Detection Methodology Based on Target Molecule-Induced Sequence-Specific Binding to a Single-Stranded Oligonucleotide

We have recently developed a mix-and-read format homogeneous antigen peptide based assay for detection of the antibodies (Tian, L.; Heyduk, T. <i>Anal. Chem.</i> <b>2009</b>, <i>81</i>, 5218–5225) that employed for target detection a simple biophysical mechanism of target antibody induced annealing between two complementary oligonucleotides attached to the antigen peptide. In this work, we propose and experimentally validate an alternative variant of this assay format in which target antibody binding to antigen peptide–oligonucleotide conjugate produces a complex with high sequence-specific binding affinity to a single-stranded capture oligonucleotide. This new assay format can be used for preparing various solid-surface based assays by immobilizing the capture oligonucleotide. This assay design is not limited to antibody detection. We demonstrate that it can also be employed for detecting proteins or pathogenic bacteria using oligonucleotide-labeled antibodies as target recognition elements. Preparation of these solid-surface based assays is simplified because all interactions with the solid surfaces are mediated by well-understood oligonucleotide–oligonucleotide interactions and because of the relative ease of immobilizing oligonucleotides on various solid surfaces. These unique aspects of the assay design also allow microarray-style multiplexing that could be most useful for multiplexed antibody profiling for diagnosis and analysis of cancer, autoimmune, and infectious diseases

Cell proliferation assay of DU145 and PNT1B cells after overexpression of the four miRNAs.

<p>Overexpression of miR-19b, miR-23b, miR-26a, or miR-92a stimulated cell proliferation in DU145 (A) and PNT1B (B). *indicates a significant difference from the control (p<0.01).</p

Loss of free amino groups and tryptophan residues in oxidized ApoE.

<p>A. Loss of free amino groups in oxidized ApoE. ApoE was oxidized by VPO1/H₂O₂/Cl⁻, MPO/H₂O₂/Cl⁻, or HOCl for 3 hrs at 37°C (n = 3). Unmodified amino groups were quantified with the method using trinitrobenzene sulfonic acid as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057571#s2" target="_blank">Materials and Methods</a>. Absorbance was measured at 340 nm. B. Loss of tryptophan residues following ApoE oxidation. ApoE was incubated with VPO1/H₂O₂/Cl⁻, MPO/H₂O₂/Cl⁻ or HOCl for 3 hrs at 37°C (n = 3) as in A. Fluorescence from free tryptophan residue (emission 335 nm/excitation 280 nm) was quantified with a BioTek Microplate Reader. Trp: tryptophan. Data are shown as means ± SEM; *p<0.05 <i>vs</i>. native ApoE; n = 3.</p

The four miRNAs co-regulate cyclin D1 expression.

<p>Overexpression of miR-19b, miR-23b, miR-26a or miR-92a up-regulated cyclin D1 expression in DU145 (A) and PNT1B (C), while neutralization of each miRNA alone repressed the cyclin D1 expression in DU145 (B) and PNT1B (D). The expression of cyclin D1 was measured by qRT-PCR and western blot. The relative quantification of PTEN protein was measured by densitometry.</p

Identification of miRNA targets in the PTEN 3’ UTR.

<p>A. Diagram of the PTEN mRNA. Predicted binding sites of four miRNAs (miR-19b, miR-23b, miR-26a and miR-92a) in the 3’ UTR region of PTEN. B. Schematic map of luciferase reporter vectors for measuring the efficacy of the miRNA regulation. C. PNT1B cells were co-transfected the luciferase reporter vectors, which harbored either miR-target positive control (PC) or full-length PTEN 3’ UTR (PU), with relevant anti-miRNA inhibitors or negative control (NC) for the luciferase reporter assay. D. Luciferase reporter vectors containing relevant truncated fragments (PUA, PUB, PUC or PUD) of PTEN 3’ UTR were co-transfected with specific anti-miRNA inhibitors or negative control for the luciferase reporter assay. *indicates a significant difference from the control (p<0.01).</p

The four miRNAs co-regulate PTEN expression.

<p>Overexpression of miR-19b, miR-23b, miR-26a or miR-92a repressed PTEN expression in DU145 (A) and PNT1B (C), while neutralization of each miRNA alone up-regulated PTEN expression in DU145 (B) and PNT1B (D). The expression of PTEN was measured by qRT-PCR and western blot. The relative quantification of PTEN protein was measured by densitometry.</p

The alteration of mRNA expression level in miRNA overexpressed DU145 and PNT1B cells.

<p>The mRNA expression level of p110δ (A), p110α (B), p85 (C) and Akt (D) was measured by qRT-PCR and appeared to be altered when the relevant miRNA was overexpressed in DU145 or PNT1B cells.</p

Effect of ox-ApoE on triglyceride, cholesterol and PC efflux from foam cells.

<p>A. Cellular triglyceride efflux by native and ox-ApoE. B. Cellular cholesterol efflux by native and ox-ApoE. C. Cellular PC efflux by native and ox-ApoE. D. Detection of triglycerides in supernatant. E. Detection of cholesterol in supernatant. F. Detection of PC in supernatant. THP-1 cells were loaded with lipids as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057571#s2" target="_blank">Materials and Methods</a>. Efflux of triglycerides, cholesterol, and PC was initiated by addition of native or ox-ApoE in cultured foam cells. After treatment of 9 hrs, triglycerides, cholesterol, and PC in supernatant were measured by the respect kit. After treatment of 24 hrs, the cellular triglycerides, cholesterol, and PC were measured with the respect kit. Tg: triglycerides; Ch: cholesterol; PC: phosphatidylcholine. Data are shown as means ± SEM; *p<0.05 vs. PBS group. n = 3.</p

Endogenous expression of p110α, p100β, p110δ, p85 and Akt in DU145 and PNT1B cell lines.

<p>A. The mRNA expressional level of the 5 genes was measured by qRT-PCR. B. the protein expressional level was measured by western blot. The relative quantification of PTEN protein was measured by densitometry.</p

Identification of the endogenous expression of four miRNAs and PTEN in multiple prostate cell lines.

<p>A. The endogenous expression of four miRNAs in prostate cancer, BPH and normal prostate epithelial cell line was measured by qRT-PCR. B. The endogenous expression of PTEN in prostate cancer, BPH and normal prostate epithelial cell line was measured by qRT-PCR and western blot. The relative quantification of PTEN protein was measured by densitometry.</p