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1
New Tools and Approaches for Discovery,
Diagnostics, and Prevention
With recent advances in the chemical sciences there has been an explosion of
information in genomics, proteomics, informatics, and high-throughput screen-
ing. This has led to an ever increasing need for new tools and approaches to
effectively create and manage the large amounts of information that are being
obtained in the postgenomic era. Concomitant with the increase in biological
information, interdisciplinary fields have emerged in the chemical sciences in
order to fully exploit all areas of research. One such field, systems biology, does
not use the traditional reductionist approach to chemical problems. Instead the
scientist looks at biological problems as the whole of its components, which is
made possible by the various technological advances in the chemical sciences.
DNA SEQUENCING
New tools and technology in the chemical sciences have advanced DNA
sequencing. The automated florescent DNA sequencer has led to the completion
of the human genome project. There has been roughly a 6,000-fold increase in the
throughput of DNA sequencing with a significant decrease in cost from its incep-
tion in 1986. There are current efforts to increase the throughput another 3,000-
to 6,000-fold, which can be accomplished by single molecule DNA sequencing.
This new approach will open the world of genomes to comparative analysis, which
in turn will allow predictive and preventative medicine based on an individual's
specific genomic makeup (see Sidebar 1.1~.
There are other new and innovative approaches to DNA sequencing in devel-
opment. The 2002 Nobel Prize winner in physiology, Sydney Brenner, has devel-
oped a technique whereby a million cDNA sequences can be affixed on separate
7
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8
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HEALTH AND MEDICINE
~ Sidebar 1.1 : ~
The DNA Sequencing Revolution
Excerpt from 'iSystems Biology and Global Analytical
Techniques"
Leroy Hood ~~ :
~ The Institute :for :Systems Biology
Since the 1986 publication of the automated fluorescent DNA proto-
type, an;~ir~crease of approximately 6,000 times the throughput of DNA
sequencing has occurred coupled to a significant decrease in cost. Ap-
plied ~Biosystems, a company set up: to~commercialize these instruments,
-then took nearly five years and $75 million to make the DNA sequencer
a robust~instr~ument. It took another 10 years and several hundred m'llio n
dollars Vito Recreate the high-throughput production Circe that led to finishing
the:human genome project. Extrapolation from today to five to seven
years in the future yields another 3,000- to 6,000-fold increase In through-
put and at least a thousand-fold decrease in cost. Therefore, fan entire
human genome could be sequenced in an afternoon for less than
$10,00O,: Ma feat that opens up the whole world of genomes for compara-
tive analyses and the possibility halve will do genetic mapping of hu-
mans by complete sequence analysis. As a result we would be able to
characterize the three billion nucleot~des of the genome instead roof just
the 300 or~3.000 features bv Genetic markers.
\
beads and amplified for 16 to 20 base pairs simultaneously in a flow cell. This
results in roughly a million sequences in about six hours. This particular tech-
nique is important in analyzing a discreet transcriptome of a particular cell type.
Another innovative approach to genome sequencing developed in the laboratory
of Leroy Hood uses inkjet technology to synthesize oligonucleotide arrays. The
current model has the ability to synthesize 60 to 70 mers in very high yield in a
short time. This technology may lead to the synthesis of large genes or even gene
families.
DNA NANOTECHNOLOGY
An emerging field that is attempting to exploit the information content of
DNA is DNA-based nanofabrication. The idea is to achieve instructed fabrication
by using the information contained in DNA to construct higher-order complexes
and to control motion by using the chemical properties of the DNA. There are a
number of examples of using DNA scaffolds to demonstrate controllable molecu-
lar motions. One such example used a DNA structural motif termed the
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NEW TOOLS AND APPROACHES FOR DISCOVERY, DIAGNOSTICS, AND PREVE=ION 9
"paranemic crossover" that was able to rotate 180 degrees depending on which
oligonucleotide was added to the solution. DNA nanotechnology is a promising
new field of science that certainly will lead to powerful new approaches to chemi-
cal and biological problems.
