Scientific Report 2008
Design of Functional Synthetic Systems
M.R. Ghadiri, M. Amorin, J. Beierle,
A. Chavochi, J. Chu, B. Frezza, N. Gianneschi, L. Leman, A. Loutchnikov, A.
Montero, C. Olsen, J. Picuri, D. Radu, Y. Ura
We are engaged
in multidisciplinary research to uncover new chemical and biochemical approaches
for the design of functional molecular, supramolecular, and complex self-organized
systems. Our efforts 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 DNA sensing, molecular
computation, and prebiotic chemistry.
Antimicrobial Peptide Nanotubes
We have shown 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 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
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, and 1-step detection of label-free DNA sequences that
does not depend on polymerase chain reactions.
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 substrate turnover.
Single-Molecule Dna Sequencing
We are interested in the study of matter
at the level of single molecules. For these studies we use the transmembrane protein
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, manipulated, and studied with high sensitivity at the single-molecule level.
We have been able to detect up to 9 consecutive DNA polymerase—catalyzed single-nucleotide
primer extensions (Fig. 3) with high sensitivity and spatial resolution (≤ 2.4
Å). The single-base resolution of this approach and the ability to control
the passage of DNA in single-base steps satisfy the 2 minimal requirements of a
nanopore-based sequencing device.
|Fig. 3. Single-molecule monitoring of DNA polymerase—catalyzed single-nucleotide primer
extensions with high sensitivity via an
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. 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.
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).
A fundamental goal of computing is to
reproduce in a molecular setting the familiar properties of microelectronics, such
as digital logic, component modularity, and hierarchical design capacity. In this
regard, significant advances have been made in the design of molecular logic gates
by using small-molecule and rotaxane complexes, deoxyribozymes, enzymatic biochemical
networks, peptide networks, and other systems. However, the molecular logic gates
must be integrated into more complex networks in which outputs from each gate can
serve as inputs to downstream gates.
We recently described the construction
of a basis set of DNA-based logic gates (AND, OR, AND-NOT) capable of communicating
with one another. These gates were rewired into a higher-order circuit that enforces
a net XOR (Exclusive OR) Boolean behavior (Fig. 5), showing that the components
can be modularly recombined to implement novel logic processing. Our results support
the notion that with a basis set of only a few logic gates and within the limits
imposed by the availability of uniquely addressable oligonucleotide sequences, design
of molecular circuits capable of performing a large variety of digital logic operations
might be within reach.
A multilevel circuit built from OR, AND, and AND-NOT gates that performs a net XOR
(Exclusive-OR) analysis on the inputs.
emergence of a polymer that could store genetic information, replicate, and exhibit
phenotypic properties subject to selective environmental pressures marked a crucial
stage in the transition from the prebiotic world to biology; however, the nature
of such a polymer remains unresolved. We have discovered an oligomer family that
quickly and efficiently self-assembles via reversible covalent anchoring of nucleobase
recognition units onto simple peptide backbones. The resulting oligomers specifically
self-pair and cross-pair with complementary strands of RNA and DNA in Watson-Crick
fashion. Moreover, the oligomers undergo dynamic component exchange, template-directed
assembly processes, and dynamic sequence modification in response to changing selective
pressures. Such oligomers could therefore have participated in a number of processes
that would be advantageous for primordial genetic systems, such as dynamic sequence
repair and adaptation.
Cockroft, S.L., Chu, J., Amorin, M.,
Ghadiri, M.R. A single-molecule
nanopore device detects DNA polymerase activity with single-nucleotide resolution.
J. Am. Chem. Soc. 130:818, 2008.
Frezza, B.M., Cockroft, S.L., Ghadiri,
M.R. Modular multi-level circuits
from immobilized DNA-based logic gates. J. Am. Chem. Soc. 129:14875, 2007.
Gianneschi, N.C., Ghadiri, M.R.
Design of molecular logic devices based on a programmable DNA-regulated semisynthetic
enzyme. Angew. Chem. Int. Ed. 46:3955, 2007.
Leman, L.J., Weinberger, D.A., Huang,
Z.-Z., Wilcoxen, K.M., Ghadiri, M.R. Functional
and mechanistic analyses of biomimetic aminoacyl transfer reactions in de novo designed
coiled coil peptides via rational active site engineering. J. Am. Chem. Soc. 129:2959,