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.
Multiplexed photoaptamer-based arrays that allow for the simultaneous measurement of multiple proteins of interest in serum samples are described. Since photoaptamers covalently bind to their target analytes before fluorescent signal detection, the arrays can be vigorously washed to remove background proteins, providing the potential for superior signal-to-noise ratios and lower limits of quantification in biological matrices. Data are presented here for a 17-plex photoaptamer array exhibiting limits of detection below 10 fM for several analytes including interleukin-16, vascular endothelial growth factor, and endostatin and able to measure proteins in 10% serum samples. The assays are simple, scalable, and reproducible. Affinity of the capture reagent is shown to be directly correlated to the limit of detection for the analyte on the array.
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.
To enable the application of protein signatures identified in our highly multiplexed biomarker discovery platform to real-world diagnostics, we developed a streamlined, plate-based assay that employs the same principles as the larger SOMAScan assay. The plate-based assay is sensitive (sub-picomolar), robust, rapid, automatable, and can multiplex upwards of 60 analytes. We have used this platform to seamlessly translate sets of biomarkers identified in our SOMAScan assay to a practical non-small cell lung cancer diagnostic.
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