NUCLEIC ACID THERAPEUTICS
Nucleic acid therapeutics was first proposed using antisense technology in
the late 1970s. This technology has since evolved into preclinical and clinical
trials. Antisense DNA binds to target mRNA through Watson-Crick pairing,
which prevents the RNA from being translated into protein. The formation of a
DNA-RNA heteroduplex initiates the enzyme RNase H to cleave the RNA por-
tion of the heteroduplex. While theoretically sound, the advancement of this tech-
nology has been very slow. A newer approach using antisense employs the mecha-
nism of RNA interference (RNAi). RNAi uses the ability of higher eukaryotic
cells to cleave double-stranded RNA, presumably in defense of a viral infection.
The double-stranded RNA is enzymatically cleaved into smaller fragments known
as small interfering RNAs (siRNAs), which in turn initiate the cleavage of the
mRNA that corresponds with the sequence of the siRNAs. RNAi technology is
still not completely elucidated. In order for this technology to be effectively in-
corporated into a viable therapeutic alternative, RNAs must be inhibited from
inducing an interferon response in the cell.
COMBINATORIAL CHEMISTRY
Combinatorial chemistry has emerged as one of the most productive new
approaches to drug discovery. The technology of creating and testing large
amounts of compounds to ascertain which ones contain the desired biological
activity has spawned many new approaches to synthesis and design. It can pro-
vide access to ligands to probe a biological process (e.g., ligands that inhibit a
target protein of interest, promote a phenotype of interest, or act as a sensor).
Many new assays have been developed to effectively screen molecular libraries.
The screens typically are immunoassays, enzyme reactions, cell-based assays, or
any number of other specialized tests chosen for the specific disease or molecule
being studied. The synthetic process in combinatorial chemistry usually employs
many automated techniques, such as robotics, to synthesize thousands of unique
chemical compounds or oligonucleotides with predetermined atomic structure
that are categorized in a database or library. The ultimate goal of this technology
in the area of health and medicine is to enable researchers to pursue rational drug
design, whether using molecular libraries to discover new drugs or in oligonucle-
otide therapeutics. Combinatorial methods are now also being employed in the
biotechnology sector in the areas of proteomics and bioinformatics.
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HEALTH AND MEDICINE
STRUCTURAL PROTEOMICS
One of the fields of science that is benefiting the most from recent advances
in the chemical sciences is structural proteomics, which is the global analysis of
proteins. There are presently only about 15,000 structures deposited in the Pro-
tein Data Bank, of which roughly 4,000 represent unique protein folds spanning
1,500 protein families. Although these numbers are growing every day, the pre-
dicted number of protein families in our proteome is on the order of 20,000-
50,000. These numbers depict the challenge that lies ahead for chemists and bi-
ologists in understanding the complex mechanisms in the human body. There are
only a limited number of proteins that have been marketed due to structure-based
discovery techniques. Timely access to molecular structures has historically been
one of the major drawbacks. Parallel processing, miniaturization, and automation
have greatly decreased the amount of time it takes from protein purification to
structure determination. In particular, the use of robotics, nano- and picoliter-
scale crystallization techniques, and high-throughput automated imaging systems
to detect viable crystals has greatly increased the speed at which protein struc-
tures are elucidated. In nuclear magnetic resonance spectroscopy there has been
limited success with automated structure determination programs. There have
also been new advances in pulse sequences that greatly reduce the number of
scans needed to obtain structural data under certain conditions, thereby decreas-
ing costly data acquisition time. In addition, innovative tandem mass spectrom-
etry techniques allow detailed characterization of proteins without having to un-
dergo the potentially painstaking task of structure determination. These advances
have dramatically improved efforts to look for biologically active compounds
that thwart disease.
PROTEIN EXPRESSION ANALYSIS
Since there is no analog to polymerase chain reaction (PCR) for proteins, it is
difficult to analyze the expression patterns of proteins in response to various phe-
notypic conditions. New techniques using mass spectrometry have been able to
help associate protein expression with genomic DNA in response to various cel-
lular stressors. A technique called "isotope coded affinity tagging" (ICAT) in
combination with mass spectrometry was used by Leroy Hood and colleagues to
analyze the galactose expression system and create a snapshot of the global inter-
actions of a series of different systems present in a yeast cell. A protein engineer-
ing technique called Expressed Protein Ligation has been developed that intro-
duces sequences of unnatural amino acids, posttranslational modifications, and
biophysical probes into proteins of any size through the chemoselective addition
of a peptide to a recombinant protein (see Sidebar 1.2~.
