Aptamer-based multiplexed proteomic technology for biomarker discovery

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

Probing the SELEX Process with Next-Generation Sequencing

Background SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. Methodology We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. Conclusions High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts

Transfer RNA intron processing in the halophilic archaebacteria

An in vitro assay system has been developed for the Halobacterium volcanii tRNA intron endonuclease using in vitro generated precursor RNAs. A partially purified enzyme preparation is capable of precise and accurate excision of the intron from the halobacterial tRNATrp precursor. The cleavage reaction produces products having 5′ hydroxyl and 2′,3′ cyclic phosphate termini. Processing of precursor molecules containing deletions within the exon regions indicates that the halobacterial endonuclease does not require intact mature tRNA structure in the substrate; this is in contrast to the eukaryotic endonuclease enzyme that has an absolute requirement for these structures. The large halobacterial tRNATrp intron does not appear to be a primary site for recognition by the endonuclease, however, its removal affects cleavage efficiency. Through a comparison of the structural and sequence features of the halobacterial substrates and the precursors of other archaebacterial intron-containing precursors, a common element is proposed for the recognition of substrates by intron endonuclease.Key words: archaebacteria, intron, tRNA, evolution, manipulation. </jats:p