Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer) reagents.

Background: Response to cetuximab (Erbitux®) and panitumumab (Vectibix®) varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer(™)) reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR).

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.

Aptamer-based multiplexed proteomic technology for biomarker discovery.

Background: The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.

Methodology/principal Findings: We 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.

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.

Charge-transfer excited state dynamics in DNA hairpins substituted with an ethylenylpyrenyl-dU electron source and halo-dU traps.

In this work nine DNA hairpins (HPs) are studied at room temperature to observe their pyrene(*+)/dU(*-) CT excited-state dynamics following photoexcitation at 355 nm with a 25 ps laser pulse. The HPs are 18-24 bases long, have a central tetra-T loop, and have a single U(PE) (5-(2-pyren-1-yl-ethylenyl)-2'-deoxyuridine) substitution in the central region of their stems. Three of the HPs are also substituted with 5-XdU traps, where X = Br or F, to learn about the effects of these traps on CT excited-state lifetimes and emission quantum yields in U(PE) substituted HPs.

T7 RNA polymerase transcription with 5-position modified UTP derivatives.

Seven UTP derivatives modified at the 5-position through an amide linkage were tested as substrates for T7 RNA polymerase (T7 RNAP) transcription. All UTP derivatives gave good yields of full-length transcript even from DNA templates that showed a significant number of abortive transcripts using unmodified UTP. A kinetic assay to determine the relative K(m) and V(max) for T7 RNAP transcription gave surprisingly similar values for UTP and the 5-position hydrophobic modifications phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl.