RNA plays a vital role in all living organisms as both a carrier of genetic information and a regulator of various cellular pathways. MicroRNAs (miRNAs) are a family of non-coding RNAs that are associated with gene regulation and developmental processes (Mirihana, 2015). Produced from their own genes or from introns of other genes, miRNAs have been shown to have a correlation in tumorigenesis (Cao, 2018). MiRNAs are transcribed into primary miRNA (pri-miRNA), folding into a stem-loop structure through Watson-Crick base pairing (Mirihana, 2015). Drosha-mediated cleavage then ensues, cleaving the stem-loop in the pri-miRNA, forming precursor miRNA (pre-miRNA) (Cao, 2018). Pre-miRNA forms a shorter stem-loop structure, which is then cleaved by an enzyme known as Dicer (Cao, 2018). About 13-16% of pre-miRNAs have G-quadruplex (G4)-forming sequences, causing an unwinding of the stem-loop and folding into this alternative structure (Tassinari, 2021). The G4 forms due to the interaction between the guanines within the RNA, forming a stacking tetrad through Hoogsteen base pairing (Koralewska, 2021). The G4 structure is notable, as it inhibits Dicer cleavage of pre-miRNA, which may have an impact on human diseases, including cancers (Tassinari, 2021). The stabilization of this structure is dependent on various monovalent cations, including sodium (Na+) and potassium (K+). It has also been shown that the structure can be stabilized through exogenous ligands, such as BRACO-19 and pyridostatin. The research presented here focuses on using Circular Dichroism (CD) to analyze the presence and stabilization of this structure in a target pre-miRNA, pre-miR-92b. Through CD, we have explored RNA-ligand binding as well as the impact of other cellular conditions on the stability of the G4, including variations in cation concentrations. CD was used to monitor the tertiary folding of the RNA at different temperatures, allowing for a better understanding of the stability of this alternative structure in pre-miR-92b. The research will give insight into the role of the G4 in miRNA processing in general.
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