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Chemo‐enzymatic treatment of RNA to facilitate analyses

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Abstract Labeling RNA is a recurring problem to make RNA compatible with state‐of‐the‐art methodology and comes in many flavors. Considering only cellular applications, the spectrum still ranges from site‐specific labeling of individual transcripts, for example, for live‐cell imaging of mRNA trafficking, to metabolic labeling in combination with next generation sequencing to capture dynamic aspects of RNA metabolism on a transcriptome‐wide scale. Combining the specificity of RNA‐modifying enzymes with non‐natural substrates has emerged as a valuable strategy to modify RNA site‐ or sequence‐specifically with functional groups suitable for subsequent bioorthogonal reactions and thus label RNA with reporter moieties such as affinity or fluorescent tags. In this review article, we will cover chemo‐enzymatic approaches (a) for in vitro labeling of RNA for application in cells, (b) for treatment of total RNA, and (c) for metabolic labeling of RNA. This article is categorized under: RNA Processing < RNA Editing and Modification RNA Methods < RNA Analyses in vitro and In Silico RNA Methods < RNA Analyses in Cells
Schematic overview of a select set of most commonly employed click reactions, sorted according to their second order rate constants (k2 [M−1s−1]). (a) Copper‐catalyzed azide‐alkyne cycloaddition (CuAAC), exhibiting rate constants from 10–200 M−1s−1 (Lang & Chin, ). (b) Reaction scheme of the strain‐promoted azide‐alkyne cycloaddition (SPAAC, upper panel) and examples for recently described cyclooctynes used in SPAAC reactions (lower panel). Fastest cyclooctynes such as aza‐dibenzocyclooctyne (DIBAC) or biarylazacyclooctynone (BARAC) show rate constants from 0.1–4 M−1s−1 (Lang & Chin, ). (c) Reaction scheme of the inverse‐electron demand Diels‐Alder cycloaddition (iEDDA, upper panel) and examples of dienophiles (lower panel). For iEDDA reactions, reaction rates generally increase from norbornenes, methylcyclopropenes, bicyclononynes to trans‐cyclooctenes and range from 10–106 M−1s−1 (Blackman, Royzen, & Fox, ; Kamber et al., ; Lang et al., ; Schoch, Staudt, Samanta, Wiessler, & Jäschke, ; Taylor, Blackman, Dmitrenko, & Fox, ; Vrabel et al., ). [4 + 2] DA, [4 + 2] Diels‐Alder‐reaction; [4 + 2] retro‐DA, [4 + 2] retro‐Diels‐Alder reaction
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Metabolic labeling with the amino acid propargyl‐selenohomcysteine (PSH) as representative for methionine analogs. (a) The wildtype enzyme methionine adenosyltransferase (MAT) uses PSH and ATP to generate the AdoMet analog SeAdoYn that is used by many MTases as cosubstrate. Here transfer of the propargyl group to RNA is depicted schematically. (b) Mammalian cells take up PSH leading to intracellular generation of SeAdoYn and transfer of propargyl groups to RNA. After isolation of total RNA, the MTase targets can be enriched via CuAAC reaction with biotin azide. The enriched sequences can be found in NGS (Hartstock et al., )
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Metabolic RNA labeling in combination with photo‐crosslinking to facilitate analyses of RNA‐binding proteins (RBPs) or sequences interacting with them. The mRNA‐protein interactions are preserved by employing either UV cCL or PAR‐CL protocols on mammalian cells. (a) Metabolic labeling with EU in combination with photo‐crosslinking enables to isolate the interactome of newly transcribed RNAs (Bao et al., ). (b) Metabolic labeling with 4SU allows photo‐crosslinking under milder conditions at 365 nm. mRNA–protein complexes are isolated by pull‐down with oligo(dT) magnetic beads, stringently washed, and then bound proteins are eluted with RNase and identified by MS
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Mechanism of m5C‐RNA‐MTases and Aza‐IP. (a) m5C‐RNA‐MTases have a cysteine in the active site and form a covalent enzyme‐substrate intermediate by attacking the C6 of the target cytidine. After transfer of a methyl group from the cosubstrate SAM to the C5, the enzyme, m5C and the coproduct S‐adenosylhomocysteine (SAH) are released. (b) Concept of 5‐azacytidine‐mediated RNA immunoprecipitation (Aza‐IP). 5‐azacytidine (5‐aza‐C) is a mechanism‐based suicide inhibitor that traps the enzyme by forming a stable RMTRNA adduct. The enzyme can be immunoprecipitated and the covalently bound target RNA can be isolated and sequenced to identify target sites of this specific m5C‐RNA‐MTase (Khoddami & Cairns, )
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Enzyme and tissue‐specific metabolic labeling via TU and 5EC (Miller et al., )
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Thiol‐selective chemistry for derivatization of 4SU. (a) HPDP‐bio leads to reversible formation of a disulfide bond between 4SU and biotin (bio‐4SU). (b) MTS‐bio is methane thiosulfonate‐biotin, an activated disulfide for more efficient biotinylation of 4SU. (c) The MTS‐reagent can also be tethered directly to a solid support (gray ball). (d) Iodacetamide modifies 4SU and is the basis of SLAM seq, where modified 4SUs in RNA lead to mutations in the sequencing readout. (e) 4SU is directly converted to cytosine (C) by osmium tetroxide (OsO4) and ammonia before sequencing in TUC‐seq. (f) 4SU is mostly converted to the trifluoroethylated cytidine using 2,2,2‐trifluorethylamine (TFEA) in combination with meta‐chloroperoxybenzoic acid (mCPBA) or sodium periodate (NaIO4). The trifluoroethylated cytidine is the basis of TimeLapse‐sequ and also results in mutations in the sequencing readout at sites originally containing 4SU. (a–c) Are used for enrichment of 4SU‐containing RNA. (d–f) Are used for RT, library preparation and NGS where 4SU‐containing sites are detected as mutations
