Researchers with Berkeley Lab’s Physical Biosciences Division (PBD) have for the first time successfully unfolded and refolded single molecules of RNA. By applying stretching forces to molecules featuring one of three representative RNA substructures, the researchers were able to observe the molecules unfold as they would in a living cell and measure the energy required to drive the folding reaction. These experiments and the results hold importance for, among other applications, the future design of antiviral and other therapeutic drugs.

DEPICTED HERE IS A FOLDED "HAIRPIN," ONE OF THE THREE TYPES OF RNA SUBSTRUCTURE EXAMINED BY RESEARCHERS

RNA -- ribonucleic acid -- is the workhorse of the genetic world, transcribing the coded instructions of DNA and assembling amino acids into proteins. What enables RNA molecules to carry out their many biological tasks is the ability of their nucleotide strands or helices to fold themselves into complex three-dimensional structures. Learning about the forces that drive and shape this folding -- by unfolding RNA molecules -- is a key to designing drugs that can enhance or inhibit the performance of a specific task. For example, retroviruses such as HIV are nothing more than protein-coated packets of RNA molecules.

"Traditionally, scientists have tried to unfold RNA by temperature melting or by denaturing the molecules with chemicals," says Carlos Bustamante, a member of the experimental team who holds a joint appointment with PBD and UC Berkeley (UCB) and is a Howard Hughes Medical Institute (HHMI) investigator.

"The problem with those approaches is that they were measuring massive numbers of molecules at a time and averaging over this vast population. Add to that the problem that every molecule might take a different pathway to unfolding."

Says team member Ignacio Tinoco, "By pulling on the ends of an RNA molecule, we are unfolding it more like it may happen in the cell. As we learn about the different paths for unfolding and refolding RNA molecules we will also learn about transient RNA species that may be good drug targets themselves."

RESEARCH TEAM MEMBERS INCLUDE CARLOS BUSTAMANTE (SEATED) AND (FROM LEFT) IGNACIO TINOCO, STEPHEN SMITH, BIBIANA ONOA, AND JAN LIPHARDT

Adds Jan Liphardt, another team member, "This is the first study in which the energetics of a three-dimensional RNA structure were investigated under the physiological conditions of temperature and ionic strength."

Like Bustamante, Tinoco and Liphardt also hold joint appointments with PBD and UCB. These three were joined by UCB researchers Bibiana Onoa and Steven Smith as co-authors on a paper published in the April 27, 2001 issue of the journal Science entitled "Reversible Unfolding of Single RNA Molecules by Mechanical Force." This work was done in part through UCB’s Health Sciences Initiative which sponsors collaborative research to address major health issues.

In their Science paper, the research team describes how they were able to unfold and refold single molecules of select RNA structure using a unique force-measuring "optical tweezers" set-up that was designed and built in Bustamante's laboratory. In this set-up, an RNA molecule is tethered between two micron-sized polystyrene beads in the middle of a chamber, one bead attached to the tip of a piezoelectric actuator and the other anchored by a laser beam. While the laser beam trapped and held one end of the molecule, the piezoelectric actuator pulled on the other end, causing the molecule to be stretched out to the point where it unfolded. The research team measured both the force required to unfold the molecule and the changing length of the molecule as it was stretched.

Says Bustamante, "This system eliminates both the problems of averaging large numbers of molecules and the multiple reaction pathways because when we are pulling, we are following a single molecule unfolding along a particular pathway."

Because the major structural units or domains that make up RNA molecules are relatively independent, it’s possible to synthesize different types of domains and pull on each to understand its distinctive characteristics. The three types of RNA domains examined in this study were a folded "hairpin," one of the most simple and common secondary RNA domains; a "helix junction," another common but more complicated secondary domain; and a molecule that forms a tertiary RNA domain, a compact "bulge" in which several secondary structures interact.

Both the hairpin and helix junction domains exhibited a phenomenon called "hopping." This occurs when molecules, held at a constant force sufficient enough to allow transitions between the folded and unfolded states, begin to hop back and forth between these two states. From that hopping behavior, the Berkeley researchers were able to measure the forces required to unfold the molecules, plus their rates of unfolding and refolding and the energy expended during the process. They found that the unfolding forces coincided with the refolding forces.

"This means the process can be carried out at equilibrium," says Bustamante. "All the mechanical work we do to pull the molecule is going to just break the bonds in the molecule that maintain the folding."

The RNA domain with the bulge displayed an unfolding phenomenon called "ripping." This occurs when the molecule partially unfolds and then pauses. Only upon a slight increase in the pulling force will the molecule abruptly unfold the rest of the way.

"When you start looking at even more complex domains, you start seeing different pathways, with similar parts of the molecule unfolding at different forces," Bustamante says.

The scientists also investigated the folding characteristics of RNA domains in the presence of magnesium ions which are known to be necessary for RNA to carry out its biological functions. They found that when an RNA molecule achieved its three-dimensional structure in the presence of magnesium it became much more difficult to unfold.

Says Tinoco, "I was surprised to learn just how important magnesium ions are to the unfolding of RNA molecules. We found that the main effect is on the kinetics of unfolding rather than on the energy needed to unfold the RNA."

Ultimately, the Berkeley researchers, through these single molecule mechanical unfolding and refolding experiments, would like to be able to provide biochemists with an energy function curve that would identify the energy barriers required to hold a given RNA molecule together and the force needed to unfold it.

Explains Liphardt, "Once you know this energy function curve, you will know (for a given RNA molecule) something about how your molecule folds, its most stable conformation, and the alternative conformations it can explore at a given temperature. You’ll also know something about how it might deform when it interacts with other molecles, such as enzymes or drugs. If you’re a biochemist trying to design an RNA molecule with a particular folding pathway and final conformation this information would help."

Members of research team say their results so far are merely a hint of the wealth of data on RNA folding that their experimental technique can provide.