Development of "Imprint-and-Report" Dynamic Combinatorial Libraries for Differential Sensing Applications.

Sensor arrays using synthetic receptors have found great utility in analyte detection, resulting from their ability to distinguish analytes based on differential signals via indicator displacement. However, synthesis and characterization of receptors for an array remain a bottleneck in the field. Receptor discovery has been streamlined using dynamic combinatorial libraries (DCLs), but the resulting receptors have primarily been utilized in isolation rather than as part of the entire library, with only a few examples that make use of the complexity of a library of receptors. Herein, we demonstrate a unique sensor array approach using "imprint-and-report" DCLs that obviates the need for receptor synthesis and isolation. This strategy leverages information stored in DCLs in the form of differential library speciation to provide a high-throughput method for both developing a sensor array and analyzing data for analyte differentiation. First, each DCL is templated with analyte to give an imprinted library, followed by in situ fluorescent indicator displacement analysis. We further demonstrate that the reverse strategy, imprinting with the fluorescent reporter followed by displacement with each analyte, provides a more sensitive method for differentiating analytes. We describe the development of this differential sensing system using the methylated Arg and Lys post-translational modifications (PTMs). Altogether, 19 combinations of 3-5 DCL data sets that discriminate all 7 PTMs were identified. Thus, a comparable sensor array workflow results in a larger payoff due to the immense information stored within multiple noncovalent networks.

A study of 2-component i, i + 3 peptide stapling using thioethers.

Peptides are promising scaffolds for use as therapeutics, targeting interactions previously considered to be "undruggable" by small molecules. While short peptides are generally unstructured in solution and rapidly degraded by proteases in the cell cytosol, peptide stapling offers an effective method to both stabilize peptides in a helical structure and increase resistance to proteolytic degradation. Most studies of peptide stapling have focused on residues with i, i + 4 and i, i + 7 spacing, while stapling of residues with i, i + 3 spacing has been understudied. Herein, we evaluated a suite of bifunctional linkers for stapling between residues with i, i + 3 spacing, comparing the ability of each compound to react with the peptide and the degree of helicity conferred. Finally, we evaluated the ability of the stapling to increase proteolytic resistance in cell lysates, comparing stapling of i, i + 3 and i, i + 4 spacing, with i, i + 3 spacing resulting in a greater increase in peptide half-life in the model system. This presents an effective stapling strategy, adding to the peptide stapling toolbox.

Using changes in speciation in a dynamic combinatorial library as a fingerprint to differentiate the methylation states of arginine.

Herein we describe the development of a sensor array that utilizes the complex response of a dynamic combinatorial library (DCL) to discriminate all of the methylation states of Arg, previously unreported in a sensor array, as well as the methylation states of Lys. We find that the use of all species in the DCL, not just those that bind, allows for discrimination of analytes that are otherwise indistinguishable, demonstrating the value of utilizing a complex network of species for differential sensing.