ABSTRACT
We describe a general strategy for the design and discovery of affinity peptides for a protein from its natural ligands. Our approach is guided by protein-protein interactions in natural systems and focuses on the hetero-trimeric complex of cardiac troponin I (cTnI), C (cTnC) and T (cTnT). A key premise of this work is that cTnC and cTnT, owing to their innate ability to bind cTnI, are potential templates for the design and discovery of cTnI-binding peptides. Relying only on the knowledge of primary sequences of cTnC and cTnT, we designed a library of short overlapping peptides that span the entirety of cTnC and cTnT and tested them for binding to cTnI. We were successful in identifying several peptides that display high affinity (1-100 nM) for cTnI. The specific implication of this work is that mimicking natural protein-protein interactions is an excellent starting point for the discovery and rational design of peptide ligands. The knowledge of secondary or tertiary structures of the proteins involved is not a necessary precondition for this approach. Nevertheless, we show that structural information can be used to validate the results of a fragment-based peptide design, and can be potentially beneficial for refining the lead candidates. Our approach is broadly applicable to any protein with at least one natural binding ligand with known primary sequence. For protein targets with multiple natural ligands, this approach can potentially yield several distinct affinity peptides capable of simultaneously binding the target protein via orthogonal modes or at complementary interfaces.
Subject(s)
Drug Design , Peptides/chemistry , Peptides/chemical synthesis , Troponin I/chemistry , Amino Acid Sequence , Animals , Humans , Ligands , Molecular Sequence Data , Peptide Library , Peptides/metabolism , Protein Binding , Protein Structure, Secondary , RatsABSTRACT
A three-dimensional microfluidic device has been successfully fabricated and the flow streams characterized for eventual use in studying communication in an in vitro network of nerve cells. The microfluidic system is composed of two layers of channels: a lower layer for the delivery of pharmacological solutions and an upper layer of channels used to direct the flow of the pharmacological solution streams and perfuse the cells with media and nutrients. Flow profiles have been characterized with computational fluid dynamics simulations, confocal fluorescence microscopy, and carbon-fiber amperometry, which have been used to map changes in flow profiles at different bulk flow rates. Ultimately, the microfluidic system and incorporated cell network will show how networked neurons adapt, compensate, and recover after being exposed to different chemical compounds.
Subject(s)
Cell Culture Techniques/instrumentation , Computer-Aided Design , Flow Injection Analysis/instrumentation , Indicators and Reagents/administration & dosage , Indicators and Reagents/chemistry , Microfluidic Analytical Techniques/instrumentation , Cell Culture Techniques/methods , Equipment Design , Equipment Failure Analysis , Flow Injection Analysis/methods , Microfluidic Analytical Techniques/methodsABSTRACT
Because variability exists within populations of cells, single-cell analysis has become increasingly important for probing complex cellular environments. Capillary electrophoresis (CE) is an excellent technique for identifying and quantifying the contents of single cells owing to its small volume requirements and fast, efficient separations with highly sensitive detection. Recent progress in both whole-cell and subcellular sampling has allowed researchers to study cellular function in the areas of neuroscience, oncology, enzymology, immunology, and gene expression.
Subject(s)
Electrophoresis, Capillary/methods , Eukaryotic Cells/chemistry , Animals , Electrophoresis, Capillary/trends , Enzymes/analysis , Enzymes/metabolism , Eukaryotic Cells/metabolism , Humans , Immunologic Techniques , Medical Oncology/methods , Neurosciences/methods , RNA, Messenger/analysis , Subcellular Fractions/chemistryABSTRACT
A microchip solid-phase extraction method for purification of DNA from biological samples, such as blood, is demonstrated. Silica beads were packed into glass microchips and the beads immobilized with sol-gel to provide a stable and reproducible solid phase onto which DNA could be adsorbed. Optimization of the DNA loading conditions established a higher DNA recovery at pH 6.1 than 7.6. This lower pH also allowed for the flow rate to be increased, resulting in a decrease in extraction time from 25 min to less than 15 min. Using this procedure, template genomic DNA from human whole blood was purified on the microchip platform with the only sample preparation being mixing of the blood with load buffer prior to loading on the microchip device. Comparison between the microchip SPE (microchipSPE) procedure and a commercial microcentrifuge method showed comparable amounts of PCR-amplifiable DNA could be isolated from cultures of Salmonella typhimurium. The greatest potential of the microchipSPE device was illustrated by purifying DNA from spores from the vaccine strain of Bacillus anthracis, where eventual integration of SPE, PCR, and separation on a single microdevice could potentially enable complete detection of the infectious agent in less than 30 min.
