Scientific Report 2006

Chemistry

Design of Functional Synthetic Systems

M.R. Ghadiri, G. Ashkenasy, N. Ashkenasy, J. Beierle, A. Chavochi, N. Gianneschi, W.S. Horne, Z.-Z. Huang, P. Imming, L. Leman, A. Loutchnikov, A. Montero, L. Motiei, D. Nicoletti, Y. Norikane, J. Picuri, N. Rahe, D. Radu, S. Rahimipour, J. Shin, R. Yamasaki, Y.S. Yoo

We are engaged in a multidisciplinary research effort to uncover new chemical and biochemical approaches for the design of functional molecular, supramolecular, and complex self-organized systems. Our endeavors span disciplines ranging from synthetic organic, bioorganic, and physical organic chemistry to nanotechnology, biophysics, enzymology, and molecular biology. Current research includes the design of synthetic peptide catalysts, antimicrobial self-assembling peptide nanotubes, semisynthetic allosteric enzymes, self-replicating molecular systems and emergent networks, single-molecule stochastic DNA sensing, molecular computation, and prebiotic chemistry.

Antimicrobial Peptide Nanotubes

We showed that appropriately designed cyclic peptide subunits can self-assemble through hydrogen bond–directed ring stacking into open-ended hollow tubular structures that have marked antibacterial and antiviral activities in vitro. The effectiveness of this novel supramolecular class of bioactive species as selective antibacterial agents was highlighted by the high efficacy of one of these antimicrobials against lethal methicillin-resistant Staphylococcus aureus infections in mice. Currently, we are exploring rational design of cyclic glycopeptides and selections from combinatorial libraries to discover novel antiviral and anticancer supramolecular compounds (Fig. 1).

Fig. 1. Antiviral agents based on self-assembling cyclic peptide nanotubes. Cyclic D,L-α-peptides act on endosomal membranes to prevent the development of low pH in endocytic vesicles, arrest the escape of virions from the endosome, and abrogate adenovirus infection.

Design Of Signal Self-Amplifying Dna Sensors

We constructed a novel sequence-specific DNA detection system based on rationally designed semisynthetic enzymes. The system is composed of covalently associated inhibitor-DNA-enzyme modules that function via DNA hybridization–triggered allosteric enzyme activation and signal amplification through substrate turnover (Fig. 2). The functional capacity of the system is highlighted by the sequence-specific detection of approximately 10 fmol of DNA in less than 3 minutes under physiologic conditions. Our studies suggest that rationally designed intrasterically regulated enzymes may be a promising new class of reagents for highly sensitive, rapid, 1-step detection of label-free DNA sequences that does not depend on polymerase chain reactions.

Fig. 2. Schematic representation of an intrasterically inactivated inhibitor-DNA-enzyme construct (left) and the DNA hybridization–triggered enzyme activation (right). The construct can be used to sense low concentrations of cDNA because of its built-in capacity for signal amplification via rapid turnover of substrate.

Stochastic Analysis of Single-Molecule DNA Rotaxanes

We are interested in the study of matter at the level of single molecules. For these studies we use the transmembrane protein α-hemolysin as a rapid and highly sensitive sensor element for stochastic analysis of the molecules lodged or trapped inside the protein pore; the analysis relies on detecting the perturbations in the conductance levels produced in the ion channel in the native protein. Using this technique, we developed an approach by which a single-stranded DNA molecule can be trapped in a specific configuration inside an α-hemolysin channel (Fig. 3), manipulated, and studied with high sensitivity at the single-molecule level. Moreover, a single adenine nucleotide at a specific location on a strand of polydeoxycytidine can be detected by its characteristic effect in reducing the ion conductance in α-hemolysin. We are extending this approach to the design of rapid single-molecule DNA sensing and sequencing.

Fig. 3. Functional supramolecular chemistry at the single-molecule level. Single strands of DNA can be captured inside an α-hemolysin transmembrane pore protein to form single-species pseudorotaxanes composed of α-hemolysin and DNA. This process can be used to identify a single adenine nucleotide at a specific location on a strand of DNA on the basis of the characteristic reductions in the α-hemolysin ion conductance.

Synthetic Networks

Living cells use complex networks of evolutionarily selected biomolecular interactions and chemical transformations to process multiple extracellular input signals rapidly and simultaneously. We are interested in understanding and experimentally modeling the organizational and functional properties of biological networks. We have developed a general strategy for the design and construction of self-organized synthetic peptide networks based on the sequence-selective autocatalytic and cross-catalytic template-directed coiled coil peptide fragment condensation reactions in aqueous solutions. The synthetic networks have some of the basic architectural and dynamic features of the living networks, reorganize in response to changes in environmental conditions and inputs (Fig. 4), and perform basic Boolean logic functions such as OR, NOR, and NOTIF logic. We suggest that the ability to rationally construct predictable chemical circuitry might be useful in advancing the modeling and better understanding of some of the basic dynamic information-processing characteristics of the more complex cellular networks.

Fig. 4. Adaptive reorganization in a synthetic peptide network. The graph structure or wiring of a synthetic peptide network responds dramatically to changes in the environmental stimuli (pH or salt content)

Prebiotic Chemistry

In almost all discussions of prebiotic chemistry, it is assumed that amino acids, nucleotides, and possibly other monomers were first formed on Earth or brought to it in comets and meteorites and that the monomers subsequently condensed nonenzymatically to form oligomeric products. Unfortunately, attempts to create plausibly prebiotic polymerization reactions have met with limited success. Direct heating of solid mixtures leads to nonspecific products, and the condensing agents that have been studied, with the possible exception of inorganic polyphosphates, are relatively inefficient and/or marginally prebiotic. We showed that carbonyl sulfide, a simple gas present in the emissions from present-day volcanoes, is a condensing agent that brings about the formation of peptides from amino acids under mild conditions in aqueous solution (Fig. 5). We have studied the carbonyl sulfide–mediated condensations of α-amino acids under aerobic and anaerobic conditions in the absence of any added reagents and in the presence of metal ions, oxidizing agents, or alkylating agents. Depending on the reaction conditions and additives used, exposure of α-amino acids to carbonyl sulfide generates peptides in yields of up to 80% in minutes to hours at room temperature.

Fig. 5. Peptide formation under plausibly prebiotic reaction conditions. Carbonyl sulfide, a volcanic gas, is the most simple and effective amino acid–condensing agent for the formation of peptides in aqueous solutions.

Publications

Askkenasy, N., Sánchez-Quesada, J., Bayley, H., Ghadiri, M.R. Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores. Angew. Chem. Int. Ed. 44:1401, 2005.

Horne, S.W., Ashkenasy, N., Ghadiri, M.R. Modulating charge transfer through cyclic D,L-α-peptide self-assembly. Chemistry 11:1137, 2005.

Horne, S.W., Wiethoff, C.M., Cui, C., Wilcoxen, K.M., Amorin, M., Ghadiri, M.R., Nemerow, G.R. Antiviral cyclic D,L-α-peptides: targeting a general biochemical pathway in viral infections. Bioorg. Med. Chem. 13:5145, 2005.

Yadav, M.K., Redman, J.E., Leman, L.J., Alvarez-Gutiérrez, J.M., Zhang, Y., Stout, C.D., Ghadiri, M.R. Structure-based engineering of internal cavities in coiled-coil peptides. Biochemistry 44:9723, 2005.