Insights into the development of chemical probes for RNA

Morgan, Brittany S; Forte, Jordan E; Hargrove, Amanda E

There are several noteworthy examples of probing ligand promiscuity and/or selectivity in human systems. Multiple studies utilized genome-wide analyses to globally assess changes in miRNA, (16, 17, 59, 60, 80) mRNA, (18, 50) or splicing (18, 21, 47, (81) (82) (83) (84) (85) ) levels compared to the target RNA or process. Toward a more direct assessment, chemical cross-linking and isolation by pull down (Chem-CLIP) was utilized to investigate proximity-based engagement of RNA (86) . In this method, the ligand is appended with a nucleic-acid reactive module (e.g. chlorambucil) and a biotin purification tag. After incubating cells with the modified ligand, the cells are lysed, the ligand is captured by streptavidin beads, and the bound targets are characterized by qRT-PCR or RNA-seq. This strategy as well as competitive Chem-CLIP (86) were used to characterize onand off-target engagement of ligands that modulate repeat expansions (45, 84, 85, 87) and miRNAs (16, 17, 60) . In another study, dual luciferase reporter assays were utilized to compare ligand binding to four G-quadruplex structures, including the RNA of interest and three other regulatory RNAs (44) . This study was one of few examples in which selectivity within a target family was assessed in cell culture. Various controls were also used to evaluate on-target effects. For example, the impacts of chemical probes have been analyzed following siRNA knockdown of FOXO1 mRNA (59) and following inhibition of the mTOR signaling pathway modulated by miRNA-544 (61) . Another example overexpressed miRNA-210 and assessed the effect of the chemical probe on the phenotype (16) . Likewise, a ligand targeting r(CUG) exp was tested with the RNA under conditional expression (88) . Another notable example used RNA immunoprecipitation (RIP) to detect RNA-binding at increasing concentrations of ligand and identified a dosedependent response (19) . For precipitation, a G-quadruplex specific antibody, BG4, was utilized and the complex was characterized by two complementary methods: dot blotting and qRT-PCR. Another important control, particularly for human systems, was to replicate on-target effects in at least two cell lines, though this control was performed for only a limited number of chemical probes (44, 53, 47, 81, 84) .