Ronald N. Zuckermann

Gold Nanoparticle Self-Similar Chain Structure Organized by DNA Origami

Here we demonstrate Au nanoparticle self-similar chain structure organized by triangle DNA origami with well-controlled orientation and <10 nm spacing. We show for the first time that a large DNA complex (origami) and multiple AuNP conjugates can be well-assembled and purified with reliable yields. The assembled structure could be used to generate high local-field enhancement. The same method can be used to precisely localize multiple components on a DNA template for potential applications in nanophotonic, nanomagnetic, and nanoelectronic devices.

Free-floating ultrathin two-dimensional crystals from sequence-specific peptoid polymers

The design and synthesis of protein-like polymers is a fundamental challenge in materials science. A biomimetic approach is to explore the impact of monomer sequence on non-natural polymer structure and function. We present the aqueous self-assembly of two peptoid polymers into extremely thin two-dimensional (2D) crystalline sheets directed by periodic amphiphilicity, electrostatic recognition and aromatic interactions. Peptoids are sequence-specific, oligo-N-substituted glycine polymers designed to mimic the structure and functionality of proteins. Mixing a 1:1 ratio of two oppositely charged peptoid 36mers of a specific sequence in aqueous solution results in the formation of giant, free-floating sheets with only 2.7 nm thickness. Direct visualization of aligned individual peptoid chains in the sheet structure was achieved using aberration-corrected transmission electron microscopy. Specific binding of a protein to ligand-functionalized sheets was also demonstrated. The synthetic flexibility and biocompatibility of peptoids provide a flexible and robust platform for integrating functionality into defined 2D nanostructures.

In vitro self-assembly of tailorable nanotubes from a simple protein building block

We demonstrate a method for generating discretely structured protein nanotubes from the simple ring-shaped building block, homohexameric Hcp1 from Pseudomonas aeruginosa. Our design exploited the observation that the crystal lattice of Hcp1 contains rings stacked in a repeating head-to-tail pattern. High-resolution detail of the ring–ring interface allowed the selection of sites for specific cysteine mutations capable of engaging in disulfide bond formation across rings, thereby generating stable Hcp1 nanotubes. Protein nanotubes containing up to 25 subunits (≈100 nm in length) were self-assembled under simple conditions. Furthermore, we demonstrate that the tube ends and interior can be independently and specifically functionalized to generate nanocapsules.

Proteolytic studies of homologous peptide and N-substituted glycine peptoid oligomers

Abstract Homologous L-amino acid, D-amino acid, and parallel and anit-parallel (retro) sequence N-substituted glycine peptide and peptoid oligomers were prepared and incubated with proteases from each major class. The L-amino acid containing peptides were readily cleaved by the appropriate enzymes, while equivalent D-amino acid containing and N-substituted glycine containing oligomers were essentially untouched. Homologous peptide and peptoid oligomers were prepared and incubated with proteases from each major class. All-L peptides were readily cleaved by the appropriate enzymes, while equivalent all-D and N-substituted glycine containing oligomers were not.

Peptoid Polymers: A Highly Designable Bioinspired Material

Bioinspired polymeric materials are attracting increasing attention due to significant advantages over their natural counterparts: the ability to precisely tune their structures over a broad range of chemical and physical properties, increased stability, and improved processability. Polypeptoids, a promising class of bioinspired polymer based on a N-substituted glycine backbone, have a number of unique properties that bridge the material gap between proteins and bulk polymers. Peptoids combine the sequence specificity of biopolymers with the simpler intra/intermolecular interactions and robustness of traditional synthetic polymers. They are highly designable because hundreds of chemically diverse side chains can be introduced from simple building blocks. Peptoid polymers can be prepared by two distinct synthetic techniques offering access to two material subclasses: (1) automated solid-phase synthesis which enables precision sequence control and near absolute monodispersity up to chain lengths of ~50 monomers, and (2) a classical polymerization approach which allows access to higher molecular weights and larger-scale yields, but with less control over length and sequence. This combination of facile synthetic approaches makes polypeptoids a highly tunable, rapid polymer prototyping platform to investigate new materials that are intermediate between proteins and bulk polymers, in both their structure and their properties. In this paper, we review the methods to synthesize peptoid polymers and their applications in biomedicine and nanoscience, as both sequence-specific materials and as bulk polymers.

