Structural Biochemistry/RNA folding

Introduction
As knowledge on the diverse functions of RNA become more and more abundant, it has become quite apparent that understanding how RNA folds and what causes it to fold is going to be key to understanding RNA itself. Since its inception as an area of study, the RNA folding problem has been compared and contrasted with the much more difficult protein folding problem and many different methods of unfolding RNA have been considered and used.

How is it done?
Originally, atomic force microscopy was considered as a method of unfolding RNA in physiological conditions of temperature and solvent in order to skip the usage of high temperatures and denaturants. Since then, the preferred method to unfold single molecules of RNA has evolved to the usage of laser tweezers, better known as optical tweezers, in order to apply force to the molecule to induce unfolding. This technique, called mechanical unfolding allows direct observation of the unfolding and refolding of the RNA sequence being studied.

As an RNA molecule unfolds from more complex tertiary structures down to single strands, force and end-to-end distance is measured. Using this information, scientists can find the amount of work that is needed to unfold the RNA and the amount of work obtained upon refolding. The work obtained while refolding is known as the Gibbs free energy change. Further study of the time dependence of the processes help scientists determine kinetics that are involved.

This unfolding of RNA is also present naturally in biological cells. Helicases unwind specific types of RNA and certain enzymes, such as RNA polymerases and ribosomes, need to unwind RNA strands before they can go about performing their assigned functions. In this case, those functions are transcription and translation of the RNA strand.

Comparing and Contrasting with Protein Folding Problem
Protein folding involves a combination of twenty amino acids in order to form a protein molecule. Also, because of its tendency to fall into different secondary structure arrangements (α-helices, parallel and anti-parallel β-sheets, β-turns, random coils, etc.) and for these secondary arrangements to share energy dependencies with its tertiary arrangements, the problem of protein folding is a formidable one.

By comparison, the RNA folding is much simpler than protein folding. Only four nucleotides are used in building the structure with each nucleotide being composed of a base, a ribose, and a phosphate. The bases themselves differ only slightly from each other because of their similarities in form and differences only in carbonyl and amino group placements. The riboses are fundamentally the same other than their two possible conformations. The phosphate's electrostatic charge, though originally seen as an obstacle, can actually be controlled through the ionic strength of the solvent. There are only four main secondary structures for RNA (helices, loops, bulges, and junctions) and since the energies for the formation of these secondary structures is actually higher than those of RNA's tertiary structures, RNA can remain stable in its secondary structure.

RNA folding software
Unlike protein folding, RNA folding is feasibly modeled with mid-range computer processing power due to the significant drop in force-introducing molecules (4 nucleotide bases compared to 20 amino acids). Modelling of the minute forces contributed by RNA bases was demonstrated by the Stanford group Simbios which is dedicated to physics modelling of biological activity. The RNA folding program adds one nucleotide at a time to the RNA which prevents the computer from becoming overloaded. Because RNA structures form relatively stable and orderly shapes, this modelling provides a realistic simulation for real-world structure. The program and source code can be found here.

The RNA folding process can be demonstrated in a game-like fashion as seen in the Carnegie Mellon and Stanford University program EteRNA. EteRNA creates a painless introduction to key forces present in RNA folding. Players can even create RNA molecules to meet certain requirements such as shape, length, and composition, desired for experimentation. If successful, the created RNA strand will be synthesized and used for testing. This sort of free-form program allows for human creativity to locate a solution to a problem where a computer's rigid method of thought would take painstaking hours to find.