Trace MicroRNA Quantification by Means of Plasmon-Enhanced Hybridization Chain Reaction

Quantifying trace microRNAs (miRNAs) is extremely important in a number of biomedical applications but remains a great challenge. Here we present an enzyme-free amplification strategy called plasmon-enhanced hybridization chain reaction (PE-HCR) for quantifying trace miRNAs with an outstanding linear range from 1 fM to 1 pM (<i>r</i>² = 0.991), along with a detection limit of 0.043 fM (1300 molecules in 50 μL of sample). The merits of the PE-HCR assay, including high sensitivity and specificity, quantitative detection, no enzyme involvement, low false positives, and easy-to-operate procedures, have been demonstrated for high-confidence quantification of the contents of miRNAs in even single cancer cells. The PE-HCR assay may open up new avenues for highly sensitive quantification of biomarkers and thus should hold great potentials in clinical diagnosis and prognosis

Genetic Variation in a MicroRNA-502 Minding Site in <i>SET8</i> Gene Confers Clinical Outcome of Non-Small Cell Lung Cancer in a Chinese Population

<div><p>Background</p><p>Genetic variants may influence microRNA-target interaction through modulate their binding affinity, creating or destroying miRNA-binding sites. SET8, a member of the SET domain-containing methyltransferase, has been implicated in a variety array of biological processes.</p><p>Methods</p><p>Using Taqman assay, we genotyped a polymorphism rs16917496 T>C within the miR-502 binding site in the 3′-untranslated region of the <i>SET8</i> gene in 576 non-small cell lung cancer (NSCLC) patients. Functions of rs16917496 were investigated using luciferase activity assay and validated by immunostaining.</p><p>Results</p><p>Log-rank test and cox regression indicated that the CC genotype was associated with a longer survival and a reduced risk of death for NSCLC [58.0 vs. 41.0 months, <i>P</i> = 0.031; hazard ratio = 0.44, 95% confidential interval: 0.26–0.74]. Further stepwise regression analysis suggested rs16917496 was an independently favorable factor for prognosis and the protective effect more prominent in never smokers, patients without diabetes and patients who received chemotherapy. A significant interaction was observed between rs16917496 and smoking status in relation to NSCLC survival (<i>P</i><0.001). Luciferase activity assay showed a lower expression level for C allele as compared with T allele, and the miR-502 had an effect on modulation of <i>SET8</i> gene <i>in vitro</i>. The CC genotype was associated with reduced SET8 protein expression based on immunostaining of 192 NSCLC tissue sample (<i>P</i> = 0.007). Lower levels of SET8 were associated with a non-significantly longer survival (55.0 vs. 43.1 months).</p><p>Conclusion</p><p>Our data suggested that the rs16917496 T>C located at miR-502 binding site contributes to NSCLC survival by altering SET8 expression through modulating miRNA-target interaction.</p></div

Patient characteristic and clinical features.

a<p>Mean survival time was provided when MST could not be calculated.</p>b<p>Other carcinomas include large cell, undifferentiated and mixed-cell carcinomas.</p

Interaction analysis between rs16917496 genotypes and smoking status.

a<p>Adjusted for age, sex, diabetes mellitus, histology, clinical stage, surgical operation and treatment status.</p>b<p>Mean survival time was provided when MST could not be calculated.</p

Stratified analysis for rs16917496 genotypes and NSCLC patients’ survival.

a<p>Adjusted for age, sex, smoking status, diabetes mellitus, histology, clinical stage, surgical operation and treatment status.</p>b<p>Other carcinomas include large cell, undifferentiated and mixed-cell carcinomas.</p

Genomic structure of <i>SET8</i>, reporter gene constructs for the 3′UTR of <i>SET8</i> and luciferase expression of the constructed plasmid in different cell lines.