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Metabolic labeling with alkyne‐containing nucleoside analogs for subsequent CuAAC. (a) Co‐transcriptional incorporation of 5‐ethynyl‐uridine (EU) by all three polymerases allows labeling of all nascent transcripts. (b) 2‐Ethynyladenosine (EA) feeding in combination with inhibition of transcription (by actinomycin D) allows labeling of poly(A) tails for imaging or enrichment and subsequent analysis
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Nucleoside and methionine analogs used for co‐ and posttranscriptional metabolic labeling. EU, 5‐ethynyl‐uridine; 4SU, 4‐thiouridine; 5‐aza‐C, 5‐aza‐cytidine; PSH, propargyl‐selenohomocysteine; N6pA, N6‐propargyl‐adenosine; EA, 2‐ethynyladenosine; s6G, 6‐thioguanosine; TU, 4‐thiouracil; DTU, 2,4‐dithiouracil; 5EC, 5‐ethynylcytosine; 6‐TG, 6‐thioguanine
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Scheme for enrichment and detection of specific RNAs or RNA modifications from total RNA. (a) NAD+ captureSeq: Trans‐glycosylation of nicotinamide adenine dinucleotide (NAD+) modified RNA by adenosine diphosphate ribosyl cyclase (ADPRC) with an alkynyl alcohol in total RNA. The introduced alkynyl group can be conjugated in a click reaction to introduce a biotin label for subsequent enrichment and determination of NAD+‐capped RNA. (b) Identification of N6‐methyladenosine (m6A): Incorporation of non‐natural N6‐modifications at free METTL3‐METTL14 target sites using AdoMet analogs. The introduced modification can be used to either perform cyclization via iodination or to add a biotin label via CuAAC for subsequent enrichment. After reverse transcription (RT) the introduced modifications result in a specific RT pattern (either termination or mutation) which allows to identify the modified position in sequencing. (c) Identification of pseudouridine (Ψ): Treatment with N3‐CMC installs a clickable moiety on N3 of many pyrimidines and N1 of many purines. Alkaline treatment removes all moieties which are not installed on the Ψ. Remaining moieties are further functionalized with biotin via click reaction, enabling enrichment. RT of the treated sample results in a specific termination pattern while untreated samples result in full length product enabling the detection of pseudouridines. (d) Identification of N1‐methyl adenosine (m1A): Alkaline treatment of m1A triggers Dimroth rearrangement resulting in the formation of m6A. RT of the untreated sample results in a specific termination pattern while rearranged samples result in full length product enabling the detection of m1A
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Schematic overview of various covalent modifications altering the properties of RNA. (a) Enzymatic installation of photo‐caging groups onto TGT recognition sites in the 5′‐UTR by TGT. Upon transfection and irradiation, translation can be turned on. (b) Enzymatic installation of various functional moieties at the 5′‐cap N7‐position by Ecm1 allow for tuning of translational efficiency of transfected RNA. (c) Installation of azido‐ATP at the 3′‐end by the poly(A)‐polymerase renders RNA more translationally active in cells. Translation efficiency is further increased, if a fluorophore label is installed in a click reaction. (d) Production of RNA equipped with photo‐cross‐linking groups at the 5′‐cap for capturing of 5′‐cap‐protein interactions through irradiation. Groups can be installed enzymatically by Ecm1 or incorporated by in vitro transcription from a corresponding cap analog. (e) Incorporation of phosphorthiolate‐modified cap‐analogs renders in vitro produced RNA resistant against Dcp2‐mediated decapping while also stimulating translation
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Chemo‐enzymatic labeling strategies for RNA in fixed and/or living cells. (a) TGT from Escherichia coli transfers fluorescent PreQ1 analogs onto a small 17 nt hairpin motif allowing for labeling in fixed cells. (b) Poly(A)‐polymerase transfers azido‐ATP onto the 3′‐end of mRNA. This strategy allowed for live and fixed cell labeling of RNA. (c) Methyltransferase (MTase) based labeling. MTases GlaTgs and Ecm1 site‐specifically modify the 5′‐cap. Here, labeling in fixed and living cells has been achieved. CD small RNP RNA 2′‐O‐MTase directs template‐dependent sequence‐specific internal modification. HEN1 MTase from Drosophila melanogaster modifies the 3′‐end of all RNAs. (d) The tRNA MTase TRM1 modifies an internal position of tRNAPhe. (e) HEN1 MTase from Arabidopsis thaliana modifies the 3′‐termini of dsRNA
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Covalent RNA labeling strategies based on nucleotide analogs and their metabolic or synthetic precursors. (a) During solid‐phase synthesis, fluorescent or functionalized nucleoside phosphoramidites are incorporated into RNA. RNA is either directly fluorescent or reacts with a reporter molecule in a subsequent click reaction after transfection. (b) Functionalized nucleotides are incorporated into RNA during in vitro transcription. After transfection, RNA reacts in a click reaction with a reporter molecule. (c) Metabolic labeling: Cells are fed with nucleosides. In cellula produced NTP analogs are then incorporated by all three RNA polymerases into nascent RNA, which can then be reacted with a reporter molecule in a click reaction
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RNA Processing > RNA Editing and Modification
RNA Methods > RNA Analyses In Vitro and In Silico
RNA Methods > RNA Analyses in Cells

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