Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications.

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field. Pertinent research was funded from mid-2015 to 2022, including an extension related to the coronavirus pandemic. Despite being a detriment to research activity in general, the pandemic has resulted in tremendously boosted interest in the field of RNA and RNA modifications as a consequence of their widespread and successful use in vaccination campaigns against SARS-CoV-2. Funded principal investigators published over 250 pertinent papers with a very substantial impact on the field. The program also helped to redirect numerous laboratories toward this dynamic field. Finally, SPP1784 spawned initiatives for several funded consortia that continue to drive the fields of nucleic acid modification.

Precision RNA base editing with engineered and endogenous effectors.

RNA base editing refers to the rewriting of genetic information within an intact RNA molecule and serves various functions, such as evasion of the endogenous immune system and regulation of protein function. To achieve this, certain enzymes have been discovered in human cells that catalyze the conversion of one nucleobase into another. This natural process could be exploited to manipulate and recode any base in a target transcript. In contrast to DNA base editing, analogous changes introduced in RNA are not permanent or inheritable but rather allow reversible and doseable effects that appeal to various therapeutic applications. The current practice of RNA base editing involves the deamination of adenosines and cytidines, which are converted to inosines and uridines, respectively. In this Review, we summarize current site-directed RNA base-editing strategies and highlight recent achievements to improve editing efficiency, precision, codon-targeting scope and in vivo delivery into disease-relevant tissues. Besides engineered editing effectors, we focus on strategies to harness endogenous adenosine deaminases acting on RNA (ADAR) enzymes and discuss limitations and future perspectives to apply the tools in basic research and as a therapeutic modality. We expect the field to realize the first RNA base-editing drug soon, likely on a well-defined genetic disease. However, the long-term challenge will be to carve out the sweet spot of the technology where its unique ability is exploited to modulate signaling cues, metabolism or other clinically relevant processes in a safe and doseable manner.

Precise and efficient C-to-U RNA base editing with SNAP-CDAR-S.

Site-directed RNA base editing enables the transient and dosable change of genetic information and represents a recent strategy to manipulate cellular processes, paving ways to novel therapeutic modalities. While tools to introduce adenosine-to-inosine changes have been explored quite intensively, the engineering of precise and programmable tools for cytidine-to-uridine editing is somewhat lacking behind. Here we demonstrate that the cytidine deaminase domain evolved from the ADAR2 adenosine deaminase, taken from the RESCUE-S tool, provides very efficient and highly programmable editing when changing the RNA targeting mechanism from Cas13-based to SNAP-tag-based. Optimization of the guide RNA chemistry further allowed to dramatically improve editing yields in the difficult-to-edit 5'-CCN sequence context thus improving the substrate scope of the tool. Regarding editing efficiency, SNAP-CDAR-S outcompeted the RESCUE-S tool clearly on all tested targets, and was highly superior in perturbing the ß-catenin pathway. NGS analysis showed similar, moderate global off-target A-to-I and C-to-U editing for both tools.

ADAR1-mediated RNA editing promotes B cell lymphomagenesis.

Diffuse large B cell lymphoma (DLBCL) is one of the most common types of aggressive lymphoid malignancies. Here, we explore the contribution of RNA editing to DLBCL pathogenesis. We observed that DNA mutations and RNA editing events are often mutually exclusive, suggesting that tumors can modulate pathway outcomes by altering sequences at either the genomic or the transcriptomic level. RNA editing targets transcripts within known disease-driving pathways such as apoptosis, p53 and NF-κB signaling, as well as the RIG-I-like pathway. In this context, we show that ADAR1-mediated editing within MAVS transcript positively correlates with MAVS protein expression levels, associating with increased interferon/NF-κB signaling and T cell exhaustion. Finally, using targeted RNA base editing tools to restore editing within MAVS 3'UTR in ADAR1-deficient cells, we demonstrate that editing is likely to be causal to an increase in downstream signaling in the absence of activation by canonical nucleic acid receptor sensing.

CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo.

RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.

Harnessing self-labeling enzymes for selective and concurrent A-to-I and C-to-U RNA base editing.

The SNAP-ADAR tool enables precise and efficient A-to-I RNA editing in a guideRNA-dependent manner by applying the self-labeling SNAP-tag enzyme to generate RNA-guided editases in cell culture. Here, we extend this platform by combining the SNAP-tagged tool with further effectors steered by the orthogonal HALO-tag. Due to their small size (ca. 2 kb), both effectors are readily integrated into one genomic locus. We demonstrate selective and concurrent recruitment of ADAR1 and ADAR2 deaminase activity for optimal editing with extended substrate scope and moderate global off-target effects. Furthermore, we combine the recruitment of ADAR1 and APOBEC1 deaminase activity to achieve selective and concurrent A-to-I and C-to-U RNA base editing of endogenous transcripts inside living cells, again with moderate global off-target effects. The platform should be readily transferable to further epitranscriptomic writers and erasers to manipulate epitranscriptomic marks in a programmable way with high molecular precision.

