Scientific Report 2008
Catalytic Antibodies, Synthetic Enzymes,
Biomolecular Computing, and Synthetic Capsids
E. Keinan, O. Reany, N. Metanis, E. Kossoy,
M. Soreni, R. Piran, M. Sinha, I. Ben-Shir, T. Shekhter, T. Ratner, T. Mejuch,
E. Solel, S. Shoshani, R. Gershoni, A. Karmakar, D. Pappo, G. Parvari
A relatively unexplored
opportunity in the science of catalytic antibodies is modifying the phenotype of
an organism by incorporating the gene for a catalytic antibody into the genome of
that organism. An attractive application of this concept would be the expression
of such a catalyst in transgenic plants to provide a beneficial trait. For example,
introduction of a herbicide-resistance trait in commercial plants is highly desirable
because plants with the trait could be grown in the presence of a nonselective herbicide
that affects only weeds and other undesired plant species.
We have shown that herbicide-resistant
plants can be engineered by designing both a herbicide and a catalytic antibody
that destroys the herbicide within the plants. Such a transgenic plant was achieved
via a 3-step maneuver: (1) development of a new carbamate herbicide, one that can
be catalytically destroyed by the aldolase antibody 38C2; (2) separate expression
of the light chain and half of the heavy chain (Fab) of the catalytic antibody in
the endoplasmic reticulum of 2 plant lines of Arabidopsis thaliana; and (3)
cross-pollination of these 2 transgenic plants to produce a herbicide-resistant
F1 hybrid (Fig. 1). In vivo expression of catalytic antibodies could
become a useful, general strategy to achieve desired phenotype modifications not
only in plants but also in other organisms.
Influence of herbicide (4) on the rooting and development of seedlings of
F1 hybrids and control A thaliana plants. The control plants are
shown in A and C; the hybrid plant lines (F1) expressing both light and
heavy chains of catalytic antibody 38C2, in B and D. Plantlets grown on medium without
the herbicide are shown in A and B; those grown with the herbicide are shown in
C and D.
Selenoenzymes have a central role in
maintaining cellular redox potential. These enzymes have selenylsulfide bonds in
their active sites that catalyze the reduction of peroxides, sulfoxides, and disulfides.
The selenol-disulfide exchange reaction is common to all of the enzymes, and the
active-site redox potential reflects the ratio between the forward and reverse rates
of this reaction. The preparation of enzymes containing selenocysteine is experimentally
challenging. As a result, little is known about the kinetic role of selenols in
enzyme active sites, and the redox potential of a selenylsulfide or diselenide bond
in a protein has not been experimentally determined.
fully evaluate the effects of selenocysteine on oxidoreductase redox potential and
kinetics, we chemically synthesized glutaredoxin 3 (Grx3) and all 3 selenocysteine
variants of its conserved 11CXX14C active site and determined
their redox potentials. The position of redox equilibrium between Grx3(C11U-C14U)
(—308 mV) and thioredoxin (—270 mV) suggests a possible role for diselenide
bonds in biological systems. Kinetic analysis showed that the lower redox potentials
of the selenocysteine variants are due primarily to the greater nucleophilicity
of the active-site selenium. The 102- to 104-fold increase
in the rate of thioredoxin reduction by the seleno-Grx3 analogs indicates that oxidoreductases
containing either selenylsulfide or diselenide bonds can have physiologically compatible
redox potentials and enhanced reduction kinetics in comparison with their sulfide
counterparts. This research was done in collaboration with P.E. Dawson, Department
of Cell Biology.
In fully autonomous molecular computing
devices, all components, including input, output, software, and hardware, are specific
molecules that interact with each other through a cascade of programmable chemical
events, progressing from the input molecule to the molecular output signal. DNA
molecules and DNA enzymes have been used as convenient, readily available components
of such computing devices because the DNA materials have highly predictable recognition
patterns, reactivity, and information-encoding features. Furthermore, DNA-based
computers can become part of a biological system, generating outputs in the form
of biomolecular structures and functions.
Our previously reported 2-symbol—2-state
finite automata computed autonomously, and all of their components were soluble
biomolecules mixed in solution. The hardware consisted of 2 enzymes, an endonuclease
and a ligase, and the software and the input were double-stranded DNA oligomers.
More recently, we designed and created 3-symbol—3-state automata that can carry
out more complex computations. In addition, we found that immobilization of the
input molecules on chips allowed parallel computation, a system that can be used
The main advantage of autonomous biomolecular
computing devices compared with electronic computers is the ability of the devices
to interact directly with biological systems. No interface is required because all
components of molecular computers, including hardware, software, input, and output,
are molecules that interact in solution along a cascade of programmable chemical
events. We showed for the first time that the output of a molecular finite automaton
can be a visible bacterial phenotype. Our 2-symbol—2-state finite automaton
uses linear double-stranded DNA inputs prepared by inserting a string of 6-bp symbols
into the lacZ gene on plasmid pUC18. The computation resulted in a circular
plasmid that differed from the original pUC18 by either a 9-bp (accepting state)
or an 11-bp (unaccepting state) insert within the lacZ gene. Upon transformation
and expression of the resultant plasmids in Escherichia coli, either blue
colonies or white colonies, respectively, were formed (Fig. 2).
|Fig. 2. Computation with aaba input in the presence (A) and absence (B) of transition molecules
results in white bacteria when the transition molecules are present. Computation
with abba input in the presence (C) and absence (D) of transition molecules results
in blue bacteria when the transition molecules are present.
Stable structures of icosahedral symmetry
can have numerous functional roles, including chemical microencapsulation and delivery
of drugs and biomolecules, a way to observe encapsulated reactive intermediates,
presentation of epitopes for efficient immunization, synthesis of nanoparticles
of uniform size, and formation of structural elements for molecular supramolecular
and molecular computing. By examining physical models of spherical virus assembly,
we developed a general synthetic strategy for producing chemical capsids at size
scales between fullerenes and spherical viruses. Such capsids can be formed by self-assembly
from a class of novel symmetric molecules developed from a pentagonal core. By designing
chemical complementarity into the 5 interface edges of the molecule, we can produce
self-assembling stable structures of icosahedral symmetry.
considered 3 different binding mechanisms: hydrogen bonding, metal binding, and
formation of disulfide bonds. These structures can be designed to assemble and disassemble
under controlled environmental conditions. We have conducted molecular dynamics
simulation on a class of corannulene-based molecules to demonstrate the characteristics
of self-assembly and to aid in the design of the molecular subunits. This research
was done in collaboration with A.J. Olson, Department of Molecular Biology.
Kossoy, E., Lavid, N., Soreni-Harari,
M., Shoham, Y., Keinan, E.
A programmable biomolecular computing machine with bacterial phenotype output. Chembiochem
Olson, A.J., Hu, Y.H.E., Keinan, E.
Chemical mimicry of viral capsid self-assembly. Proc. Natl. Acad. Sci. U. S. A.