BackgroundThe interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.Methodology/Principal FindingsWe 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.Conclusions/SignificanceWe 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.

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.

Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community.

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average 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 DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique 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 discover unique protein signatures characteristic of various disease states. More generally, 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.

Seven UTP derivatives modified at the 5-position through an amide linkage were tested as substrates for T7 RNA polymerase (T7 RNAP) transcription. All UTP derivatives gave good yields of full-length transcript even from DNA templates that showed a significant number of abortive transcripts using unmodified UTP. A kinetic assay to determine the relative K(m) and V(max) for T7 RNAP transcription gave surprisingly similar values for UTP and the 5-position hydrophobic modifications phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl. The 5-position modifications imidazole and amino, which could both be positively charged, gave K(m) values significantly higher than UTP. All seven UTP derivatives gave relative V(max) values similar to UTP, indicating that insertion of these modified bases into the transcript did not impede its elongation.