Controlling Site-Directed RNA Editing by Chemically Induced Dimerization.

Various RNA-targeting approaches have been engineered to modify specific sites on endogenous transcripts, breaking new ground for a variety of basic research tools and promising clinical applications in the future. Here, we combine site-directed adenosine-to-inosine RNA editing with chemically induced dimerization. Specifically, we achieve tight and dose-dependent control of the editing reaction with gibberellic acid, and obtain editing yields up to 20 % and 44 % in the endogenous STAT1 and GAPDH transcript in cell culture. Furthermore, the disease-relevant MECP2 R106Q mutation was repaired with editing yields up to 42 %. The introduced principle will enable new applications where temporal or spatiotemporal control of an RNA-targeting mechanism is desired.

New Frontiers for Site-Directed RNA Editing: Harnessing Endogenous ADARs.

RNA editing activity can be exploited for the restoration of disease-causing nonsense and missense mutations and as a tool to manipulate the transcriptome in a simple and programmable way. The general concept is called site-directed RNA editing and has high potential for translation into the clinics. Due to its different mode of action RNA editing may well complement gene editing and other gene therapy options. In this method chapter, we particularly highlight RNA editing strategies that harness endogenous ADARs. Such strategies circumvent the delivery and expression of engineered editases and are notably precise and simple. This is particularly true if endogenous ADARs are recruited with chemically modified antisense oligonucleotides, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). To foster the research and development of RESTORE we now report a detailed protocol for the procedure of editing reactions, and a protocol for the generation of partly chemically modified RESTORE ASOs with a combination of in vitro transcription and ligation.

Protocols for the generation of caged guideRNAs for light-triggered RNA-targeting with SNAP-ADARs.

The SNAP-tag technology offers a convenient way to assemble guideRNA-protein conjugates for transcript-specific RNA editing in vitro, in cell culture and in vivo. In contrast to other methods, including CRISPR/Cas-based, the SNAP-tag is small, well expressed and of human origin. Furthermore, the SNAP-ADAR approach enables the ready inclusion of photo control by caging/decaging of the benzylguanine moiety required for the conjugation reaction with the SNAP-tag. Beyond site-directed RNA editing, the method has high potential for various applications in the field of RNA targeting. However, the generation of the required guideRNAs includes some basic chemistry. Here, we provide step-by-step protocols for (a) conduction of photo controlled RNA editing reaction, (b) the generation of photo activatable guideRNAs, and (c) the synthesis of the caged benzylguanine moiety. With this we hope to foster a broader application of these attractive methods to researchers with less experience in chemistry.

Chemistry Helps to Bump Off-Target Edits Away.

Side-directed RNA editing is an emerging tool to manipulate genetic information. Unfortunately, most approaches suffer from off-target editing. In this issue of Cell Chemical Biology, Monteleone et al. (2019) present a clever strategy that improves specificity by combining a mutant editase with a compensating chemical modification in the guiding RNA.

Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides.

Site-directed RNA editing might provide a safer or more effective alternative to genome editing in certain clinical scenarios. Until now, RNA editing has relied on overexpression of exogenous RNA editing enzymes or of endogenous human ADAR (adenosine deaminase acting on RNA) enzymes. Here we describe the engineering of chemically optimized antisense oligonucleotides that recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). We observed almost no off-target editing, and natural editing homeostasis was not perturbed. We successfully applied RESTORE to a panel of standard human cell lines and human primary cells and demonstrated repair of the clinically relevant PiZZ mutation, which causes α1-antitrypsin deficiency, and editing of phosphotyrosine 701 in STAT1, the activity switch of the signaling factor. RESTORE requires only the administration of an oligonucleotide, circumvents ectopic expression of proteins, and represents an attractive approach for drug development.

Critical review on engineering deaminases for site-directed RNA editing.

The game-changing role of CRISPR/Cas for genome editing draw interest to programmable RNA-guided tools in general. Currently, we see a wave of papers pioneering the CRISPR/Cas system for RNA targeting, and applying them for site-directed RNA editing. Here, we exemplarily compare three recent RNA editing strategies that rely on three distinct RNA targeting mechanisms. We conclude that the CRISPR/Cas system seems not generally superior to other RNA targeting strategies in solving the most pressing problem in the RNA editing field, which is to obtain high efficiency in combination with high specificity. However, once achieved, RNA editing promises to complement or even outcompete DNA editing approaches in therapy, and also in some fields of basic research.

Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs.

Molecular tools that target RNA at specific sites allow recoding of RNA information and processing. SNAP-tagged deaminases guided by a chemically stabilized guide RNA can edit targeted adenosine to inosine in several endogenous transcripts simultaneously, with high efficiency (up to 90%), high potency, sufficient editing duration, and high precision. We used adenosine deaminases acting on RNA (ADARs) fused to SNAP-tag for the efficient and concurrent editing of two disease-relevant signaling transcripts, KRAS and STAT1. We also demonstrate improved performance compared with that of the recently described Cas13b-ADAR.

