chemical force microscopy, transmission electron microscopy, and confocal fluorescence microscopy. Finally, fluorescence resonance energy transfer between adjacent fluorophores confirmed the programmed spatial arrangement between two different nanomaterials. DNA templates that bring together multiple nanostructures with precise spatial control have applications in catalysis, biosensing, and nanomaterials design.
Subject(s)
DNA, Catenated/chemical synthesis , Nanostructures/chemistry , Nanotechnology/methods , Adsorption , DNA, Single-Stranded , Microscopy, Electron, TransmissionABSTRACT
Circular DNA is used as a template for the amplified detection of M13 phage ssDNA by a rolling circle amplification (RCA) process that synthesizes DNAzyme chains, thus enabling the colorimetric or chemiluminescent detection of the analyte.
Subject(s)
DNA Viruses/genetics , DNA, Catalytic/chemistry , DNA, Circular/chemistry , DNA, Single-Stranded/genetics , Bacteriophage M13/genetics , Base Sequence , Colorimetry/methods , DNA/chemistry , DNA, Catalytic/metabolism , Electrophoresis, Agar Gel , Hydrogen Peroxide/chemistry , Luminescence , Molecular Sequence Data , Nucleic Acid Hybridization , Oxygen/chemistry , Time FactorsABSTRACT
The ultrasensitive detection of DNA is achieved by PCR-induced evolution of a DNAzyme.
Subject(s)
DNA Primers/genetics , DNA Primers/metabolism , DNA, Catalytic/metabolism , Base Sequence , Molecular Sequence Data , Oxidation-Reduction , Polymerase Chain Reaction , Sensitivity and SpecificitySubject(s)
Bacteriophage M13/genetics , Bacteriophage M13/isolation & purification , DNA, Single-Stranded/chemistry , DNA, Viral/chemistry , Nucleic Acid Amplification Techniques/methods , Base Sequence , Catalysis , DNA, Catalytic/metabolism , DNA, Single-Stranded/analysis , DNA, Single-Stranded/genetics , DNA, Viral/analysis , DNA, Viral/genetics , Nucleic Acid ConformationSubject(s)
DNA Replication , DNA/genetics , Templates, Genetic , Base Pairing , Base Sequence , Molecular Sequence DataABSTRACT
Here we describe a protocol for the amplified detection of a target DNA using a DNA/FokI-based replicating cutting machine. The protocol is based on the design of a sensing hairpin oligonucleotide that is opened upon hybridization with the analyte DNA. The endonuclease FokI binds to the double-stranded complex and cleaves it to a "cutter" unit. The "cutter" unit reacts with a fuel oligonucleotide to generate and amplify the signal. The fuel molecule is an oligonucleotide in a hairpin configuration with a fluorophore/quencher pair attached to the 5' and 3' ends. Formation of the duplex between the cutter and the fuel leads to the scission of the duplex by FokI, leading to a second, replicated "cutter", a fluorescent waste product, and to the regeneration of the original "cutter" unit. The autonomous replication of the "cutter" unit, as a result of the primary recognition of the analyte DNA, leads to the amplified fluorescent detection of the analyte DNA with a sensitivity limit of 1 x 10(-14) M. The operation of the machine and the sensing process are monitored by the fluorescence generated by the waste product. Here we apply the protocol, which takes about 2 h to complete, to analyze a Tay-Sachs genetic disorder mutant DNA.
