[R35] Defining and Controlling Protein-RNA interactions in editing pathways
Ente: National Institute of General Medical Sciences
Scadenza: 2031-02-28
Importo max: 674.487 EUR
Paese: US
Descrizione
This renewal application for the Maximizing Investigators Research Award (MIRA)
R35GM141907 is proposed to continue support for research in the Beal lab focused on defining and
controlling protein-RNA interactions in RNA editing pathways. The RNA editing ADAR enzymes
convert adenosines (A) to inosines (I) in duplex RNA. Since inosine behaves similarly to guanosine
(G) in RNA, this modification can have profound effects on the structure and function of the modified
RNA including changes in the meaning of specific codons. Mutations in the human ADAR genes can
cause disease and ADAR1 upregulation and hyper editing have been observed in cancer. While our
understanding of the ADAR reaction mechanism and specificity have advanced considerably, it is still
not clear how all the domains present in full length ADAR proteins coordinate during substrate
recognition and catalysis. Also, ADAR isoform-specific selectivity for different editing sites is not fully
understood. Furthermore, ADARs’ ability to produce functional A-to-G transitions in RNA makes
directed ADAR editing an attractive approach for therapeutic applications. Indeed, therapeutic
oligonucleotide-directed RNA editing was realized for the first time in humans in 2024. While this
approach is clearly promising, identification of the optimal guide RNA sequence and chemical
modification pattern for a specific therapeutic target RNA is challenging and typically requires the
synthesis and testing of hundreds of candidate oligonucleotides. In addition, given our understanding
of ADAR-RNA interactions, additional opportunities exist for the development of novel ADAR-
activating guide strand modifications. Finally, high levels of off-target editing have limited the use of
exogenously expressed ADAR fusion proteins for directed editing purposes.
Key questions in this field that will be addressed in this project are: 1) What are the design
rules for chemically modified ADAR-guiding oligonucleotides? To address this question, we will
use a novel high throughput screen developed in our lab that allows for hundreds of thousands of
chemically modified guide strand sequences to be tested in parallel. These screens will be used to
generate large data sets that will serve as training inputs for predictive modeling. This approach is
expected to deliver a computational tool that can predict efficient guide strand sequences for a given
modification pattern. 2) Can we generate new and useful ADAR-guide strand contacts with novel
chemical modifications? We will use a combination of in silico screening, chemical synthesis of
ADAR guide strands bearing new nucleosides along with editing activity assays to discover new ways
to control ADAR-RNA interactions. These efforts will advance the development of ADAR directing
therapeutics by providing new guide strand modifications that stimulate ADAR activity. 3) Can we
define the role of dsRBDs in ADAR reactions? Covalent crosslinking will be used to identify dsRBD
binding si
Istituzione: UNIVERSITY OF CALIFORNIA AT DAVIS
PI: PETER A. BEAL
Progetto: 2R35GM141907-06
Settori: National Institute of General Medical Sciences
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