Npom-Protected NONOate Enables Light-Triggered NO/cGMP Signalling in Primary Vascular Smooth Muscle Cells.

Diazeniumdiolates (NONOates) are a class of nitric-oxide-releasing substances widely used in studies of NO/cGMP signalling. Because spatiotemporal control is highly desirable for such purposes, we have synthesised a new Npom-caged pyrrolidine NONOate. A kinetic analysis together with a Griess assay showed the photodependent release of NO with high quantum yield (UV light). In primary vascular smooth muscle cells (VSMCs), our compound was reliably able to induce fast increases in cGMP, as measured with a genetically encoded FRET-based cGMP sensor and further validated by the phosphorylation of the downstream target vasodilator-stimulated phosphoprotein (VASP). Thanks to their facile synthesis, good decaging kinetics and capability to activate cGMP signalling in a fast and efficient manner, Npom-protected NONOates allow for improved spatiotemporal control of NO/cGMP signalling.

Switching Protein Localization by Site-Directed RNA Editing under Control of Light.

Site directed RNA editing is an engineered tool for the posttranscriptional manipulation of RNA and proteins. Here, we demonstrate the inclusion of additional N- and C-terminal protein domains in an RNA editing-dependent manner to switch between protein isoforms in mammalian cell culture. By inclusion of localization signals, a switch of the subcellular protein localization was achieved. This included the shift from the cytoplasm to the outer-membrane, which typically is inaccessible at the protein-level. Furthermore, the strategy allows to implement photocaging to achieve spatiotemporal control of isoform switching. The strategy does not require substantial genetic engineering, and might well complement current optogenetic and optochemical approaches.

Applying Human ADAR1p110 and ADAR1p150 for Site-Directed RNA Editing-G/C Substitution Stabilizes GuideRNAs against Editing.

Site-directed RNA editing is an approach to reprogram genetic information at the RNA level. We recently introduced a novel guideRNA that allows for the recruitment of human ADAR2 to manipulate genetic information. Here, we show that the current guideRNA design is already able to recruit another human deaminase, ADAR1, in both isoforms, p110 and p150. However, further optimization seems necessary as the current design is less efficient for ADAR1 isoforms. Furthermore, we describe hotspots at which the guideRNA itself is edited and show a way to circumvent this auto-editing without losing editing efficiency at the target. Both findings are important for the advancement of site-directed RNA editing as a tool in basic biology or as a platform for therapeutic editing.

The notorious R.N.A. in the spotlight - drug or target for the treatment of disease.

mRNA is an attractive drug target for therapeutic interventions. In this review we highlight the current state, clinical trials, and developments in antisense therapy, including the classical approaches like RNaseH-dependent oligomers, splice-switching oligomers, aptamers, and therapeutic RNA interference. Furthermore, we provide an overview on emerging concepts for using RNA in therapeutic settings including protein replacement by in-vitro-transcribed mRNAs, mRNA as vaccines and anti-allergic drugs. Finally, we give a brief outlook on early-stage RNA repair approaches that apply endogenous or engineered proteins in combination with short RNAs or chemically stabilized oligomers for the re-programming of point mutations, RNA modifications, and frame shift mutations directly on the endogenous mRNA.

Harnessing human ADAR2 for RNA repair - Recoding a PINK1 mutation rescues mitophagy.

Site-directed A-to-I RNA editing is a technology for re-programming genetic information at the RNA-level. We describe here the first design of genetically encodable guideRNAs that enable the re-addressing of human ADAR2 toward specific sites in user-defined mRNA targets. Up to 65% editing yield has been achieved in cell culture for the recoding of a premature Stop codon (UAG) into tryptophan (UIG). In the targeted gene, editing was very specific. We applied the technology to recode a recessive loss-of-function mutation in PINK1 (W437X) in HeLa cells and showed functional rescue of PINK1/Parkin-mediated mitophagy, which is linked to the etiology of Parkinson's disease. In contrast to other editing strategies, this approach requires no artificial protein. Our novel guideRNAs may allow for the development of a platform technology that requires only the administration or expression of a guideRNA to recode genetic information, with high potential for application in biology and medicine.

Site-Directed RNA Editing in Vivo Can Be Triggered by the Light-Driven Assembly of an Artificial Riboprotein.

Site-directed RNA editing allows for the manipulation of RNA and protein function by reprogramming genetic information at the RNA level. For this we assemble artificial RNA-guided editases and demonstrate their transcript repair activity in cells and in developing embryos of the annelid Platynereis dumerilii. A hallmark of our assembly strategy is the covalent attachment of guideRNA and editing enzyme by applying the SNAP-tag technology, a process that we demonstrate here to be readily triggered by light in vitro, in mammalian cell culture, and also in P. dumerilii. Lacking both sophisticated chemistry and extensive genetic engineering, this technology provides a convenient route for the light-dependent switching of protein isoforms. The presented strategy may also serve as a blue-print for the engineering of addressable machineries that apply tailored nucleic acid analogues to manipulate RNA or DNA site-specifically in living organisms.