Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity

Katrekar, Dhruva; Xiang, Yichen; Palmer, Nathan; Saha, Anushka; Meluzzi, Dario; Mali, Prashant

doi:10.7554/elife.75555

Cited by 30 publications

(18 citation statements)

References 52 publications

(64 reference statements)

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“…It is clear that ADARs have 5′, 3′, and opposite base preferences within base paired, A-form duplexes. , Indeed, these preferences are well understood, and they can be used to inform the design of ADAR guide strands for site-directed RNA editing applications. ,,,,, However, these preferences are not absolute as ADARs can deaminate adenosines in natural substrate RNAs that contain suboptimal nearest neighbors and/or are adjacent to helix defects, etc . ,, Furthermore, many therapeutically relevant target adenosines for directed RNA editing applications do not conform to ADAR’s known preferred nearest neighbors. , In such cases where rational design based on current knowledge of ADAR-RNA recognition is insufficient, it is sensible to screen for sequences that can enable editing by forming beneficial, non-Watson–Crick structural features in the RNA. Different screening strategies for ADAR-RNA combinations have been published, but each requires transfection of plasmid libraries, limiting the size of libraries that can be practically screened. ,, Here we describe a type of screen that does not require plasmid transfections, and that allows one to query very large libraries. By linking an editing site covalently through a hairpin loop to a site where the sequence is randomized, we could use NGS to quantify the number of reads of G or A associated with each specific sequence within the randomized region.…”

Section: Resultsmentioning

confidence: 99%

“…Different screening strategies for ADAR-RNA combinations have been published, but each requires transfection of plasmid libraries, limiting the size of libraries that can be practically screened. 14,20,23 Here we describe a type of screen that does not require plasmid transfections, and that allows one to query very large libraries. By linking an editing site covalently through a hairpin loop to a site where the sequence is randomized, we could use NGS to quantify the number of reads of G or A associated with each specific sequence within the randomized region.…”

Section: Development Andmentioning

confidence: 99%

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Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites

et al. 2023

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Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs' selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR's main limitations is its preference to edit adenosines with specific 5′ and 3′ nearest neighbor nucleotides (e.g., 5′ U, 3′ G). Current rational design approaches are wellsuited for this ideal sequence context, but limited when applied to difficult-to-edit sites. Here we describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates (En Masse Evaluation of RNA Guides, EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. We used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. A guide RNA bearing one of these sequence motifs enabled the cellular repair of a premature termination codon arising from mutation of the MECP2 gene associated with Rett Syndrome. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs' specific RNA−protein interactions.

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Section: Resultsmentioning

confidence: 99%

Section: Development Andmentioning

confidence: 99%

Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites

et al. 2023

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“…A common problem with delivering the naked DD with a gRNA is that it tends to create many off-target edits, not only within the targeted message but also in unrelated messages across the transcriptome (Vallecillo-Viejo et al 2018;Buchumenski et al 2021). Approaches such as localizing the DD to the nucleus, or splitting it into two units that only form a functional deaminase enzyme when they come together, have significantly reduced unwanted edits (Vallecillo-Viejo et al 2018;Katrekar et al 2022a). These engineered systems have been used to good effect in vivo.…”

Section: Site-directed Rna Editing Systemsmentioning

confidence: 99%

Site-directed A → I RNA editing as a therapeutic tool: moving beyond genetic mutations

Quiroz

Siskel

Rosenthal

2023

RNA

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Adenosine deamination by the ADAR family of enzymes is a natural process that edits genetic information as it passes through messenger RNA. Adenosine is converted to inosine in mRNAs, and this base is interpreted as guanosine during translation. Realizing the potential of this activity for therapeutics, a number of researchers have developed systems that redirect ADAR activity to new targets, ones that aren’t normally edited. These site-directed RNA editing (SDRE) systems can be broadly classified into two categories: ones that deliver an antisense RNA oligonucleotide to bind opposite a target adenosine, creating an editable structure that endogenously expressed ADARs recognize, and ones that tether the catalytic domain of recombinant ADAR to an antisense RNA oligonucleotide that serves as a targeting mechanism, much like with CRISPR-Cas or RNAi. To date, SDRE has been used mostly to try and correct genetic mutations. Here we argue that these applications are not ideal SDRE, mostly because RNA edits are transient and genetic mutations are not. Instead, we suggest that SDRE could be used to tune cell physiology to achieve temporary outcomes that are therapeutically advantageous, particularly in the nervous system. These include manipulating excitability in nociceptive neural circuits, abolishing specific phosphorylation events to reduce protein aggregation related to neurodegeneration or reduce the glial scarring that inhibits nerve regeneration, or enhancing G protein-coupled receptor signaling to increase nerve proliferation for the treatment of sensory disorders like blindness and deafness.

