Special Report: The Peptide-Oligonucleotide Partnership

Molecular biology has come a long way in the nearly 40 years since James Watson and Francis Crick published their classic paper on the three-dimensional structure of DNA (Nature, 171:737-738, 1953). Since then, the intimate relationship between nucleic acids and proteins has not only been deciphered, but also harnessed and manipulated to fuel a whole new industry. Armed with sophisticated equipment, today's molecular biologists can easily extract, purify, and sequence DNA, RNA, and proteins, and even create short stretches of DNA bases (oligonucleotides) and short proteins (peptides) to their own specifications. The effects of these new developments have reverberated far beyond the biochemical research arena to such diverse fields as agriculture, horticulture, human and veterinary medicine, and forensics. The functional connections among DNA, proteins, and traits began to be unraveled in 1909, when English physician Archibald Garrod established a key link between what he called "inborn errors of metabolism" (such as alkaptonuria and phenylketonuria) and a biochemical build-up reflecting impaired enzyme function. In 1928, noted microbiologist Fred Griffith showed that one strain of bacteria could pick up a trait from another, courtesy of a not-yet-defined "transforming principle," which would turn out to be DNA (Journal of Hygiene, 27:113-159, 1928). Meanwhile, several groups found that chromosomes consisted of DNA and small, positively charged proteins. Before the 1940s, proteins, with their varied biological functions, were the No. 1 candidate to be the genetic material now known as DNA. But in 1944, O.T. Avery, C.M. MacCleod, and M. McCarty (Journal of Experimental Medicine, 79:137-158) identified Griffith's transforming principle as DNA, which was confirmed by A.D. Hershey and M. Chase's famous "blender" experiments (Journal of General Physiology, 36:39-56, 1952), in which DNA was labeled in one batch of bacteria, and protein in another. Only the DNA label was passed to the next generation. This showed that DNA, not protein, is the genetic material. The dawn of nucleic acid synthesis arrived with the discovery of the DNA double helix, as lab-made oligonucleotides were needed to help crack the genetic code (Nature, 192:1227-1232, 1961). Early on, DNA was synthesized in solution, which required purification after every step. Today, with the advent of solid phase phosphoramidite chemistry, purification between steps is not necessary. In this process, the first nucleotide in the chain is anchored to a solid support, usually glass beads. The researcher then adds an activated nucleotide, which bonds with the anchored molecule. The investigator continues the process, sequentially, to build an oligonucleotide. The length and sequence of the product is controlled by the investigator. Solid-phase synthesis is used in manufacturing peptides as well. The researcher anchors the amino acid by its carboxyl terminus, or end, to an insoluble support, such as polystyrene beads. Its exposed end is activated ("deprotected"), and the next amino acid in the chain, in protected form, is added along with dicyclo-carbodiimide, which couples the amino acids so that a peptide bond forms between them. The beads are washed to remove excess reagents, and the next amino acid is added. The completed oligopeptide is released from the resin. Synthesizing oligonucleotides and peptides is costly, especially in today's climate of dwindling grant support. A handful of companies offer manual and automated synthesizers, which range greatly in price (see accompanying list). Ralph Walker, a customer service representative for Rockville, Md.-based Synthecell Corp., one of several companies offering custom synthesized oligonucleotides and peptides, advises researchers to consider the cost of the machine as well as the expense of operating it. "If you make lots of oligonucleotides or peptides, continuously, it is worthwhile to purchase a machine," he says. "But most people work with only a few sequences over a year, and ordering a machine is not even realistic." Firms such as Synthecell generally charge a small setup fee, and then charge per base. Prices increase with the purity of the product. At Synthecell, for example, the machine grade cost is $5 per DNA base for greater than 90 percent purity; $10 per base for greater than 95 percent purity (reagent grade), and $16 per base for greater than 98 percent purity (research grade). Richard Zitomer, professor of biology at the State University of New York, Albany, agrees that researchers should consider the nature of their projects when making purchasing decisions. He points out that synthesizers have hidden costs. Although many are easy to operate, frequently "there are problems in logistics," he says. "The chemicals have short lifetimes on the machine, and require completely anhydrous conditions. It is impossible to keep it [the machine] in this condition for more than three weeks." Zitomer offers his colleagues at SUNY an alternative: He synthesizes oligonucleotides for them, charging their grants $10 for the first residue and $2.50 for each subsequent one. Arnold Lippa, marketing specialist for Vega Biotechnologies Inc., a Tucson, Ariz., firm that manufactures both DNA and peptide synthesizers, says that although most purchasers are now from research labs, the picture will change--and soon--as the value of oligonucleotides and peptides in diagnosis and therapeutics becomes more widely recognized, something that is sure to happen as the trek through the