Assembly of Multiple DNA Components through Target Binding toward Homogeneous, Isothermally Amplified, and Specific Detection of Proteins

We describe a strategy of utilizing specific target binding to trigger assembly of three DNA components that are otherwise unable to spontaneously assemble with one another. This binding-induced DNA assembly forms a three-arm DNA junction, subsequently initiating nicking endonuclease-assisted isothermal fluorescence signal amplification. Real-time monitoring of fluorescence enables amplified detection of specific protein targets. The implementation of the strategy necessitates the simultaneous binding of a single target molecule with two affinity ligands each conjugated to a DNA motif. Simple alternation of affinity ligands enables different protein targets to induce the formation of the DNA junction and subsequent isothermal amplification. The use of the strategy allowed us to develop a sensitive assay for proteins with three appealing features: homogeneous analysis without the need for separation, isothermal amplification, and high specificity. Streptavidin was chosen as an initial target to establish and optimize the assay. Sensitivity of protein detection was improved by 1000-fold upon the application of isothermal amplification. A limit of detection of 10 pM was achieved for detection of prostate-specific antigen in buffer and diluted serum. The combination of its three appealing features makes the assay attractive for potential applications in molecular diagnosis, point-of-care testing, and on-site analysis

Enzyme-Powered Three-Dimensional DNA Nanomachine for DNA Walking, Payload Release, and Biosensing

Herein, we report a DNA nanomachine, built from a DNA-functionalized gold nanoparticle (DNA–AuNP), which moves a DNA walker along a three-dimensional (3-D) DNA–AuNP track and executes the task of releasing payloads. The movement of the DNA walker is powered by a nicking endonuclease that cleaves specific DNA substrates on the track. During the movement, each DNA walker cleaves multiple substrates, resulting in the rapid release of payloads (predesigned DNA sequences and their conjugates). The 3-D DNA nanomachine is highly efficient due to the high local effective concentrations of all DNA components that have been co-conjugated on the same AuNP. Moreover, the activity of the 3-D DNA nanomachine can be controlled by introducing a protecting DNA probe that can hybridize to or dehybridize from the DNA walker in a target-specific manner. This property allows us to tailor the DNA nanomachine into a DNA nanosensor that is able to achieve rapid, isothermal, and homogeneous signal amplification for specific nucleic acids in both buffer and a complicated biomatrix

A Target-Triggered DNAzyme Motor Enabling Homogeneous, Amplified Detection of Proteins

We report here the concept of a self-powered, target-triggered DNA motor constructed by engineering a DNAzyme to adapt into binding-induced DNA assembly. An affinity ligand was attached to the DNAzyme motor via a DNA spacer, and a second affinity ligand was conjugated to the gold nanoparticle (AuNP) that was also decorated with hundreds of substrate strands serving as a high-density, three-dimensional track for the DNAzyme motor. Binding of a target molecule to the two ligands induced hybridization between the DNAzyme and its substrate on the AuNP, which are otherwise unable to spontaneously hybridize. The hybridization of DNAzyme with the substrate initiates the cleavage of the substrate and the autonomous movement of the DNAzyme along the AuNP. Each moving step restores the fluorescence of a dye molecule, enabling monitoring of the operation of the DNAzyme motor in real time. A simple addition or depletion of the cofactor Mg²⁺ allows for fine control of the DNAzyme motor. The motor can translate a single binding event into cleavage of hundreds of substrates, enabling amplified detection of proteins at room temperature without the need for separation

Label-Free and Separation-Free Atomic Fluorescence Spectrometry-Based Bioassay: Sensitive Determination of Single-Strand DNA, Protein, and Double-Strand DNA

