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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
8 / 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
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.
10 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~.
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
12 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.