48 research outputs found

    Inhibitory Impact of 3′-Terminal 2′-O-Methylated Small Silencing RNA on Target-Primed Polymerization and Unbiased Amplified Quantification of the RNA in <i>Arabidopsis thaliana</i>

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    3′-terminal 2′-O-methylation has been found in several kinds of small silencing RNA, regarded as a protective mechanism against enzymatic 3′ → 5′ degradation and 3′-end uridylation. The influence of this modification on enzymatic polymerization, however, remains unknown. Herein, a systematic investigation is performed to explore this issue. We found these methylated small RNAs exhibited a suppression behavior in target-primed polymerization, revealing biased result for the manipulation of these small RNAs by conventional polymerization-based methodology. The related potential mechanism is investigated and discussed, which is probably ascribed to the big size of modified group and its close location to 3′-OH. Furthermore, two novel solutions each utilizing base-stacking hybridization and three-way junction structure have been proposed to realize unbiased recognition of small RNAs. On the basis of phosphorothioate against nicking, a creative amplified strategy, phosphorothioate-protected polymerization/binicking amplification, has also been developed for the unbiased quantification of methylated small RNA in <i>Arabidopsis thaliana</i>, demonstrating its promising potential for real sample analysis. Collectively, our studies uncover the polymerization inhibition by 3′-terminal 2′-O-methylated small RNAs with mechanistic discussion, and propose novel unbiased solutions for amplified quantification of small RNAs in real sample

    Target-Triggered Three-Way Junction Structure and Polymerase/Nicking Enzyme Synergetic Isothermal Quadratic DNA Machine for Highly Specific, One-Step, and Rapid MicroRNA Detection at Attomolar Level

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    MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3′-end, the 5′-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3′ end modification (2′-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay

    Charge Transport within a Three-Dimensional DNA Nanostructure Framework

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    Three-dimensional (3D) DNA nanostructures have shown great promise for various applications including molecular sensing and therapeutics. Here we report kinetic studies of DNA-mediated charge transport (CT) within a 3D DNA nanostructure framework. A tetrahedral DNA nanostructure was used to investigate the through-duplex and through-space CT of small redox molecules (methylene blue (MB) and ferrocene (Fc)) that were bound to specific positions above the surface of the gold electrode. CT rate measurements provide unambiguous evidence that the intercalative MB probe undergoes efficient mediated CT over longer distances along the duplex, whereas the nonintercalative Fc probe tunnels electrons through the space. This study sheds new light on DNA-based molecular electronics and on designing high-performance biosensor devices

    A Graphene Oxide-Based Fluorescent Biosensor for the Analysis of Peptide–Receptor Interactions and Imaging in Somatostatin Receptor Subtype 2 Overexpressed Tumor Cells

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    Analysis of peptide–receptor interactions provides insights for understanding functions of proteins in cells. In this work, we report the development of a fluorescent biosensor for the analysis of peptide–receptor interactions using graphene oxide (GO) and fluorescein isothiocyanate (FITC)-labeled octreotide (FOC). Octreotide is a synthesized cyclic peptide with somatostatin-like bioactivity that has been clinically employed. FOC exhibits high adsorption affinity for GO, and its binding results in efficient fluorescence quenching of FITC. Interestingly, the specific binding of the antibody anti-octreotide (AOC) with FOC competitively releases FOC from the GO surface, leading to the recovery of fluorescence. By using this GO-based fluorescent platform, we can detect AOC with a low detection limit of 2 ng/mL. As a step further, we employ this GO–FOC biosensor to image somatostatin receptor subtype 2 overexpressed AR42J tumor cells, which demonstrates high promise for molecular imaging in cancer diagnosis

    Rational Design of pH-Controlled DNA Strand Displacement

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    Achieving strategies to finely regulate with biological inputs the formation and functionality of DNA-based nanoarchitectures and nanomachines is essential toward a full realization of the potential of DNA nanotechnology. Here we demonstrate an unprecedented, rational approach to achieve control, through a simple change of the solution’s pH, over an important class of DNA association-based reactions. To do so we took advantage of the pH dependence of parallel Hoogsteen interactions and rationally designed two triplex-based DNA strand displacement strategies that can be triggered and finely regulated at either basic or acidic pHs. Because pH change represents an important input both in healthy and pathological biological pathways, our findings can have implication for the development of DNA nanostructures whose assembly and functionality can be triggered in the presence of specific biological targets