Based on selective and sensitive determination of Hg²⁺ released from mercury complex by cold vapor generation (CVG) atomic fluorescence spectrometry (AFS) using SnCl₂ as a reductant, a novel label-free and separation-free strategy was proposed for DNA and protein bioassay. To construct the DNA bioassay platform, an Hg²⁺-mediated molecular beacon (hairpin) without labeling but possessing several thymine (T) bases at both ends was employed as the probe. It is well-known that Hg²⁺ could trigger the formation of the hairpin structure through T–Hg²⁺–T connection. In the presence of a specific target, the hairpin structure could be broken and the captured Hg²⁺ was released. Interestingly, it was found that SnCl₂ could selectively reduce only free Hg²⁺ to Hg⁰ vapor in the presence of T–Hg²⁺–T complex, which could be separated from sample matrices for sensitive AFS detection. Three different types of analyte, namely, single-strand DNA (ssDNA), protein, and double-strand DNA (dsDNA), were investigated as the target analytes. Under the optimized conditions, this bioassay provided high sensitivity for ssDNA, protein, and dsDNA determination with the limits of detection as low as 0.2, 0.08, and 0.3 nM and the linear dynamic ranges of 10–150, 5–175, and 1–250 nM, respectively. The analytical performance for these analytes compares favorably with those by previously reported methods, demonstrating the potential usefulness and versatility of this new AFS-based bioassay. Moreover, the bioassay retains advantages of simplicity, cost-effectiveness, and sensitivity compared to most of the conventional methods

Strand Displacement-Induced Enzyme-Free Amplification for Label-Free and Separation-Free Ultrasensitive Atomic Fluorescence Spectrometric Detection of Nucleic Acids and Proteins

In previous work, we have developed a simple strategy for a label-free and separation-free bioassay for target DNA and protein, with the limit of detection at the nM level only. Herein, taking advantage of atomic fluorescence spectrometric detection of metal ions and amplification of DNA, a label-free and separation-free ultrasensitive homogeneous DNA analytical platform for target DNA and protein detection was developed on the basis of an enzyme-free strand displacement signal amplification strategy for dramatically improved detectability. Using the T–Hg²⁺–T hairpin structure as the probe, the target DNA binds with HP (T–Hg²⁺–T hairpin structure) and released the Hg²⁺ first; then, the P4 (help DNA) hybridizes with target–P3 complex and free the target DNA, which is used to trigger another reaction cycle. The cycling use of the target amplifies the mercury atomic fluorescence intensity for ultrasensitive DNA detection. Moreover, the enzyme-free strand displacement signal amplification analytical system was further extended for protein detection by introducing an aptamer–P2 arched structure with thrombin as a model analyte. The current homogeneous strategy provides an ultrasensitive AFS detection of DNA and thrombin down to the 0.3 aM and 0.1 aM level, respectively, with a high selectivity. This strategy could be a promising unique alternative for nucleic acid and protein assay

Lanthanide Encoded Logically Gated Micromachine for Simultaneous Detection of Nucleic Acids and Proteins by Elemental Mass Spectrometry

DNA-based logic computing potentially for analysis of biomarker inputs and generation of oligonucleotide signal outputs is of great interest to scientists in diverse areas. However, its practical use for sensing of multiple biomarkers is limited by the universality and robustness. Based on a proximity assay, a lanthanide encoded logically gated micromachine (LGM-Ln) was constructed in this work, which is capable of responding to multiplex inputs in biological matrices. Under the logic function controls triggered by inputs and a Boolean “AND” algorithm, it is followed by an amplified “ON” signal to indicate the analytes (inputs). In this logically gated sensing system, the whole computational process does not involve strand displacement in an intermolecular reaction, and a threshold-free design is employed to generate the 0 and 1 computation via intraparticle cleavage, which facilitates the computation units and makes the “computed values” more reliable. By simply altering the affinity ligands for inputs’ biorecognition, LGM-Ln can also be extended to multi-inputs mode and produce the robust lanthanide encoded outputs in the whole human serum for sensing nucleic acids (with the detection limit of 10 pM) and proteins (with the detection limit of 20 pM). Compared with a logically gated micromachine encoded with fluorophores, the LGM-Ln has higher resolution and no spectral overlaps for multiple inputs, thus holding great promise in multiplex analyses and clinical diagnosis

Heteromultivalent DNA Enhances the Assembly Yield of Hybrid Nanoparticles and Facilitates Dynamic Disassembly for Bioanalysis Using ICP–MS

