Development
of Amplifiable and Evolvable Unnatural Molecules
Combinatorial approaches towards the isolation of
molecules with new function have been employed with great
success over the past decade. These efforts have lead to
the isolation of nucleic acids, proteins, and small
molecules with novel binding, catalytic, or medicinal
properties out of libraries of hundreds to trillions of
variants.[1] Nucleic acids and proteins, unlike their
small molecule counterparts, share a critical feature
which explains their dominance among solutions to complex
chemical problems: these molecules can be amplified. This
feature allows, in theory, a single molecule out of
billions to be isolated by iterated selection and
amplification, providing exquisite sensitivity. Equally
important, amplification is a prerequisite for molecular
evolution, and researchers have demonstrated extensively
that nucleic acids and proteins which initially lack or
only weakly possess desired activities can be mutated,
amplified, and reselected to afford molecules with
greatly enhanced properties.[1] Yet nucleic acids and
proteins, while amplifiable, lack the flexibility which
chemists routinely exert on synthetic small molecules and
thus are restricted to a limited set of chemical and
biological properties. We are interested in designing
amplifiable unnatural polymers, thus combining the major
benefits of natural molecules with the design flexibility
of synthetic molecules. DNA-templated synthesis provides
a promising starting point towards this goal.[2-9]
Libraries of amplifiable unnatural polymers would be
suitable for sequencing, solid-phase or solution-phase
selections, random or directed mutagenesis, and
homologous recombination using DNA shuffling[1,10,11] and
therefore can be evolved over iterated rounds of
selection and amplification using the same extremely
powerful genetic methods employed by Nature during the
evolution of life. This strategy may lead to the
isolation of entirely new classes of unnatural molecules
which selectively bind molecules or which catalyze
chemical reactions. Characterization of these molecules
will provide important insight into the ability of
unnatural polymers such as polycarbamates, polyureas,
polyesters, polysaccharides, or peptide nucleic acids
bearing novel side chains to form higher-order structures
with efficient binding or catalytic properties. We are
also applying this approach to the generation of
amplifiable non-polymeric molecules through the
development of chemistry for general DNA-templated bond
formation on solid support as well as in solution. The
development of such systems may allow the direct
evolution of new generations of small molecule ligands
and drugs, an approach which may prove more effective
than traditional cycles of compound screening and analog
synthesis.
Molecular Evolution of Proteins
We are also interested in the evolution of natural
molecules (proteins and nucleic acids) using in vivo
selections by coupling the survival of a cell with the
solution to a chemical problem of interest. Initial
efforts will focus on (i) the evolution of nucleases with
arbitrary single site per genome cleavage specificity,
(ii) the evolution of new trans intein pairs with
exclusive pairwise protein splicing activity, (iii) the
evolution of artificial allosteric proteins which are
activated or inactivated by arbitrarily chosen small
molecules, (iv) the evolution of recombinases with new
substrate specificities, (v) the evolution of
fibril-forming and fibril-resistant prions and (vi) the
design and evolution of artificial transcription factors
with tailor made specificities. In each case, the
evolution of proteins with desired properties will both
answer basic science questions in protein-DNA,
protein-protein, or protein-small molecule recognition as
well as provide powerful new research tools. Evolved
nucleases, for example, may lead to virus-resistant cell
lines and enzymes capable of selective allelic
destruction in vivo. Inteinevolution may provide a
general method of intracellular protein recombination to
create libraries of more than 10^(16) protein variants in
vivo. Artificial allosteric proteins may serve as
chemical sensors for the detection of small molecules
inside or outside of living cells. Engineered and evolved
transcription factors may be used to activate or repress
the expression of any gene in vivo or in vitro. We are
also interested in defining the scope of biopolymeric
catalysis and in expanding our ability to generate
complex protein- and RNA-based catalysts by the stepwise
evolution of new protein catalysts from catalytic RNAs
and of new ribozymes from existing protein enzymes. We
are currently developing both an in vitro and an in vivo
method for selecting catalytically active protein-RNA
complexes.
Biologically Inspired Synthetic Methodologies
A third area of focus lies in the development of
synthetic methodologies which employ new approaches,
often inspired by Nature, to address existing problems in
interdisciplinary areas spanning chemistry and biology.
For example, we are developing a new
"protecton-deprotecton" strategy for synthetic
protecting group chemistry that links short and
unreactive nucleic acid analogs to protecting groups
which are then sequence-specifically deprotected with
appropriate reagents linked to complementary nucleic acid
analogs. In addition, we are interested in applying
combinatorial approaches coupled with gene chip analysis
(typically employed by the field of genomics) towards the
comprehensive characterization and refinement of
sequence-specific DNA binders[12] as well as towards the
discovery of entirely new classes of DNA-binding
molecules. Finally, we are interested in the design and
implementation of triplet gene synthesis system that may
allow the one-pot generation of DNA libraries encoding
all possible alanine scanning, truncation, insertion,
deletion, or recombined mutants of a protein or protein
family of interest.
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