MicroRNAs (miRNAs) are RNAs that act as gene regulators by binding to and silencing complementary mRNAs. Some of these mRNA targets produce tumor-suppressing proteins. When there is an overexpression of certain miRNAs, elevated levels can lead to the silencing of tumor suppressor genes. Before miRNA can function, it is matured from its precursor pre-miRNA. This occurs by cleavage of the pre-miRNA by the enzyme Dicer. Because Dicer is only able to recognize the normal, stem-loop structure of pre-miRNAs, the presence of alternative structures causes inhibition of processing. Recent studies have shown that a structure known as a G-quadruplex (GQ) can form in some pre-miRNAs, inhibiting Dicer cleavage and miRNA function.
Our focus has been on two projects aimed at investigating these distinct structures that exist in some pre-miRNAs. In the first project, we are optimizing the labeling of a pre-miRNA with a fluorophore and a quencher on the 5’ and 3’ ends. The goal is to apply the labeled pre-miRNA in a FRET assay to investigate how cellular stimuli affect the GQ and stem-loop structures. Labeling of pre-miR-92b at the 5’ end was done via phosphorylation followed by a dye labeling reaction. Labeling of the 3’ end begins with sodium periodate to oxidize the 3’ terminal ribose to form an aldehyde followed by a reaction with an aldehyde-reactive quencher. The process for 3’ end labeling was successfully optimized with 80% efficiency. Despite numerous attempts to optimize the 5’ end labeling, the highest percent labeling with fluorophore was only 40%. Modifications to this reaction are still underway to improve efficiency.
In the second project, we are assessing the ability of small molecules to bind to pre-miR-92b, to determine if small molecule binding can stabilize the GQ conformation. To do this, we used a fluorescence titration assay with Cy-3 labeled pre-miR-92b. Upon titration of the RNA with a compound of interest, binding is observed by changes to the overall fluorescence intensity. The goal of this project is to determine whether stabilization of the GQ in pre-miRNAs through small molecule binding can inhibit miRNA maturation and function, thereby impacting the production of tumor-suppressing target genes. If so, pre-miRNAs that contain G-quadruplex structures could be valuable targets for therapeutic discovery.