RT Journal Article
SR Electronic
T1 RT-qPCR as a screening platform for mutational and small molecule impacts on structural stability of RNA tertiary structures
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.01.16.476525
DO 10.1101/2022.01.16.476525
A1 Zafferani, Martina
A1 Muralidharan, Dhanasheel
A1 Hargrove, Amanda E.
YR 2022
UL http://biorxiv.org/content/early/2022/01/17/2022.01.16.476525.abstract
AB The exponential increase in the discovery and characterization of RNA tertiary structures has highlighted their active role in a variety of human disease, yet often their interactome and specific function remain unknown. Small molecules offer opportunities to both decode these cellular roles and develop therapeutics, yet there are few examples of small molecules that target biologically relevant RNA tertiary structures. While RNA triple helices are a particular attractive target, discovery of triple helix modulators has been hindered by the lack of correlation between small molecule affinity and effect on structural modulation, thereby limiting the utility of affinity-based screening as a primary filtering method. To address this challenge, we developed a high-throughput RT-qPCR screening platform that reports on the effect of mutations and additives, such as small molecules, on the structuredness of triple helices. Using the 3’-end of the oncogenic non-coding RNA MALAT1 as an example, we demonstrated the applicability of both a two-step and a one-pot method to assess the impact of mutations and small molecules on the stability of the triple helix. Employment of a functional high-throughput assay as a primary screen will significantly expedite the discovery of probes that modulate RNA triple helices structural landscape and, consequently, help gain insight into the roles of these pervasive structures.Competing Interest StatementThe authors have declared no competing interest.