Cuiping Ma

Exponential strand-displacement amplification for detection of micrornas

MicroRNAs (miRNAs) are promising targets for disease diagnosis. However, miRNA detection requires rapid, sensitive, and selective detection to be effective as a diagnostic tool. Herein, a miRNA-initiated exponential strand-displacement amplification (SDA) assay was reported. With the Klenow fragment, nicking enzyme Nt.AlwI, and two primers, the miRNA target can trigger two cycles of nicking, polymerization, and displacement reactions. These reaction cycles amplified the target miRNA exponentially and generated dsDNAs detectable with SYBR Green I in real-time PCR. As low as 16 zmol of the target miRNA was detected by this one-pot assay within 90 min, and the dynamic range spanned over 9 orders of magnitude. Negligible impact from the complex biological matrix was observed on the amplification reaction, indicating the assays capability to directly detect miRNAs in biofluids.

An aptamer-based fluorescent biosensor for potassium ion detection using a pyrene-labeled molecular beacon

A novel and sensitive biosensor based on aptamer and pyrene-labeled fluorescent probes for the determination of K+ was developed. The aptamer was used as a molecular recognition element and a partially complementary oligonucleotide with the aptamer was labeled by pyrene moieties at both ends to transduce the binding event of K+ with aptamer. In the presence of K+, the complementary oligonucleotides were displaced from aptamers, which was accompanied by excimer fluorescence of pyrenes because the self-hairpin structure of the complementary oligonucleotide brought pyrene moieties into close proximity. However, it gave only monomer emission in the absence of K+. Under optimum conditions, the relative fluorescence intensity of pyrene was proportional to the concentration of K+ in the range of 6.0 x 10(-4) to 2.0 x 10(-2) M. A detection limit of 4.0 x 10(-4) M was achieved. Moreover, this method was able to detect K+ with high selectivity in the presence of Na+, NH4+, Mg2+, and Ca2+ ions of biological fluids. In brief, the assay may have great potential applications, especially in a biological environment because of its simplicity, sensitivity, and specificity.

Simple colorimetric DNA detection based on hairpin assembly reaction and target-catalytic circuits for signal amplification.

A simple strategy of colorimetric DNA detection is presented based on a hairpin assembly reaction and target-catalytic DNA circuits to achieve enzyme-free signal amplification. The method employed two hairpin species (H1 and H2), which were stable and unable to hybridize in the absence of target. In the presence of target, the target hybridized with hairpin H1 and the opened hairpin H1 hybridized with hairpin H2, allowing the target to be displaced. H1 and H2 were respectively attached to gold nanoparticles, allowing the duplex formed from H1 and H2 to be visualized with the naked eye. The displaced target again triggered the next round of strand exchange reaction to achieve signal amplification. The method may have a wide range of sensor applications because it is enzyme-free and simple to perform.

Innate Reverse Transcriptase Activity of DNA Polymerase for Isothermal RNA Direct Detection.

RNA detection has become one of the most robust parts in molecular biology, medical diagnostics and drug discovery. Conventional RNA detection methods involve an extra reverse transcription step, which limits their further application for RNA rapid detection. We herein report a novel finding that Bst and Klenow DNA polymerases possess innate reverse transcriptase activities, so that the reverse transcription step and next amplification reaction can be combined to one step in isothermal RNA detection. We have demonstrated that Bst and Klenow DNA polymerases could be successfully used to reverse transcribe RNA within 125-nt length by real time RT-PCR and polyacrylamide gel electrophoresis (PAGE). Our findings will spur the development of a myriad of simple and easy to use RNA detection technologies for isothermal RNA direct detection. This will just meet the future needs of bioanalysis and clinical diagnosis to RNA rapid detection in POC settings and inspection and quarantine.

Three-dimensional DNA nanostructures for colorimetric assay of nucleic acids

Here, we propose a kinetically controlled DNA self-assembly pathway based on exponential hairpin assembly (EHA) to obtain a novel DNA network-like structure. This method is very simple over existing DNA self-assembly techniques, only requiring the input of four DNA hairpins to form dendritic nanostructures. AFM imaging reveals the expected dendritic nanostructures, and they interweave to form a regular nanoporous structure that has a mesh size ranging from 200 to 400 nm. The network-like structure is very large and almost isotropic along all directions. At present, so large and regular self-assembly nanomaterials are very rare. The DNA network can potentially be used as a nanoporous material and a general signal carrier for bioanalytical application. As a model, the DNA nanomaterial has been successfully applied to detect nucleic acids coupled with the AuNP colorimetric strategy with a detection limit of 25 pM for the naked eye within 15 min.

Rapid detection of foodborne pathogen Listeria monocytogenes by strand exchange amplification

A strand exchange amplification (SEA) method to detect foodborne pathogen Listeria monocytogenes was developed. SEA is a novel nucleic acid amplification method that only requires one pair of primers. The specie-specific primers were designed by targeting the 16S rRNA gene and the amplification reaction was performed as short as 60 min at 61 °C. Notably, SEA method could not only detect genomic DNA but also detect RNA by one step without requiring extra reverse transcription. The result could be visualized by naked eyes so that water bath pot would be the only equipment needed. Moreover, culture fluids and bacteria colony could be successfully detected without any pretreatment and the method displayed good specificity and strong anti-jamming capacity. These features greatly simplified the operating procedure and made SEA method be potential for developing point-of-care testing (POCT) devices to detect viable L. monocytogenes.

Isothermal amplification detection of nucleic acids by a double-nicked beacon

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.

DNA Self-assembly Catalyzed by Artificial Agents

Nucleic acids have been shown to be versatile molecules and engineered to produce various nanostructures. However, the poor rate of these uncatalyzed nucleic acid reactions has restricted the development and applications. Herein, we reported a novel finding that DNA self-assembly could be nonenzymatically catalyzed by artificial agents with an increasing dissociation rate constant K2. The catalytic role of several artificial agents in DNA self-assembly was verified by real-time fluorescent detection or agarose gel electrophoresis. We found that 20% PEG 200 could significantly catalyze DNA self-assembly and increase the reaction efficiency, such as linear hybridization chain reaction (HCR) and exponential hairpin assembly (EHA). Therefore, we foresee that a fast and efficient DNA self-assembly in structural DNA nanotechnology will be desirable.

Combinatorial Library Based on Restriction Enzyme-mediated Modular Assembly

Combinatorial approaches in directed evolution were proven to be more efficient for exploring sequence space and innovating function of protein. Here, we presented the modular assembly of secondary structures (MASS) for constructing a combinatorial library. In this approach, secondary structure elements were extracted from natural existing protein. The common linkers were flanking secondary structure elements, and then secondary structure elements were digested by Hinf I restriction endonuclease that was used in the construction of combinatorial library for the first time. The digested DNA fragments were randomly ligated in the sense orientation, then in sequence to be amplified by PCR and transformation. This approach showed that different DNA fragments without homologous sequences could be randomly assembled to create significant sequence space. With the structure analysis of recombinants, it would be beneficial to the rational design, even to the design of protein de novo, and to evolve any genetic part or circuit.