Transcriptome-Wide, Unbiased Profiling of Ribonuclease Targeting Chimeras

Various approaches have been developed
to target RNA and modulate
its function with modes of action including binding and cleavage.
Herein, we explored how small molecule binding is correlated with
cleavage induced by heterobifunctional ribonuclease targeting chimeras
(RiboTACs), where RNase L is recruited to cleave the bound RNA target,
in a transcriptome-wide, unbiased fashion. Only a fraction of bound
targets was cleaved by RNase L, induced by RiboTAC binding. Global
analysis suggested that (i) cleaved targets generally form a region
of stable structure that encompasses the small molecule binding site;
(ii) cleaved targets have preferred RNase L cleavage sites nearby
small molecule binding sites; (iii) RiboTACs facilitate a cellular
interaction between cleaved targets and RNase L; and (iv) the expression
level of the target influences the extent of cleavage observed. In
one example, we converted a binder of LGALS1 (galectin-1)
mRNA into a RiboTAC. In MDA-MB-231 cells, the binder had no effect
on galectin-1 protein levels, while the RiboTAC cleaved LGALS1 mRNA, reduced galectin-1 protein abundance, and affected galectin-1-associated
oncogenic cellular phenotypes. Using LGALS1, we further
assessed additional factors including the length of the linker that
tethers the two components of the RiboTAC, cellular uptake, and the
RNase L-recruiting module on RiboTAC potency. Collectively, these
studies may facilitate triangulation of factors to enable the design
of RiboTACs.