<p>(A) The sequence complementarity of has-miR-502 and <i>SET8</i> 3′UTR is shown here. (B) Direct sequencing and Schematic drawing of the reporter constructs containing 1650 bp 3′UTR of <i>SET8</i> with rs16917496 T or C allele. (C) Luciferase expression of constructs containing rs16917496 T or C allele in A549 and 293T cells. Each transfection was performed with pRL-SV40 plasmids as normalized controls. <i>SET8</i> 3′UTR luciferase reporter plasmids were cotransfected with chemically synthesized mature hsa-miR-502 with or without miR-502 inhibitors in A549 and 293T cell lines. 3′UTR, 3′-untranslated region.</p

Protein Detection Based on Small Molecule-Linked DNA

Based on small molecule-linked DNA and the nicking endonuclease-assisted amplification (NEA) strategy, a novel electrochemical method for protein detection is proposed in this work. Specifically, the small molecule-linked DNA (probe 1) can be protected from exonuclease-catalyzed digestion upon binding to the protein target of the small molecule, so the DNA strand may hybridize with another DNA strand (probe 2) that is previously immobilized onto an electrode surface. Consequently, the NEA process is triggered, resulting in continuous removal of the DNA strands from the electrode surface, and the blocking effect against the electrochemical species [Fe­(CN)₆]^3–/4– becomes increasingly lower; thus, increased electrochemical waves can be achieved. Because the whole process is activated by the target protein, an electrochemical method for protein quantification is developed. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 0.3 to 15 ng/mL with a detection limit of 0.19 ng/mL. Furthermore, because the method can be used for the assay of FR in serum samples and for the detection of other proteins such as streptavidin by simply changing the small molecule moiety of the DNA probes, this novel method is expected to have great potential applications in the future

A General Way to Assay Protein by Coupling Peptide with Signal Reporter via Supermolecule Formation

Protein-binding peptide is recently recognized as an effective artificial affinity reagent for protein assays. However, its application is hampered by the limited choices of available signal readout methods. Herein, we report a general electrochemical signal readout method for protein-binding peptides exploiting the host–guest chemistry of cucurbituril. Via the formation of supermolecules among cucurbituril, electrochemical reporter, and the peptide, a protein-binding peptide can be noncovalently coupled with the electrochemical reporter. To assay the target protein, the protein-binding peptides are first self-assembled in the sensing layer, and after the capturing of the target protein, a portion of the peptides become protein-bound. The protein-free peptides are then coupled with the electrochemical reporter to yield a signal readout inversely proportional to the amount of the captured target proteins. Since the only requirement of supermolecule formation is the incorporation of aromatic amino acids in the peptide sequence, this strategy is universally applicable to many protein-binding peptides. The generality and target specificity of the proposed method are successfully demonstrated in the assays of two kinds of target proteins: tumor necrosis factor-α and amyloid β 1-42 soluble oligomer, respectively. The feasibility of our method is also tested in the monitoring of tumor necrosis factor-α secretion activity of HL-60 cells. These results indicate that our method can have great use in protein detection in the future

Combining Peptide and DNA for Protein Assay: CRIP1 Detection for Breast Cancer Staging

In this work, a novel method for a protein assay is proposed which uses the specific protein-binding peptide of the target protein and sequence-specific DNA to interact with the target as the capture and detection probe, respectively. Meanwhile, since the DNA sequence can be coupled with gold nanoparticles to amplify the signal readout, a sensitive and easily operated method for protein assay is developed. We have also employed a transcription factor named as cysteine-rich intestinal protein 1 (CRIP1), which has been identified as an ideal biomarker for staging of breast cancer, as the model protein for this study. With the proposed method, CRIP1 can be determined in a linear range from 1.25 to 10.13 ng/mL, with a detection limit of 1.25 ng/mL. Furthermore, the proposed method can be directly used to assay CRIP1 in tissue samples. Owing to its desirable sensitivity, excellent reproducibility, and high selectivity, the proposed method may hold great potential in clinical practice in the future

Results of multivariate Cox regression analysis on NSCLC patients’ survival.

<p>Results of multivariate Cox regression analysis on NSCLC patients’ survival.</p