Aptamer-based multiplexed proteomic technology for biomarker discovery

Abstract

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.

Aptamers from Cell-Based Selection for Bioanalytical Applications

“…Gold et al proposed a method for developing aptamers for any protein through SELEX using a new class of aptamers termed “slow off-rate modified aptamer” (SOMAmer). 35 Similar alterations like these may be applied to cell-SELEX. These aptamers increase their chemical diversity by adding protein-like properties to them, such as functional groups that mimic amino acid side chains.…”

“…The group utilizes an array to discover molecular properties in proteomics that are able to transform the complex proteomic samples, that is, plasma, serum, or cell lysates, into a quantifiable protein structure. 35 This solution-based array takes advantage of equilibrium binding, as well as the kinetics of binding and dissociation. In solution, the SOMAmers contain biotin, a photocleavable group, and a fluorescent tag.…”

“…The platform is capable of measuring approximately 800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. 35 This design allows complex protein matrices to be transformed into manageable, quantifiable signatures of aptamer complexes. DNA microarrays are very useful in protein analysis based on their ability to quantify results.…”

Molecular Elucidation of Disease Biomarkers at the Interface of Chemistry and Biology

“…97–99 (Figure 3) Briefly, a mixture of biotinylated aptamer having a photocleavable group and a fluorescent tag is incubated with the test samples, followed by capture using streptavidin beads (Catch-1). The beads are stringently washed to remove unbound protein before a secondary biotin is labeled on each aptamer-captured protein.…”

“…97 Two well-known CKD biomarkers plus 58 potential biomarkers were identified, demonstrating the potential utility of this technology to rapidly discover unique protein signatures characteristic of various disease states. This technology has now been used to identify biomarkers from bronchoalveolar lavage fluid of pediatric patients with cystic fibrosis (CF) lung disease, 100 from serum of malignant pleural mesothelioma (MM) patients, 101 from blood of non-small cell lung cancer (NSCLC) patients, 102 from blood of myocardial injury patients, 103 from serum of tuberculosis (TB) patients, 104 and from serum of Duchenne muscular dystrophy (DMD) patients.…”