Protein catalyzed capture agents with tailored performance for in vitro and in vivo applications.

We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T1/2 ) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

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.

A chemically synthesized capture agent enables the selective, sensitive, and robust electrochemical detection of anthrax protective antigen.

We report on a robust and sensitive approach for detecting protective antigen (PA) exotoxin from Bacillus anthracis in complex media. A peptide-based capture agent against PA was developed by improving a bacteria display-developed peptide into a highly selective biligand through in situ click screening against a large, chemically synthesized peptide library. This biligand was coupled with an electrochemical enzyme-linked immunosorbent assay utilizing nanostructured gold electrodes. The resultant assay yielded a limit of detection of PA of 170 pg/mL (2.1 pM) in buffer, with minimal sensitivity reduction in 1% serum. The powdered capture agent could be stably stored for several days at 65 °C, and the full electrochemical biosensor showed no loss of performance after extended storage at 40 °C. The engineered stability and specificity of this assay should be extendable to other cases in which biomolecular detection in demanding environments is required.

Genetically engineered peptides for inorganics: study of an unconstrained bacterial display technology and bulk aluminum alloy.

The first-ever peptide biomaterial discovery using an unconstrained engineered bacterial display technology is reported. Using this approach, we have developed genetically engineered peptide binders for a bulk aluminum alloy and use molecular dynamics simulation of peptide conformational fluctuations to demonstrate sequence-dependent, structure-function relationships for metal and metal oxide interactions.

Accumulation of the cyclobutane thymine dimer in defined sequences of free and nucleosomal DNA.

Photochemical cyclobutane dimerization of adjacent thymines generates the major lesion in DNA caused by exposure to sunlight. Not all nucleotide sequences and structures are equally susceptible to this reaction or its potential to create mutations. Photostationary levels of the cyclobutane thymine dimer have now been quantified in homogenous samples of DNA reconstituted into nucleosome core particles to examine the basis for previous observations that such structures could induce a periodicity in dimer yield when libraries of heterogeneous sequences were used. Initial rate studies did not reveal a similar periodicity when a homogenous core particle was analyzed, but this approach examined only formation of this photochemically reversible cyclobutane dimer. Photostationary levels result from competition between dimerization and reversion and, as described in this study, still express none of the periodicity within two alternative core particles that was evident in heterogeneous samples. Such periodicity likely arises from only a limited set of sequences and structural environments that are not present in the homogeneous and well-characterized assemblies available to date.

Assembly of DNA Architectures in a Non-Aqueous Solution.

In the present work, the procedures for the creation of self-assembled DNA nanostructures in aqueous and non-aqueous media are described. DNA-Surfactant complex formation renders the DNA soluble in organic solvents offering an exciting way to bridge the transition of DNA origami materials electronics applications. The DNA retains its structural features, and these unique geometries provide an interesting candidate for future electronics and nanofabrication applications with potential for new properties. The DNA architectures were first assembled under aqueous conditions, and then characterized in solution (using circular dichroism (CD) spectroscopy) and on the surface (using atomic force microscopy (AFM)). Following aqueous assembly, the DNA nanostructures were transitioned to a non-aqueous environment, where butanol was chosen for optical compatibility and thermal properties. The retention of DNA hierarchical structure and thermal stability in non-aqueous conditions were confirmed via CD spectroscopy. The formation and characterization of these higher order DNA-surfactant complexes is described in this paper.

Metabolite analysis of Clostridium acetobutylicum: fermentation in a microbial fuel cell.

Microbial fuel cells (MFCs) were used to monitor metabolism changes in Clostridium acetobutylicum fermentations. When MFCs were inoculated with C. acetobutylicum, they generated a unique voltage output pattern where two distinct voltage peaks occurred over a weeklong period. This result was markedly different to previously studied organisms which usually generate one sustained voltage peak. Analysis of the fermentation products indicated that the dual voltage peaks correlated with glucose metabolism. The first voltage peak correlated with acidogenic metabolism (acetate and butyrate production) and the second peak with solventogenic metabolism (acetone and butanol production). This demonstrates that MFCs can be applied as a novel tool to monitor the shift from acid production to solvent production in C. acetobutylicum.

Modulating the ground- and excited-state oxidation potentials of diaminonaphthalene by sequential N-methylation.

A series of 1,5-diaminonaphthalene derivatives were synthesized and characterized to provide ground- and excited-state electron donors of similar structure but varying potential. Electrochemical and spectroscopic properties of the series are reported and together illustrate two opposing consequences of alkyl substitution on the aryl amines. Inductive effects of methylation are evident from the decrease in ground-state oxidation potential for derivatives containing monomethylamino substituents. In contrast, steric effects seem to dominate the increase in the ground-state oxidation potential of derivatives containing dimethylamino substituents since the conformational constraints created by dimethylation suppress delocalization of the nonbonding electrons. Absorption and emission properties also respond to increasing levels of N-methylation, and the excited-state oxidation potentials of the parent 1,5-diaminonaphthalene and its monomethylamine derivatives (ca. -3.2 V) are approximately 200 mV lower than the corresponding dimethylamino derivatives (-3.0 V).