Excerpts: By applying precise, mechanical forces
to the ends of individual RNA molecules, researchers have
successfully unfolded and refolded the molecules. According to the
scientists, measurements of the forces needed to unfold and refold
these molecules will yield new information about how RNA molecules
achieve their stable three-dimensional structures. (…)
To measure the force needed to unfold a single RNA molecule,
Bustamante and his colleagues attached each end of a test molecule
to a single microscopic plastic bead via RNA/DNA "handles." They
used an "optical trap" consisting of a laser beam that held and
measured the force on one bead, as a piezoelectric actuator
attached to the other bead supplied the minute forces required to
unfold the molecule. The scientists also used the technique to
measure the change in the length of the molecule as it was
unfolded.
"This system eliminated both the problems of averaging large
numbers of molecules and the multiple reaction pathways," said
Bustamante, "because when we are pulling, we are following a
single molecule unfolding along a particular pathway."
In their experiments, the scientists unfolded three kinds of
RNA molecules, each more complex than the previous one:
- the simplest, a folded "hairpin" RNA structure
- an RNA that also contained a more complicated "helix
junction" found in many folded RNAs
- the most complex, an RNA that had a "bulge" in its
structure, and can attain tertiary structure
"Originally, we wanted to study a full ribozyme," said
Bustamante, "but it became clear to us that the complexity we were
observing when we pulled the ribozyme was going to make it
impossible to understand everything from the beginning.
Ultimately, we realized that because RNA molecules are
hierarchical in their structure, with each domain being relatively
independent, it would be possible to synthesize the different
domains of the molecule and then pull on each type of domain to
understand their characteristics."
The results of the scientists' experiments revealed that
each of the three types of RNA molecules had distinctive
characteristics. For example, the hairpin and helix junction RNAs
both exhibited a phenomenon the scientists called "hopping." In
these instances, if the molecules were held at a constant force
that was great enough to cause the molecule to transition to the
unfolded state, they would hop back and forth between the unfolded
and folded states. From that hopping state, the scientists could
pinpoint the forces required to unfold the molecules, measure the
rates of folding and unfolding and the energy of the process.
They also found that the unfolding forces coincided with the
refolding forces. "This means that all the work that we are doing
mechanically, to pull the molecule, to unfold the molecule, is
going to just break the bonds in the molecule that maintain the
folding," said Bustamante. "This means you can carry the process
at equilibrium."