human genome continues. "Everybody is focusing on cloning this gene or that gene," says Lippa. "Yet there is so much more to the field than that. For example, the explosion in peptide technology alone is immense." Oligonucleotides have many applications. According to Zitomer, who studies regulatory genes in yeast, "one use is for DNA sequencing, where oligonucleotides serve as primers in the sequencing reaction, which saves time and energy because you don't have to subclone fragments into vectors before sequencing. You can sequence from both ends of one subclone, using oligonucleotide primers." Oligonucleotides can also be used to create specific mutations by site-directed mutagenesis. "You can create oligonucleotides with single base changes or small deletions," says Zitomer. Such tailor-made snippets of DNA can be used, for example, as diagnostic probes for mutant disease-causing genes. Oligonucleotides are also an integral part of the polymerase chain reaction (PCR), in which short sequences bracketing a particular gene are used to direct rapidly repeated rounds of replication. Zitomer says that sequences of 12 to 15 nucleotides are generally sufficient for priming sequencing, about 20 to 30 residues are needed for site-directed mutagenesis, and about 25 are used for PCR. Another exciting application of specific short DNA base sequences-- and sometimes RNA sequences or analogs--is in antisense technology. This application involves the manufacturing of oligonucleotides that, owing to complementary base pairing, bind to the sense strand (the transcribed strand) of a gene or to its mRNA, halting gene expression. This approach has been used to retard ripening in fruits by squelching enzyme production, to halt viral replication, and to slow activity of oncogenes. Although the concept of antisense technology evolved along with the genetic discoveries of the 1960s and 1970s, it awaited synthesizers to jump from the theoretical to the practical. "This work has been facilitated enormously by solid state synthesis of oligodeoxynucleotides within the last six years," says Paul Zamecnik, of the Worcester Foundation for Experimental Biology, an independent research institute in Shrewsbury, Mass. "With gene machines, we can now synthesize a 20-mer [an oligonucleotide consisting of 20 building blocks] with infinitely less effort than was possible previously." Zamecnik used synthetic oligo-deoxynucleotides in 1978 to inhibit transforming ability, replication, and translation of Rous sarcoma virus in chick embryo fibroblasts, and, more recently, to inhibit HIV replication and expression of some of its highly conserved genes in human-cultured lymphocytes. It's easy to see why geneticists in the first half of the century were so enamored of proteins--these molecules are both ubiquitous and incredibly variable. Peptide research ranges from the fascinatingly theoretical to the highly practical. With the aid of computers, many researchers are scrutinizing protein sequence "motifs"--the loops and ladders, barrels and sheets, zippers and fingers formed by amino acid interactions. Using a peptide synthesizer, Robert Hodges, Paul Semchuk, and their colleagues at the University of Alberta built two identical 35-amino-acid-long alpha helices, incorporating a particular overall conformation. They are now altering them, amino acid by amino acid, to decipher the influences of each residue on folding and stability. At the University of Colorado Medical School in Denver, John Steward and his coworkers are taking a more specific approach to deciphering protein folding rules. They started by displaying the 274-amino-acid structure of the enzyme chymotrypsin on a computer screen. They then deleted all of the molecule except for a triad of amino acids forming the catalytic site to which ester substrates bind. Next, they built a 73-amino-acid-long peptide, chymohelizyme-1, that consists of four short, parallel helices. This peptide mimics the native protein in many ways. Approaches such as this may lead to improved industrial enzymes, such as detergents. The simplicity of peptides that make them useful models of larger proteins is also an advantage in the clinic. Peptide drugs and vaccines may be small enough to be effective when taken orally. On a more fundamental level, very short peptides allow exploration of the interactions between antigens and receptors that underlie various disease states. For example, Steven J. Burakoff and colleagues at the Dana-Farber Cancer Institute in Boston have synthesized a class of phenylalanine-containing dipeptides, called CPFs, that block the CD4 receptor on T cells to which the gp120 molecule on the surface of the AIDS virus attaches. This minute peptide not only protects the T cell from infection, but also allows it to continue to receive signals from the immune system, enabling it to remain active, producing lympho-kines. In their recent paper, the researchers call the CPFs "promising forerunners to an effective inhibitor of HIV-1 infection" (Science, 249:287-90, 1990). The beauty of the gene-protein relationship is that it is identical among all species, yet retains a virtually limitless plasticity that has enabled life to evolve. With the aid of powerful machines to synthesize these molecules of life, researchers can tap into this treasure chest of possibilities to better understand life as well as to eliminate suffering. Holly Ahern and Ricki Lewis teach biology at the State University of New York, Albany.

Special Report: The Peptide-Oligonucleotide Partnership

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