Contact Us
Center for Catalysis |
Combinatorial Catalysis
Aptamers are single stranded nucleic acids that fold to form unique structures with specificity for selected target molecules. A process of evolution in the test tube is used to select for these aptamers. The process starts with a large combinatorial library of randomized nucleic sequences from which are selected the rare nucleic acid sequences that fold appropriately to bind the target molecule. The process involves sequential selection of nucleic acids that bind to a column to which the target molecule is attached. The few aptamers that bind to the column are collected and amplified by PCR to produce a new group of molecules that have some affinity for the target molecule. After 6-12 rounds of selection and amplification, the resulting nucleic acid aptamers are cloned. The Nilsen-Hamilton laboratory is selecting for aptamers to specific targets. One project is to select for aptamers that bind to the molecule sitosterol which is a byproduct of the extraction of oil from the soybean. The project is being done in collaboration with the Kraus laboratory where the sitosterol is isolated and derivatized to produce a product that can be used for aptamer selection (figure 1). The aptamer will be used for detecting sitosterol and will be developed as a possible means of selecting for sitosterol from a mix of related steroids. Sitosterol has beneficial effects on the heart and so would be an excellent value-added product for soy-oil manufacturers.
Figure 1 Other uses for aptamers come in the medical sciences. The most important challenge in fighting almost any disease is to be able to detect and treat it in the early stages. For most diseases there are changes in gene expression and subsequent protein products that could be used for detection. However, disease-initiated changes often occur in the depths of our tissues and are not visible from the surface. Therefore another challenge for the developing new technology to fight disease is to find ways of non-invasive imaging (e.g. no biopsy or surgery) of the body's status and to find ways to target small sites inside the body where the disease process is initiating. For non-invasive imaging and targeted disease treatment, reagents are needed that will specifically recognize the result of new gene expression. Two types of reagents could be used for this purpose:
To develop such reagents the Nilsen-Hamilton lab is using nucleic acids called aptamers. Aptamers are single stranded nucleic acids that are selected to have a high affinity and specificity for the target molecular structure. Aptamers are being developed in two ways to detect changes in gene expression in vivo. The first is to prepare probes in which the aptamer is part of a regulated nucleic acid sequence that is complementary to a particular mRNA. The second is to develop allosteric aptamers that will bind specific cell surface proteins that are the result of altered gene expression. Binding the cell surface protein brings about a structural change in the aptamer that results in its binding an imaging agent or drug to treat the disease. Nilsen-Hamilton and her collaborators are developing probes, called "targeted reversibly activated probes" (TRAPs) are inactive in the absence of the target mRNA and then become activated when the TRAP hybridizes with the target mRNA. The activity consists of the ability of the aptamer in the TRAP to bind to its ligand; the ligand, which is linked to a radioisotope is the means by which the cells that express the mRNA are imaged (figure 2). When a cell expresses the target mRNA then the TRAP will be activated and the aptamer ligand will be concentrated in the cell. The TRAP design can also be used to incorporate a ribozyme where the released activity can convert the target to another form - such as cleaving a prodrug to produce a drug that will treat the cell.
Figure 2 Aptamers can also be used to detect a change in gene expression by binding the protein product of the gene. Many aptamers have been selected to recognize specific proteins. Some of these aptamers are currently in use clinically or are being investigated in clinical trials for treating disease. Allosteric aptamers called "cis-linked aptamers for medical procedures" (CLAMPs) are being developed that will recognize a particular cell surface protein and respond by binding another ligand once it has bound to the surface of a diseased cell. The other ligand could be a radiolabeled ligand that can be used to detect the diseased cell. Or it could be a prodrug that will release a toxic compound when brought near to the diseased cell. Thus, allosteric aptamers being developed by Nilsen-Hamilton and her colleagues to recognize specific cell surface proteins for use as imaging agents or for cell killing.
Funded ProjectsCombinatorial Development of Homogeneous Transition Metal Catalysts Using Molecular EvolutionKeith Woo, Iowa State University Department of Chemistry
Graduate student Goudong Du (left) with Keith Woo in his laboratory. This proposal entails the use of high-throughput, in vitro evolution to develop new classes of DNA enzymes that also require a transition metal ion as a key co-factor. As a model system, we will develop metalla-deoxyribozymes for the catalysis of cyclopropanation. This reaction joins a carbon fragment from a diazo reagent to an olefin to produce a three-membered ring organic compound, but requires a transition metal complex to catalyze the process. Once our model system has been tested, a representative target to illustrate the utility of this approach is the synthesis of chrysanthemic acid, a key component of agriculturally important insecticides, pyrethroids, that have negligible mammalian toxicity. |
Effective catalysis involves the specific recognition
of a target molecule with high affinity. Although many catalytic functions
have evolved naturally, mostly in proteins, the need for new catalytic functions
is growing; for example to convert industrial byproducts to useful products
To develop chemicals that recognize new targets such as industrial byproducts,
Dr. Nilsen-Hamilton and her group are using combinatorial molecular biology.
Center for Catalysis
Copyright © 2008, Iowa State University of Science and Technology. All rights reserved.