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.

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.

Background: IL-6 signaling is a key component of inflammatory diseases.Results: Modified DNA aptamers that inhibit IL-6 signaling were discovered and optimized.Conclusion: Modified aptamers are stable in serum and block the interaction of IL-6 with its receptor IL-6Rα.Significance: Modified aptamers are a new class of antagonist with properties potentially suitable for clinical treatment of inflammation.

SignificanceAptamers are now used ubiquitously as binding agents for a broad range of applications. Natural (unmodified) DNA and RNA aptamers have considerably less chemical diversity than protein-based ligands such as antibodies, limiting their utility. Aptamers possessing a single chemical modification have helped bridge this diversity gap. We report the selection and identification of aptamers with two diversity-enhancing chemical modifications that bind and inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9), a representative human therapeutic protein target. The addition of a second modification, especially in certain pairwise combinations, resulted in significant improvements in affinity, ligand efficiency, epitope coverage, metabolic stability, and inhibitory activity. Extensively chemically functionalized aptamers have the potential to become the next generation of nucleic-acid–based ligands.

The Drosophila master sex-switch protein Sex-lethal (SXL) regulates the splicing and/or translation of three known targets to mediate somatic sexual differentiation. Genetic studies suggest that additional target(s) of SXL exist, particularly in the female germline. Surprisingly, our detailed molecular characterization of a new potential target of SXL, enhancer of rudimentary (e(r)), reveals that SXL regulates e(r) by a novel mechanism-polyadenylation switching-specifically in the female germline. SXL binds to multiple SXL-binding sites, which include the GU-rich poly(A) enhancer, and competes for the binding of CstF64 in vitro. The SXL-binding sites are able to confer sex-specific poly(A) switching onto an otherwise nonresponsive polyadenylation signal in vivo. The sex-specific poly(A) switching of e(r) provides a means for translational regulation in germ cells. We present a model for the SXL-dependent poly(A) site choice in the female germline.