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Chaperone-Assisted RNA Crystallography (CARC)

Example of a crystal formed by CARC using a Fab

Chaperone-Assisted RNA Crystallography (CARC) is a method of RNA Crystallography that utilizes a binding protein (chaperone) to facilitate crystal formation. Chaperones may include antibodies, DARPins, and other types of proteins.

Purpose of a chaperone

While crystallography is a popular method for determining the structures of biological molecules (see the PDB's website on number of structures obtained by experimental method here), less than 1,500 of the approximately 130,600 structures in the Protein data bank are of RNA. RNA is negatively charged and has extensive secondary but not tertiary structure, thus RNA does not form appropriate crystal contacts to make a lattice structure. While adding ions help to stabilize the negatively charged backbone, the issue still remains of locking RNA into one rigid conformation so that crystals form and phasing is good.

To combat this problem, chaperones are used to help stabilize RNA and form a regular lattice structure. Common chaperones used in research are Fabs, the region of an antibody that binds the antigen (in this case, RNA), including the paratope.

Fab CARC Process

Phage display cycle

A common method to select appropriate chaperones is by Phage display. This method utilizes Fab-bound phage in order to allow for easy selection and subsequent amplification. Tight-binding Fabs are selected for against the desired RNA, and the unbound Fab-phage complexes are washed away. Fab-phage complex is then eluted and cell culture is infected with the phage in order to amplify the Fabs that passed that round of selection. Then, another round of selection and amplification can be performed and after sufficient rounds, the Fab can be sequenced to determine the primary structure sequence of Fabs that bind most efficiently.

However, Phage display alone is insufficient to find a chaperone for crystallography. Simply because the Fab may have high affinity for the RNA, this does not necessarily mean that the Fab-RNA complex will crystallize. To test if crystals are capable of forming, small-scale crystal trials are performed under various conditions. If crystals form, large scale trials can be set up and structure can be determined by X-ray diffraction.

Another method to be used that selects instead for an RNA against a specific chaperone is called SELEX. This works in the opposite way of phage display, in that a large pool of RNA is repeatedly selected for against a binding molecule and then amplified between each round by reverse transcription and PCR.

Applications

CARC can be used in many different branches of research, including medicine, biochemistry, biology, and other related fields. CARC, along with other methods, allows researchers to determine the 3-D structure of RNA and see activity such as stalled RNA machinery (see Ribosomes, Ribozymes, Riboswitches, Spliceosome) to determine the mechanisms by which they work and allow us to better understand their functions and how they work.