Allosteric Inhibitor of KRas Identified Using a Barcoded Assay Microchip Platform.

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC50) of ∼24 µM.

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

Fast metabolic response to drug intervention through analysis on a miniaturized, highly integrated molecular imaging system.

UNLABELLED: We report on a radiopharmaceutical imaging platform designed to capture the kinetics of cellular responses to drugs. METHODS: A portable in vitro molecular imaging system comprising a microchip and a ß-particle imaging camera permitted routine cell-based radioassays of small numbers of either suspended or adherent cells. We investigated the kinetics of responses of model lymphoma and glioblastoma cancer cell lines to (18)F-FDG uptake after drug exposure. Those responses were correlated with kinetic changes in the cell cycle or with changes in receptor tyrosine kinase signaling. RESULTS: The platform enabled direct radioassays of multiple cell types and yielded results comparable to those from conventional approaches; however, the platform used smaller sample sizes, permitted a higher level of quantitation, and did not require cell lysis. CONCLUSION: The kinetic analysis enabled by the platform provided a rapid (≈ 1 h) drug screening assay.

Complementary symmetry nanowire logic circuits: experimental demonstrations and in silico optimizations.

Complementary symmetry (CS) Boolean logic utilizes both p- and n-type field-effect transistors (FETs) so that an input logic voltage signal will turn one or more p- or n-type FETs on, while turning an equal number of n- or p-type FETs off. The voltage powering the circuit is prevented from having a direct pathway to ground, making the circuit energy efficient. CS circuits are thus attractive for nanowire logic, although they are challenging to implement. CS logic requires a relatively large number of FETs per logic gate, the output logic levels must be fully restored to the input logic voltage level, and the logic gates must exhibit high gain and robust noise margins. We report on CS logic circuits constructed from arrays of 16 nm wide silicon nanowires. Gates up to a complexity of an XOR gate (6 p-FETs and 6 n-FETs) containing multiple nanowires per transistor exhibit signal restoration and can drive other logic gates, implying that large scale logic can be implemented using nanowires. In silico modeling of CS inverters, using experimentally derived look-up tables of individual FET properties, is utilized to provide feedback for optimizing the device fabrication process. Based upon this feedback, CS inverters with a gain approaching 50 and robust noise margins are demonstrated. Single nanowire-based logic gates are also demonstrated, but are found to exhibit significant device-to-device fluctuations.