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“…Current directed RNA editing methods either employ endogenous human ADARs or overexpress engineered ADAR proteins with improved efficiency and target specificity. 22,23 To achieve better editing outcomes for therapeutic RNA editing using endogenous ADARs, chemically modified guide oligonucleotides (∼30−40 nt long) are presently utilized. 3,22 An example of a chemically modified guide that has been previously used by our lab is shown in Figure 2B, 3 and a detailed discussion of the chemical modifications that specifically enhance ADAR catalysis is presented in section 3.…”

Section: Introductionmentioning

confidence: 99%

“…ADAR performs the corrective A-to-I edit, resulting in a transcript that would now translate to a functional protein (Figure A). Current directed RNA editing methods either employ endogenous human ADARs or overexpress engineered ADAR proteins with improved efficiency and target specificity. , To achieve better editing outcomes for therapeutic RNA editing using endogenous ADARs, chemically modified guide oligonucleotides (∼30–40 nt long) are presently utilized. , An example of a chemically modified guide that has been previously used by our lab is shown in Figure B, and a detailed discussion of the chemical modifications that specifically enhance ADAR catalysis is presented in section . However, other backbone (phosphorothioate (PS) and phosphoryl DMI amidate (PN) linkages) and sugar (2′-deoxy, 2′- O -methyl (2′-OMe), 2′-fluoro (2′-F), and locked nucleic acid (LNA)) modifications are also being employed to improve the metabolic stability, target binding kinetics, and specificity of guide oligonucleotides (Figure B). ,, …”

Section: Introductionmentioning

confidence: 99%

Chemical Modifications in RNA: Elucidating the Chemistry of dsRNA-Specific Adenosine Deaminases (ADARs)

Mendoza,

Beal

2023

Acc. Chem. Res.

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Conspectus The term RNA editing refers to any structural change in an RNA molecule (e.g., insertion, deletion, or base modification) that changes its coding properties and is not a result of splicing. An important class of enzymes involved in RNA editing is the ADAR family (adenosine deaminases acting on RNA), which facilitates the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Inosines are decoded as guanosines (G) in most cellular processes; hence, A-to-I editing can be considered to be an A-to-G substitution. Among the RNA editing enzymes, ADARs are of particular interest because a large portion of RNA editing events are due to A-to-I editing by the two catalytically active human ADARs (ADAR1 and ADAR2). ADARs have diverse roles in RNA processing, gene expression regulation, and innate immunity; and mutations in the ADAR genes and dysregulated ADAR activity have been associated with cancer, autoimmune diseases, and neurological disorders. A-to-I editing is also currently being explored for correcting disease-causing mutations in the RNA, where therapeutic guide oligonucleotides complementary to the target transcript are used to form a dsRNA substrate to site-specifically direct ADAR editing. Knowledge of the mechanism of the ADAR-catalyzed reaction and the origin of its substrate selectivity will lead to a broader understanding of ADAR’s role in disease biology and expedite the process of developing ADAR-targeted therapeutics. Chemically modified oligonucleotides provide a versatile platform for modulating the activity and interrogating the structure, function, and selectivity of nucleic acid binding and modifying proteins. In this Account, we provide an overview of oligonucleotide modifications that have allowed us to gain a deeper understanding of ADAR’s molecular mechanisms, which we utilize in the rational design and optimization of ADAR activity modulators. First, we describe the use of the nucleoside analog 8-azanebularine (8-azaN) to generate high-affinity ADAR-RNA complexes for biochemical and biophysical studies with ADARs, with a particular emphasis on X-ray crystallography. We then discuss key observations derived from the crystal structures of ADAR2 bound to 8-azaN-modified RNA duplexes and describe how these findings provided insight into ADAR editing optimization by introducing nucleoside modifications at various positions in the synthetic guide strands. We also present the informed design of 8-azaN-modified RNA duplexes that selectively bind and inhibit ADAR1 but not the closely related ADAR2 enzyme. Finally, we conclude with some open questions on ADAR structure and substrate recognition and share our current endeavors in the development of ADAR guide oligonucleotides and inhibitors.

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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity

Cited by 30 publications

References 52 publications

Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites

Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites

Site-directed A → I RNA editing as a therapeutic tool: moving beyond genetic mutations

Chemical Modifications in RNA: Elucidating the Chemistry of dsRNA-Specific Adenosine Deaminases (ADARs)

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