Synthesis of N-substituted glycine peptoid libraries.

Publisher Summary Oligomeric N-substituted glycines (NSG) or “peptoids” are a novel class of polymer that is ideally suited for the generation of diverse molecular libraries. The compounds are stable, easy to synthesize, and have potent biological activities. This chapter describes straightforward detailed protocols, for synthesizing diverse peptoid libraries, without any sophisticated equipment and with only a minimum of effort. In order for a class of molecules to be suitable for the generation of diverse libraries, ideally several criteria should be met. First, the compounds should be accessible, by solid-phase synthesis, to allow the resin-splitting methods of equimolar mixture synthesis to be used. Second, molecules with an oligomeric architecture allow a variety of chemical functionalities to be incorporated into a molecule, with a single linking chemistry. This greatly simplifies the synthesis of libraries, especially with respect to automation. The efforts here are focused on the synthesis of NSG peptoid oligomers, because they fit the criteria just listed. These oligomers are structurally similar to peptides, but have several major differences.

Designing polymers that mimic biomolecules

A new field is emerging. Chemists are beginning to synthesize polymers with properties that are similar to those of proteins and RNA. Recent studies have identified oligomer backbones that form stable secondary structures. It is now possible to assemble specific sequences of diverse monomer sets into chain lengths that are nearly sufficient for tertiary structure formation. Such molecules will teach us how natural biopolymers fold; they will also enable us to design synthetic heteropolymers with novel structures and desirable functions.

Biomimetic Nanostructures: Creating a High-Affinity Zinc-Binding Site in a Folded Nonbiological Polymer

One of the long-term goals in developing advanced biomaterials is to generate protein-like nanostructures and functions from a completely nonnatural polymer. Toward that end, we introduced a high-affinity zinc-binding function into a peptoid (N-substituted glycine polymer) two-helix bundle. Borrowing from well-understood zinc-binding motifs in proteins, thiol and imidazole moieties were positioned within the peptoid such that both helices must align in close proximity to form a binding site. We used fluorescence resonance energy transfer (FRET) reporter groups to measure the change of the distance between the two helical segments and to probe the binding of zinc. We systematically varied the position and number of zinc-binding residues, as well as the sequence and size of the loop that connects the two helical segments. We found that certain peptoid two-helix bundles bind zinc with nanomolar affinities and high selectivity compared to other divalent metal ions. Our work is a significant step toward generating biomimetic nanostructures with enzyme-like functions.

Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage

An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors. This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd. A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed. The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1. Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described. Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames. Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides.

Improving SH3 domain ligand selectivity using a non-natural scaffold.

BACKGROUND Src homology 3 (SH3) domains bind sequences bearing the consensus motif PxxP (where P is proline and x is any amino acid), wherein domain specificity is mediated largely by sequences flanking the PxxP core. This specificity is limited, however, as most SH3 domains show high ligand cross-reactivity. We have recently shown that diverse N-substituted residues (peptoids) can replace the prolines in the PxxP motif, yielding a new source of ligand specificity. RESULTS We have tested the effects of combining multiple peptoid substitutions with specific flanking sequences on ligand affinity and specificity. We show that by varying these different elements, a ligand can be selectively tuned to target a single SH3 domain in a test set. In addition, we show that by making multiple peptoid substitutions, high-affinity ligands can be generated that completely lack the canonical PxxP motif. The resulting ligands can potently disrupt natural SH3-mediated interactions. CONCLUSIONS Peptide-peptoid hybrid scaffolds yield SH3 ligands with markedly improved domain selectivity, overcoming one of the principal challenges in designing inhibitors against these domains. These compounds represent important leads in the search for orthogonal inhibitors of SH3 domains, and can serve as tools for the dissection of complex signaling pathways.