[Show abstract][Hide abstract] ABSTRACT: Immunohistochemistry is used in both research and clinical settings to identify proteins in tissue samples. Despite the power and versatility of immunohistochemistry, limitations are imposed by the slow diffusion of antibodies through tissue and the need for secondary staining or signal amplification. Aptamers can circumvent these limitations, but their application has been hindered by nonspecific binding to cellular components, particularly in the nucleus. Here we describe unique slow off-rate modified aptamers that facilitate rapid and selective binding to target proteins in tissue. Specifically, we have developed a fluorescent aptamer that binds to the human epidermal growth factor receptor 2 (HER2) in breast carcinomas quickly and specifically, and we have shown that the slow off-rate of the aptamer from the HER2 protein contributes to its selectivity. These findings open the door to aptamer histochemistry applications in both research and clinical settings, including intraoperative diagnostics in which speed and accuracy are paramount.
Full-text · Article · May 2011 · Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry
[Show abstract][Hide abstract] ABSTRACT: The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.
We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.
We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.
[Show abstract][Hide abstract] ABSTRACT: Cumulative probability functions (cdfs) for limits of quantification computed from precision for 356 analytes measured in buffer. A. Distribution of LLOQs; median 0.9 pM; inter‐quartile range 0.3 pM–3.9 pM; lowest 10 fM. B. Distribution of ULOQs; median 1.5 nM; inter‐quartile range 0.7 nM–4.5 nM. C. Distribution of log ROQ; median quantification range ∼3 logs. (TIF)
[Show abstract][Hide abstract] ABSTRACT: Reproducibility of measurements in plasma and serum. The cumulative distribution function (cdf) of intra‐run coefficients of variation (CVs) and inter‐run CVs for plasma and serum are shown for the three dilutions mixes: 10% (red), 1% (green), and 0.03% (blue). (TIF)
[Show abstract][Hide abstract] ABSTRACT: Precision profile for a2‐Antiplasmin. A. Representative dose‐response curve calculated with a four‐parameter fit to average concentration (blue circles) of eight replicate protein measurements (red circles). B. Standard deviation σx of calculated concentration (blue circles) with quadratic fit (solid line) and 95% confidence (dashed lines). C. Standard deviation of assay response shown as σlogRFU (red circles) with quadratic fit (solid line) and the 95% confidence (dashed lines). D. Precision profiles for assay response computed by modeling σx (blue) and σlogRFU (red). (TIF)
[Show abstract][Hide abstract] ABSTRACT: Six new 5-position modified dUTP derivatives connected by a unique amide linkage were synthesized and tested for compatibility with the enzymatic steps of in vitro selection. Six commercially available DNA polymerases were tested for their ability to efficiently incorporate each of these dUTP derivatives during PCR. It was not possible to perform PCR under standard conditions using any of the modified dUTP derivatives studied. In contrast, primer extension reactions of random templates, as well as defined sequence templates, were successful. KOD XL and D. Vent DNA polymerases were found to be the most efficient at synthesizing full-length primer extension product, with all of the dUTP derivatives tested giving yields similar to those obtained with TTP. Several of these modified dUTPs were then used in an in vitro selection experiment comparing the use of modified dUTP derivatives with TTP for selecting aptamers to a protein target (necrosis factor receptor superfamily member 9, TNFRSF9) that had previously been found to be refractory to in vitro selection using DNA. Remarkably, selections employing modified DNA libraries resulted in the first successful isolation of DNA aptamers able to bind TNFRSF9 with high affinity.
No preview · Article · Mar 2010 · Journal of the American Chemical Society