Insights into the development of chemical probes for RNA

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

When using a chemical probe in a biological system, the quality and specificity of the probe must be well characterized to draw accurate and meaningful conclusions, as recently highlighted by several preeminent chemical biologists (11, 12, 74, 75) . Evaluation of the chemical probes revealed a diverse spectrum of characterization techniques with few ligands meeting the traditional criteria for robust chemical probes, and not all of these gaps can be attributed to a lack of relevant tools. For example, characterization inconsistencies include incomplete reports of cytotoxicity and a lack of attention to cell permeability and localization. In addition, many in vitro assays are accessible to help establish the potency and selectivity of a probe in multiple experiments, and these assays should include evaluation against both specifically mutated targets and a number of other structured RNAs. Any cell-based observations should be reproducible in multiple cell lines and validated in the absence of the target by utilizing siRNA or CRISPR-Cas technologies. Further, on-target effects should be established by using a number of spatial-based experiments (12) . These include the biochemical methods described herein (e.g. Chem-CLIP or RIP) and novel applications of other technologies such as in-cell chemical probing (104, 105) to observe changes in RNA secondary structure upon ligand binding or photoaffinity labeling (106) to assess target engagement under temporal control. If plausible, serial passage experiments followed by deep sequencing should also be performed, even in human systems (107) , to identify ligandescaping mutations to confirm target engagement and/or off-target effects. Lastly, it is critical to use an inactive analog and an active analog from a different chemical class, if feasible, to draw conclusions regarding the targeted biology. Moving toward these standards will be crucial for the RNA-targeting field to avoid the scientific pollution that has plagued many others (11, 12, 74) .