# Structural Biochemistry/Nucleic Acid/RNA/RNA Folding
## How RNA Unfolds and Refolds

In general, RNA unfolds from the tertiary structure to secondary structure to single stranded RNA and vice-versa is true for how RNA folds. RNA unfolding depends on temperature to denature RNA or sometimes enzymes such as RNA-dependent RNA polymerase (RdRps) or helicases. Moreover, scientists use the techniques called optical tweezers, which is also called laser tweezers, and fluorescence resonance energy transfer, also known as FRET, to study how secondary and tertiary RNA structures unfold and refold. Furthermore, scientists use cation binding to study how ribozymes fold and unfold.

## Secondary Structure RNA

Unfolding:
Secondary RNA structure can unfold by increasing the temperature or using chemical reagents to denature RNA. Another technique used to study how RNA unfolds is optical tweezers. This technique applies a force that causes RNA to unfold in physiological temperature and buffer solutions (79). For example, the ends of a hairpin RNA have two beads—one that has an optical trap and the other has a micropipette strap. From this, RNA can be pulled and unzipped as the micropipette moves.
Refolding: RNA refolding occurs in the reverse process of RNA unfolding. When micropipette moves, RNA can be pushed back which makes RNA relaxed and refolds RNA. However, if the relaxation force applied by optical tweezers increases, this can cause RNA to misfold (81).
Misfolding in RNA can be corrected by increasing the force. When force is increased, the RNA will try to refold into an active and functional form.

## Tertiary Structure RNA

Unfolding
Tertiary RNA structure is relatively weak therefore, by changing the temperature or solutions that are not much different from the physiological state can destabilize RNA interaction.
Refolding
A technique called FRET, fluorescence resonance energy transfer, can be used to understand how RNA folds (78). Scientists label two-dyed nucleotides on RNA strand and through observations of RNA folding, FRET signal allows scientists to measure the distance and motif between those two-dyed labeled nucleotides with respect to time. Furthermore, scientists can also use FRET to understand the changes in RNA conformation when RNA is bound to Mg2+ or ribosomal proteins.

## Single-molecule of RNA Enzymes

To study the single-molecule of RNA enzymes, scientists use ribozymes and FRET technique. The difference between the study of how ribozyme unfolds and folds and that of the secondary or tertiary RNA structure is that scientists add a series of Mg2+ and they observe the FRET signals in order to tell whether ribozyme is docked (folded) or undocked (unfolded). From this Mg2+ “pulse-chase experiments,” scientists can find the “kinetic fingerprints” of the hairpin ribozymes’ enzymatic states (84). Based on this, scientists were able to figure out that ribozymes participate in chemical reactions such as oxidation or reduction, synthesis of nucleotides, and formation of peptides. Thus, the study of ribozymes reinforces the RNA World hypothesis, which stated that RNA preceded DNA (85).

## Effects of Ligand and Protein Binding to RNA

Another way that RNA unfolds is through the appearance or the lack of ligands and/or proteins. Specific proteins and/or ligands bind to RNA and cause it to unzip. By using a technique called single-molecule fluorescence, scientists studied ribonucleoproteins (RNP) and its effect on RNA (88). In this technique, scientists can count RNP subunits in bacteriophage through “electron cryomicroscopy and crystallography” (89). Then, when RNA hairpins unfold, RNPs are assembled and proteins bind to RNA causing RNA to change conformation.
There are three commonly used applications of single-molecule fluorescence techniques. The first is simply counting the subunits in an ribonucleoprotein (RNP). The second common technique is annealing two hairpins, requiring the unfolding of both. As of current, the specific protein role is still not entirely clear. The third technique is to use the fact the RNP assembly is sequential. Because RNP assembly is sequential, this is an indication that events that occur early on in protein binding result in conformational changes in the RNA. By labeling a pair of fluorophores at different positions of telomeric RNA scientists have identified the binding of p65 protein can induce conformational change.
In DNA, argininine is the component used to bind and stabilize the molecule. However, in RNA, it is argininamide, and not arginine that stabilizes and binds the TAR hairpin.

