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.

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.

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.

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.

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.

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.

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.

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.

Optimal guideRNAs for re-directing deaminase activity of hADAR1 and hADAR2 in trans.

Adenosine deaminases that act on RNA (ADAR) are a class of enzymes that catalyze the conversion of adenosine to inosine in RNA. Since inosine is read as guanosine ADAR activity formally introduces A-to-G point mutations. Re-addressing ADAR activity toward new targets in an RNA-dependent manner is a highly rational, programmable approach for the manipulation of RNA and protein function. However, the strategy encounters limitations with respect to sequence and codon contexts. Selectivity is difficult to achieve in adenosine-rich sequences and some codons, like 5'-GAG, seem virtually inert. To overcome such restrictions, we systematically studied the possibilities of activating difficult codons by optimizing the guideRNA that is applied in trans. We find that all 5'-XAG codons with X = U, A, C, G are editable in vitro to a substantial amount of at least 50% once the guideRNA/mRNA duplex is optimized. Notably, some codons, including CAG and GAG, accept or even require the presence of 5'-mismatched neighboring base pairs. This was unexpected from the reported analysis of global editing preferences on large double-stranded RNA substrates. Furthermore, we report the usage of guanosine mismatching as a means to suppress unwanted off-site editing in proximity to targeted adenosine bases. Together, our findings are very important to achieve selective and efficient editing in difficult codon and sequence contexts.

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.

The selenocysteine incorporation machinery allows the dual use of sense codons: a new strategy for expanding the genetic code?

Sense and Secis: Even though selenocysteine, Sec, is naturally incorporated by suppressing UAG stop codons, it was recently shown that sense codons such as UAC can efficiently code for Sec. This article highlights the implications of using such a strategy to introduce unnatural amino acids site-selectively into proteins.

Pyrene chromophores for the photoreversal of psoralen interstrand crosslinks.

Applying psoralen interstrand crosslinks for the photoactivation of nucleic acids is a new concept. To find chromophores that can efficiently stimulate crosslink repair we screened several pyrenes and appended them to peptide nucleic acids for their site-selective addressing. Even though pyrenes conjugated to uracil revealed desirable spectroscopic properties they were not effective in crosslink reversal. In contrast, bare pyrenes are well suitable for crosslink repair with 350 nm light showing an uncaging efficiency similar to classical photocaging groups.

Improving site-directed RNA editing in vitro and in cell culture by chemical modification of the guideRNA.

Adenosine-to-inosine deamination can be re-addressed to user-defined mRNAs by applying phosphothioate/2'-methoxy-modified guideRNAs. Dense chemical modification of the guideRNA clearly improves performance of the covalent conjugates inside the living cell. Furthermore, careful positioning of a few modifications controls editing selectivity in vitro and was exploited for the challenging repair of the Factor 5 Leiden missense mutation.

Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA.

Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5'-UAN triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo.

Photoactivation of a psoralen-blocked luciferase gene by blue light.

A single psoralen cross-link completely blocks expression of a gene. An aminopyrene derivative has been found that allows the efficient light-triggered activation of cross-linked genes by UV or blue light. This paves the way to apply such psoralen cross-links for the photocleavable protection of nucleic acids.

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.

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.

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.

A FlAsH reporter for protein-dimerization triggers.

A FlAsH of potential: the specific binding of the biarsenical probe to the tetracysteine motif has matured as a tool for cell biology studies. Combining two such binders in one probe generates a useful reporter of protein dimerization events. The current state of art and the perspective for future developments are highlighted.

An RNA-deaminase conjugate selectively repairs point mutations.

Checking for mistakes: By conjugating a catalytic domain with a guide RNA, deamination activity can be harnessed to repair a specific codon on mRNA. This method can be used for the highly selective repair of point mutations in mRNA by site-selective editing.