The evolvability of lead peptides from small library screens.

Very little is known about the evolvability of lead peptides that are isolated from small library screens. Here we begin to explore this question by comparing the directed evolution of two peptides previously isolated from a small library screen to new ligands generated de novo by in vitro selection.

Synthesis of peptide-oligonucleotide conjugates using a heterobifunctional crosslinker.

Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which enables amine-modified oligonucleotides to become covalently linked to cysteine-modified polypeptides.

Random mutagenesis by error-prone PCR.

In vitro selection coupled with directed evolution represents a powerful method for generating nucleic acids and proteins with desired functional properties. Creating high-quality libraries of random sequences is an important step in this process as it allows variants of individual molecules to be generated from a single-parent sequence. These libraries are then screened for individual molecules with interesting, and sometimes very rare, phenotypes.

Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds.

A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps.

Recognition imaging of acetylated chromatin using a DNA aptamer.

Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target.

Evolution of a histone H4-K16 acetyl-specific DNA aptamer.

We report the in vitro selection of DNA aptamers that bind to histone H4 proteins acetylated at lysine 16. The best aptamer identified in this selection binds to the target protein with a K(d) of 21 nM and discriminates against both the nonacetylated protein and histone H4 proteins acetylated at lysine 8. Comparative binding assays performed with a chip-quality antibody reveal that this aptamer binds to the acetylated histone target with similar affinity to a commercial antibody but shows significantly greater specificity (15-fold versus 2400-fold) for the target molecule.

Creating protein biocatalysts as tools for future industrial applications.

Background: Biocatalysts provide an economical and energy-efficient alternative to traditional chemical manufacturing processes. For processes where biocatalysts currently do not exist or existing protein catalysts function poorly, there is a tremendous need to discover new protein catalysts that function in industrial settings. The protein engineering community has traditionally relied on cell-based techniques in 96-well format to evolve new catalysts or improve existing enzymes.