When Ronald Breaker, associate professor of molecular, cellular and developmental biology, wanted to develop a high-tech tool for detecting everything from infectious agents such as HIV in the blood to contaminants such as arsenic in water, he figured the point to start was about 3.5 billion years ago, back at the origins of life.
Some scientists theorize that the earth's first life forms were composed of RNA, the active component in genes, and not DNA, the genetic library. If that theory is true, Breaker conjectured, then RNA would have needed to act like a simple switch when it came into contact with another molecule, sending out signals in order to control metabolism.
He and his team put that evolutionary theory to work in a test tube to "back-engineer" RNA-based molecular switches. As he reported in a paper published in Nature Biotechnology last April, the RNA molecular switches worked.
Because they can be engineered to indicate the presence of different molecules, RNA molecular switches potentially have a wide range of uses. The biosensors can be designed to detect contaminants in food, pollutants in water, metabolites in clinical samples or biological warfare agents on the battle field.
In fact, some scientists believe the RNA switches developed in Breaker's laboratory could be key to developing small, easy-to-use biosensors that someday may become common features in homes and workplaces -- just as home pregnancy tests and home blood tests for diabetics, which use other kinds of biosensors, are already widely available.
Yale licensed the RNA molecular-switch technology to Archemix, Inc., a Cambridge, Massachusetts, biotechnology company that Breaker helped found. The company is developing possible commercial applications of the biosensor technology. In his own laboratory, the Yale researcher is continuing to explore the science behind molecular switches.
Breaker recently spoke to the Yale Bulletin & Calendar about the potential of biosensors to detect biochemical warfare agents. An edited transcript of that conversation follows:
What is a biosensor?
A biosensor is a device that uses biological molecules, usually proteins, to detect chemicals or other biological molecules. Each of our cells holds a complex mixture of chemical compounds, and yet all these compounds are created and used in a highly orchestrated fashion. To do this, our cells mostly use intricately folded proteins to selectively recognize many of these compounds and to make use of them in very specific ways. Scientists found that they could remove one or more of these proteins from their natural setting, and use them as biological components of sensor devices.
Perhaps the best known target for a biosensor is glucose. Many diabetics must frequently monitor the concentration of glucose in their blood. However, glucose is only one type of molecule within the very complex mixture of chemical and biological compounds that are present in blood. One way to "see" glucose in this vast sea of compounds is to use an enzyme called glucose oxidase, which identifies a glucose molecule and combines it with oxygen to produce hydrogen peroxide. Then a second enzyme called "catalase" destroys hydrogen peroxide in a process that can be monitored electronically. Higher levels of glucose in a drop of blood will give higher electronic signals, revealing precisely how much glucose is present.
How difficult is it to detect the presence of a potentially harmful biological agent, such as anthrax or salmonella, in the water or air or various surfaces?
Detecting infectious agents such as anthrax, salmonella or HIV is very difficult, particularly if the contamination is low and if you want the results fast. The challenge largely centers on the number of targets you want to see. Biological agents such as bacteria and viruses can make billions of copies from a single infectious particle. There might be only a few HIV particles in a pint of blood, but they need to be detected in order to ensure that donated blood is safe for transfusion.
How, then, do you detect these dangerous agents?
Detection of biological infections has routinely been achieved by growing cultures from samples taken from patients, or by looking for the production of antibodies. More recently, scientists have exploited two technologies to expand the power of biosensors and bioanalytical methods.
Sometimes natural proteins are not available for a particular target, so we have no readily available protein to recognize the new agent. However, the immune system of animals can be used to produce new antibodies that selectively bind to target molecules. These antibodies are typically used in biosensor tests called ELISA, for Enzyme-Linked Immuno-Sorbent Assays.
More recently, PCR -- or "polymerase chain reaction" -- tests have become commonplace. These tests rely on the power of DNA-making enzymes called DNA polymerase to selectively amplify genetic fragments of infectious agents such as anthrax or HIV in order to make billions of copies, which can then be observed by one of several different methods.
Are there problems or limitations to these new methods?
Speed, shelf-life and cost are of significant concern with all existing methods. ELISA and PCR assays take time to run and are usually labor-intensive. Automating some aspects of these tests can reduce the time needed to set up and interpret the tests, but the biochemical processes themselves also take time. Antibodies and many other proteins are notoriously unstable, and most biosensor kits have a shelf-life that is measured in months -- far too short to be of use to most consumers. Finally, proteins can be expensive to produce and store, which drives up the cost of making most biosensors.
What is being done in your Yale laboratory and elsewhere to develop better biosensors?
Several science funding agencies embarked on efforts to accelerate biosensor development even before recent events made bioterrorism a reality.
Our laboratory took a rather unusual path to enter the area of biosensor research. We were testing a theory for how life began some three-and-a-half billion years ago. It is believed that an "RNA world" once existed where all enzymes and molecular recognition components of early life were made of ribonucleic acid, or RNA. Although this entire way of life has long since gone extinct, we can use evolution in a test tube to perhaps recreate many of these long-lost RNA molecules.
Through these efforts, we invented "RNA switches" that can be used as biosensor elements. For example, we have made many types of RNAs that self-destruct only when they come in contact with a specific target molecule. We recently assembled these on a prototype "RNA biochip" that can be used to detect toxic metals such as cobalt, drug compounds such as theophylline, and natural compounds such as cyclic AMP and cyclic GMP.
How could these RNA switches be used?
We expect that this new type of biological switch will be used to make next-generation biosensor devices that detect a variety of chemical and biological agents in a single assay. Scientists have recently developed "gene chips" that can be used to see thousands of genes on one miniature platform. We imagine similar platforms that see genes, metabolites, drugs, toxins, biohazards and any other targets of interest -- all in a single assay.
Biosensor technology of this advanced type could be used to diagnose patients in a doctor's office, help discover new treatments for disease, detect industrial contaminations and even aid in monitoring for chemical or biological attacks.
Will we eventually have low-cost, easy-to-use biosensors in our homes and workplaces?
Without question. I think that biosensor technology will continue to advance as scientists begin to employ the latest knowledge in enzyme engineering and nanotechnology to this very important challenge. Glucose tests and pregnancy tests have already become routinely self-administered. If the technology continues to advance, I imagine that home diagnostic tests for diseases such as cancer and viral infections might become the first line of defense for health care.
What would these biosensors look like, and how would they be used?
Perhaps not surprisingly, we need to look at our science fiction stories to give us some sense of the possibilities. Although biosensors of the near future might look more like home pregnancy tests than a tricorder from "Star Trek," I think that hand-held biosensor devices that can detect thousands of important targets are being envisioned by many in the field. They will contain some sophisticated electronics and some savvy computer algorithms for interpreting various signals -- and perhaps for giving courses of action to the user. But at their core, they are likely to contain an advanced form of biochip that is engineered to recognize and report the presence of thousands of targets.
-- By Marc Wortman
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