Recent work from many labs has underlined the critical importance of RNA-controlled cellular pathways, and
clinically relevant connections of specific RNAs to human health. As a result, the development of small-
molecule ligands for RNAs, in contrast to traditional protein targets, is now offering a promising approach for
future targeting of diseases. In addition to this designed approach, it seems likely that many current protein-
targeted drugs may well bind to RNAs off-target and cause unintended biological effects there. Thus, the
analysis of RNA-small molecule interactions transcriptome-wide is critical. Unfortunately, methods for
analyzing RNA-ligand interactions directly in the cell lag far behind those for protein-ligand binding analysis.
Preliminary experiments from this laboratory have established multiple new molecular tools for analysis of
biologically and clinically relevant RNAs. New RNA Seq-based approaches have been developed for
identifying RNA base modifications directly. Central to this new project was the recent development of the
first cell-permeable RNA acylating agents, based on a nicotinyl scaffold, that react with accessible 2'-OH
groups transcriptome-wide. This allows unprecedented measurement of RNA structure and protein-RNA
interactions in vivo at nucleotide resolution. In very recent work, new acylating reagents that can polyacylate
RNAs in vitro, temporarily inactivating the RNA have been developed. These groups can then be removed
chemically or photochemically to control RNA activity temporally or locally. Overall, this recent new work
suggests a suite of new molecular reagents, tools and sequencing methods that can be used directly in cells
to analyze ligand-RNA interactions for the whole transcriptome in one experiment.
The proposed project will develop a new set of functionalized RNA-reactive reagents, including acylating
and alkylating species, that can enter cells and provide specific, quantitative information about ligand binding
in the transcriptome. Combined with next-gen sequencing, the methods will pinpoint binding sites in specific
transcripts. These methodologies, together termed Reactivity-Based RNA Profiling (RBRP), will be applied to
analyzing off-target binding by known drugs with clinically limiting toxicity. Further, new reactivity-based
approaches - involving reactive druglike fragments – will be used to identify ligands for cancer-related RNAs.
This work is significant because it will develop enabling molecular technologies that will greatly enhance
the study of RNA biology and biomedicine, analyzing drug interactions transcriptome-wide and directly in the
cell. In addition, it will outline how serious is the phenomenon of protein-targeted drugs binding off target to
RNAs. The research program is innovative because it develops a suite of new molecular probes and novel
molecular strategies, making use of RNA reactivity. It will develop unprecedented data regarding existing
clinically useful but toxic drugs. It will also develop novel cell-permeable ligands for RNAs upregulated in
cancer, which can be broadly useful as molecular tools for cancer research.