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From the landscape of an ancient “RNA world” springs an idea
that could lead to the creation of ultrasensitive biosenors based on molecular
switches. Marc Wortman talks to biology professor Ron Breaker about the
potential of so-called RNA switches.
Biosensors exploit the natural tendency of proteins to recognize and
react to other molecules. Ron Breaker theorized that the RNA in proteins
could be isolated and serve as a “switch” that targets specific
molecules. The central image at left represents a binary RNA switch as
it goes through the chemical machinations necessary to recognize a molecule—it
must bind to two different molecules before the ribozyme can cleave. Biosensors
could be used to diagnose patients, monitor for bioterror weapons and
detect metabolites in clinical samples, contaminants in food or pollutants
in water.
When Ronald R. Breaker, Ph.D., associate professor of molecular, cellular
and developmental biology, wanted to develop a high-tech tool for detecting
everything from infectious agents like HIV in the blood to contaminants
such as arsenic in water, he figured that the place to start was about
3.5 billion years ago, when life began.
Some have theorized that RNA, the active component in genes, and not
DNA, the genetic library, composed the first life forms. Based on that
theory, Breaker supposed that 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
in April 2001, the switches worked. In fact, they worked so well that
some believe they could be key to one day developing small, easy-to-use
biosensors for detecting everything from tumor cells to toxic chemicals.
RNA molecular switches could be used to construct a kind of dashboard
panel to detect when sought-after molecules are present. Such biosensors
might be developed to detect contaminants in food, pollutants in water,
metabolites in clinical samples or biological-warfare agents on the battlefield.
Yale licensed the RNA molecular-switch technology to a Cambridge, Mass.,
biotechnology company that Breaker helped found, Archemix Inc., which
is developing possible commercial applications of the biosensor technology.
In his own laboratory, he is continuing to explore the science behind
molecular switches. Contributing Editor Marc Wortman asked Breaker to
explain how biosensors work and to discuss the challenges of developing
the technology.
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 all of 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. For many diabetics,
frequent monitoring of the concentration of glucose in their blood is
necessary. However, glucose is only one type of molecule within the very
complex mixture of chemical and biological compounds present in blood.
One way to “see” glucose in this vast sea of other compounds
is to use an enzyme called glucose oxidase. This enzyme can identify glucose
among all the other compounds and cause it to react 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, and
thus one can determine 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 on the surface of things?
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. Glucose concentrations are typically measured
in “millimolar” units, which means that there are more than
a billion trillion glucose molecules in a pint of blood. There are so
many glucose molecules that it is quite easy to detect and accurately
measure them. However, HIV concentrations could be present in “zeptomolar”
units, where only a few hundred virus particles might be present but still
able to cause disease. 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.
What are the usual methods?
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—enzyme-linked
immunosorbent assays (ELISA) and polymerase chain reaction (PCR)—to
expand the power of biosensors and bioanalytical methods.
Sometimes natural proteins are not available for a particular target;
interestingly, the immune systems of animals can be used to produce new
antibodies that selectively bind to new targets of interest. These antibodies
are typically used in ELISA tests.
More recently, PCR tests have become commonplace. These tests rely on
the power of DNA-making enzymes (DNA polymerase) to selectively amplify
genetic fragments of infectious agents such as anthrax and HIV. DNA fragments
of just a few infectious particles in a sample are used in PCR to make
billions of copies, which can then be observed by any one of several different
methods.
What are the problems or limitations of those current 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.
Automation of some aspects of these tests can reduce the time needed to
set up and interpret multiple 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.
Given concerns about bioterrorism, what efforts are under way in your
laboratory and elsewhere to develop better biosensors?
Several science funding agencies had embarked on efforts to accelerate
biosensor development even before recent events brought attention to bioterrorism.
I think that this was a recognition that improvements were needed in detection
technology, and also that recent advances in biological research indeed
make possible great advances in biosensor sophistication.
Our laboratory took a rather unusual path to enter the area of biosensor
research. We were testing a theory of how life began some 3.5 billion
years ago. It is believed that an “RNA world” once existed,
where all enzymes and other molecular components of primitive cells were
made of ribonucleic acid, or RNA. Although this entire way of life has
long since become extinct, we can perhaps use evolution in a test tube
to 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.
What types of applications could they have?
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.
Are RNA molecules stable enough to serve as biosensor elements?
Nature has chosen DNA to store genetic information because each link in
the chain is extraordinarily stable. In contrast, each link in RNA’s
chain is about 100,000 times more likely to break. If this occurs in one
of our switches (which are about 100 links in size), then its function
is likely to be destroyed. Fortunately, RNA is sufficiently stable to
provide a shelf life of several years. After immobilization, the RNA array
can be stored for long periods of time simply by letting the surface air
dry and storing it at room temperature. Of course this is assuming that
the RNA is not being digested by contaminating enzymes. Even the oils
covering our skin carry enzymes that can degrade RNA very rapidly. Again,
we are fortunate because chemists have created many modified RNA links
that resist destruction by nucleases. In other words, the science to create
modified RNA switches that resist attack by chemicals and enzymes is already
well developed.
Will we eventually have low-cost, easy-to-use biosensors in our homes
and workplaces?
Without question. 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 might they 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 handheld 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. YM
Marc Wortman is a contributing editor of Yale Medicine.
James Yang is an illustrator in New York City.
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