Sheri K. Wilcox

Expanding the Chemistry of DNA for in Vitro Selection

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.

From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

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.

Aptamers and the RNA World, Past and Present

Aptamers and the SELEX process were discovered over two decades ago. These discoveries have spawned a productive academic and commercial industry. The collective results provide insights into biology, past and present, through an in vitro evolutionary exploration of the nature of nucleic acids and their potential roles in ancient life. Aptamers have helped usher in an RNA renaissance. Here we explore some of the evolution of the aptamer field and the insights it has provided for conceptualizing an RNA world, from its nascence to our current endeavor employing aptamers in human proteomics to discover biomarkers of health and disease.

Protein signature of lung cancer tissues.

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment.

Rapid Histochemistry Using Slow Off-rate Modified Aptamers With Anionic Competition

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.

Consequences Of Natural Perturbations In The Human Plasma Proteome

Proteins are the primary functional units of biology and the direct targets of most drugs, yet there is limited knowledge of the genetic factors determining inter-individual variation in protein levels. Here we reveal the genetic architecture of the human plasma proteome, testing 10.6 million DNA variants against levels of 2,994 proteins in 3,301 individuals. We identify 1,927 genetic associations with 1,478 proteins, a 4-fold increase on existing knowledge, including trans associations for 1,104 proteins. To understand consequences of perturbations in plasma protein levels, we introduce an approach that links naturally occurring genetic variation with biological, disease, and drug databases. We provide insights into pathogenesis by uncovering the molecular effects of disease-associated variants. We identify causal roles for protein biomarkers in disease through Mendelian randomization analysis. Our results reveal new drug targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.

Abstract 2501: Differential protein signatures in cancer cell lines after dosing with anticancer molecules in vitro and in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Using a sensitive proteomics platform that can precisely and simultaneously measure 1129 distinct proteins (SOMAscan™), we profiled the proteomic changes in cancer cell lines and xenograft models following exposure to several oncological agents. Our goals were to identify novel biomarkers and drug targets, and to map out pharmacological MOA, with the ultimate goal of assessing the translatability of in vitro and preclinical models to human patients. We have generated multiple successful examples of using our platform in this manner. We treated a breast cancer cell line MCF-7 with eight pharmacological agents, including TamoxifenTM and TaxolTM. We have assessed PKC-δ resistant and sensitive lung cancer cell lines with a PKC-δ inhibitor, and profiled protein changes in response to these compounds. To assess how well in vitro models translate to preclinical models, we profiled protein changes in HCC827 (sensitive) or H1299 (resistant) cell lines treated with erlotinib (TarcevaTM), and NOD/SCID mice implanted with HCC827 cells and dosed with erlotinib. We observed good concordance of the protein changes in the HCC827 cell-lines and the xenograft models, which included cyclin A and p27KIP1 expression levels (proteins previously associated with erlotinib sensitivity) as well as additional protein changes with highly plausible connections to erlotinibs mode of action. These newly identified proteins may have implications for treating patients that develop resistance to erlotinib in the clinic, and suggest new targets for drug development. A human clinical study is underway to explore how the proteomic changes seen in this study translate to erlotinib treatment of NSCLC patients in the clinic. Citation Format: Robert Swift, Deb Ayers, Ying Chang, Darryl Perry, Sheri Routt, Sheri Wilcox, Bridget Lollo, Nick Saccomano. Differential protein signatures in cancer cell lines after dosing with anticancer molecules in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2501. doi:10.1158/1538-7445.AM2013-2501 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Highly multiplexed SOMAmer assays as a flexible platform for protein biomarker discovery research

SomaLogic presents a transformative proteomic biomarker discovery technology that measures ~1000 human proteins in low volumes (~15 of uL biological sample) with a high-performance, high-throughput, and economical assay. Limits of detection average 1 pM and the overall dynamic range spans 7 logs with ~5% average coefficient of variation. This technology is enabled by a new class of DNA aptamers — “SOMAmers” — that contain novel chemicallymodified nucleotides, which greatly expand the physicochemical diversity of the large combinatorial SELEX libraries from which they are selected. Proteins are measured with a process that transforms a signature of protein concentrations into a representative DNA concentration signature, which is quantified with a DNA microarray. We demonstrate the utility of this technology in a large clinical biomarker discovery study of non-small cell lung cancer, as well as in alternate biological matrices.