To obtain enhanced physical and biological properties, various nanoparticles are typically assembled into hybrid nanoparticles through the binding of multiple homologous DNA strands to their complementary counterparts, commonly referred to as homomultivalent assembly. However, the poor binding affinity and limited controllability of homomultivalent disassembly restrict the assembly yield and dynamic functionality of the hybrid nanoparticles. To achieve a higher binding affinity and flexible assembly choice, we utilized the paired heteromultivalency DNA to construct hybrid nanoparticles and demonstrate their excellent assembly characteristics and dynamic applications. Specifically, through heteromultivalency, DNA-functionalized magnetic beads (MBs) and gold nanoparticles (AuNPs) were efficiently assembled. By utilizing ICP–MS, the assembly efficiency of AuNPs on MBs was directly monitored, enabling quantitative analysis and optimization of heteromultivalent binding events. As a result, the enhanced assembly yield is primarily attributed to the fact that heteromultivalency allows for the maximization of effective DNA probes on the surface of nanoparticles, eliminating steric hindrance interference. Subsequently, with external oligonucleotides as triggers, it was revealed that the disassembly mechanism of hybrid nanoparticles was initiated, which was based on an increased local concentration rather than toehold-mediated displacement of paired heteromultivalency DNA probes. Capitalizing on these features, an output platform was then established based on ICP–MS signals that several Boolean operations and analytical applications can be achieved by simply modifying the design sequences. The findings provide new insights into DNA biointerface interaction, with potential applications to complex logic operations and the construction of large DNA nanostructures

miR-21 stimulates HCV replication and attenuates the HCV response to IFN-α treatment.

<p>(<i>A</i>) Huh7 hepatocytes were transfected with control RNA (miR-ctrl) or miR-21 mimics (final concentration, 50 nM). After 48 h, cells were infected with HCV (MOI = 1) for 2 h and washed before fresh medium was added. After 72 h, intracellular HCV RNA replicates were quantified by qPCR and normalized to the GAPDH internal control. (<i>B</i>) Huh7 hepatocytes were transfected as described in (<i>A</i>) and infected with HCV (MOI = 1) for 2 h. After 48 h, HCV core expression was analyzed by Western blot (top panel) using β-actin as a loading control (bottom panel). (<i>C</i>) Huh7 hepatocytes were transfected with miR-21 mimics or control RNA (miR-ctrl) (final concentration, 50 nM). After 48 h, cells were infected with HCV (MOI = 1) for 2 h and washed before adding fresh medium with or without recombinant human IFN-α (100 U/ml). After 72 h, intracellular HCV RNA replicates were quantified by qPCR. (<i>D and E</i>) Huh7 hepatocytes were transfected with miR-21 inhibitors or control inhibitor (ctrl) (final concentration, 50 nM). After 48 h, cells were infected with HCV (MOI = 1) for 2 h and washed before adding fresh medium with or without recombinant human IFN-α (100 U/ml) or anti-IFN-α-neutralizing antibody (100 neutralizing units/ml) as indicated. After 72 h, RNA was isolated from the cell culture medium, and supernatant HCV replicates (<i>D</i>) were measured by qPCR. Intracellular HCV RNA replicates (<i>E</i>) were quantified by qPCR using GAPDH as internal control. Data are presented as the meansSD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.</p

miR-21 regulates components of the Toll-like receptor 7 signaling cascade.

<p>Huh7 hepatocytes were transfected with miR-21 mimics or control RNA, miR-21 inhibitor or control inhibitor (final concentration, 50 nM). After 48 h, MyD88, IRAK1, IRAK4, and TRAF6 mRNA levels were determined by qPCR (<i>A</i>, <i>B</i>, <i>C</i>, and <i>D</i>) and RT-PCR (<i>E</i> and <i>F</i>), respectively. Data are presented as the meansSD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.</p

Investigation of the roles of ERK, JNK, and PKC in the regulation of miR-21 expression upon HCV infection.

<p>(A) Huh7 cells were co-transfected with miPPR21 and pCMV-NS5A (<i>left panel</i>) or pCMV-NS3/4A (<i>right panel</i>) for 24 h, and then signal pathway specific inhibitors (20 µM each) were then added, as indicated. The cells were lysed and luciferase activity was measured. (B) Cells were transfected with pCMV-NS5A (<i>left panel</i>) or pCMV-NS3/4A (right panel) for 24 h, and then treated with the signal pathway inhibitors (20 µM each) as indicated. The phosphorylation and total protein levels of c-Jun (<i>left panel</i>) and c-Fos (<i>right panel</i>) were determined by Western blot (<i>upper panel</i>), and miR-21 expression was measured by qPCR (<i>lower panel</i>). (C) Huh7 cells were co-transfected with miPPR21 and dominant-negative mutants of ERK1 (mERK1), ERK2 (mERK2), JNK (mJNK) or control vectors at different concentrations, as indicated and the resultant luciferase activities were measured. All experiments were repeated at least three times with similar results. Bar graphs represent the means ± SD, n = 3.</p