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NEW TOOLS AND APPROACHES FOR DISCOVERY, DIAGNOSTICS, AND PREVENTION 1l
. . .
:
: i:: ::: : ~ a: :
Sidebar 1.2
Genetic Code Expansion
Excerpt from "~Biotechnology"
Peter G. :Schultz
The Scripps Research Institute
Every known form~of life has the same genetic code containing the
same common 20 amino~acids, with a few rare exceptions. Logical ques-
tions from chemists may follow: "Why these 20? What would it be like if it
were not these 20? If we find life on another planet, will the same ~20 be
present7" With only 20~amino acids there is a limitation with what can be
done with proteins. Presently enough is known about these systems that
they are actually amenable to chemical synthesis, but different~starting
materials in the cell need to be dealt with. Wolf one wants to make proteins
and change the genetic code of a living cell, the components of the pro-
tein biosynthetic machinery must be used: the DNA that transcribes the
message that is translated on the ribosome by ~ set of adapter mol-
ecules, the tRNAs ~that~translate the triplet codons in the genetic code,
the polypeptide, and the amino acids in the polypeptide sequence.
Peter Schultz and colleagues have been successful at expanding
the genetic code by incorporating additional amino acids. An E. icon that
has a 21 -amino-acid genetic code, in which the additional amino acid is
benzophenone, is one example. A second example is the introduction of
a keto amino acid: an important functional group in chemistry that was
missing from the genetic code. Schultz and colleagues successfully
added the keto group with fidelity of 99.99 percent. There is no difference
between the benzophenone and keto amino acids and alanine, since the
yields of protein are identical. Kilograms of proteins can be produced with
these unnatural amino acids, allowing selective modification of proteins
in the case of the keto due to the unique functional group. Furthermore,
the unique genetic:code can be modified~with fluorescein to create a
molecular-level probe Schultz would like to create molecular resolution
tags and probes that can be used to image Any event in a living cell. J
_ ~ ~ ~ ~
PROTEIN STRUCTURE ANALYSIS
Although protein structure determination is critical to understanding the pro-
cess of an enzyme, it often does not adequately address real-time protein-protein
interactions in viva, which is imperative when attempting to decipher the inner
workings of the cell. Co-expression tags can aid in detecting protein localization
and interactions in the cell. Barbara Imperiali and colleagues have developed a
new, less obtrusive class of expression probes that bind lanthanide ions to impart
luminescent effects. This enables these co-expressed proteins to be monitored by
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HEALTH AND MEDICINE
protein-protein interaction assays. The small size of these probes aids in minimiz-
ing any steric interactions between the probe and the proteins of interest.
MICROFLUIDICS
To circumvent the increasing cost and increase the efficiency of research and
development in health and medicine, there is an increasing trend toward minia-
turizing reactions and reaction conditions. Microfluidics is the study of reaction
conditions and fluid flow in microenvironments. Many scientists are investigat-
ing this type of "lab on a chip" technology where compounds can undergo com-
plicated reaction schemes in a microenvironment. This new technology affords
the possibility of having multiple laboratory functions, such as purification, im-
mobilization, sorting, and detection, carried out on a single chip, enabling the
capacity to perform multiple parallel analyses in a faster and often more accurate
microreaction.
MOLECULE DELIVERY
One of the challenges in studying intracellular interactions is successfully
delivering the molecule of interest to its site of action. There have been many new
approaches and advances in this arena. One such advance is in the area of caged
phosphopeptides. Phosphopeptides that represent phosphorylation sites in vari-
ous kineses have been designed to examine the effect of the liberated phospho-
peptide on cell migration. Typically, the phosphopeptides can be cleaved by pho-
tolysis of the cage upon migrating to the site of action in the cell. Although
promising, there is still much research to be done before this approach can be
widely used in medicine. There are many new and innovative approaches to drug
delivery that are currently under investigation.
Representative terms from entire chapter:
genetic code