## Enzymes used to unfold RNA

Scientists learned that RNA needs energy input in order to unfold itself however, RNA folding does not require energy because this is a spontaneous reaction. According to the authors in the “How RNA Unfolds and Refolds,” in order to unfold three to four base pairs in RNA, one ATP is used (89). Therefore, enzymes such as helicases or RNA-dependent RNA polymerase help RNA unfold by using chemical energy that is present when nucleoside triphosphates undergo a hydrolysis reaction. For example, helicases take the energy from the ATP hydrolysis reaction to extract the proteins bound on RNA and unfold the double-stranded RNA (90). Therefore, as the concentration of ATP goes up, the faster this step will be. Although RNA-dependent RNA polymerase has not completely explored, scientists believed and expected that it is similar to how helicases work.
Another way that RNA can be unfolded is by binding single stranded RNA to a single-strand specific protein. However, in this situation, the binding must be strong so that it can overcome the forces seen in base pair bonding.
In viral RNA replication, RNA must be single-stranded in order for its sequence to be interpreted during replication and translation. The RNA molecule is first unfolded by an RNA-dependent RNA-polymerase or ribosome. Through the hydrolysis of nucleoside triphosphates, the enzymes can use that energy to be able to unfold the RNA substrates. The RNA needs to have varying sequences.
Work Cited: Li, Pan T.X., Tinoco Jr, Ignacio, and Vieregg, Jeffrey. “How RNA Unfolds and Refolds.” Annual Review of Biochemistry. 2008. 77-100. Print.

## Tertiary Structure Folding

The tertiary folding refers to the interactions between the distal domains that form the structure needed for the RNA to carry out catalytic and regulatory functions. These interactions are fairly weak and can be easily unfolded using small change in temperature and solutions. Specifically, the FRET technique is utilized to observe the tertiary folding of RNA. This technique is performed by measuring the distance between the two florescence-dyed nucleotides. This enables observation of specific tertiary motifs in real time which consists of distinctive folding of the interacting RNA strands. The FRET technique was crucial in studying the RNA folding by measuring the conformational change during the binding of salt (Mg2+) or ribosomal protein. One prominent motif that was studied extensively is the tetraloop-receptor interaction which is present in many large folded RNAs and has been used to create synthetic RNA “building blocks.”
Using optical tweezers and increasing the force, RNA can be unfolded into four distinct conformations in the following order: kissing complex, two linked hairpins, one hairpin, and single strand. Similarly, when the force is decreased, the single strand can be refolded in the reverse order into the kissing complex. The kissing interaction is defined as the base pairing (complementary sequences) between the two hairpin loops and the hairpin loops is created when two complementary sequences in a single RNA meet and bind.

## Salt effect on tertiary structures

In tertiary structures folding and stability are highly dependent on ionic conditions, especially of Mg$^{2+}$. Thus metal ions have a greater effect on tertiary structures than on secondary structures of RNA. Mg$^{2+}$ slows down the kinetics of breaking tertiary interactions, but only moderately affects the folding rates.
Common motifs that demonstrate salt effects include intron ribozymes, pseudoknots, and loop-loop interactions: In intron ribozymes distinct rips were observed in MgCl$_2$, indicating the unfolding of a structural domain. When there was no Mg$^{2+}$, no rips were observed.
In pseudoknots compact structures are formed, and have increased stability with bound Mg$^{2+}$.
In loop-loop interactions force manipulation is used to see how an intramolecular kissing complex changes. This can be seen from the unfolding and refolding of secondary structures. The base pair sequence affects the salt dependence of kissing interactions.

## References

Work Cited: Li, Pan T.X., Jeffrey Vieregg, and Ignacio Tinoco. "How RNA Unfolds and Refolds." Annual Review of Biochemistry 77.1 (2008): 77-100. Print.
* ↑ Li PTX, Vieregg J, Tinoco I Jr. How RNA Unfolds and Refolds. Annu Rev Biochem. 2008;77:77-100.
* ↑ Li PTX, Vieregg J, Tinoco I Jr. How RNA Unfolds and Refolds. Annu Rev Biochem. 2008;77:77-100.
* ↑ Li PTX, Vieregg J, Tinoco I Jr. How RNA Unfolds and Refolds. Annu Rev Biochem. 2008;77:77-100.
* ↑ Li PTX, Vieregg J, Tinoco I Jr. How RNA Unfolds and Refolds. Annu Rev Biochem. 2008;77:77-100.
* ↑ Li PTX, Vieregg J, Tinoco I Jr. How RNA Unfolds and Refolds. Annu Rev Biochem. 2008;77:77-100.