Chemical Labeling and Affinity Capture of Inosine-Containing RNAs Using Acrylamidofluorescein

Knutson, Steve D.; Ayele, Tewoderos M; Heemstra, Jennifer M.

doi:10.1021/acs.bioconjchem.8b00541

Cited by 29 publications

(30 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In assembling reaction mixtures, we also noted improved solubility of EPhAA compared to acrylamidofluorescein, and we were able to double our normal working concentrations to ≈500 m m . As expected, this resulted in more rapid overall reaction kinetics, and when compared to our previous reagent, we observed a ≈2–3‐fold increase in conversion percentages at similar reaction times (Figure d) . Next, we incubated EPhAA with the remaining ribonucleosides uridine (U), guanosine (G), adenosine (A), and cytidine (C) (Figures d, S6).…”

Section: Methodssupporting

confidence: 70%

“…While acrylonitrile is a promising scaffold for chemical detection of inosine, derivatizing these reagents is difficult and requires several synthetic and purification steps. Alternatively, we recently reported an acrylamidofluorescein reagent that enables fluorescent detection and enrichment of inosine in RNA . While our initial study demonstrated feasibility, acrylamidofluorescein also displayed poor solubility and was restricted to fluorescein addition.…”

Section: Methodsmentioning

confidence: 90%

See 1 more Smart Citation

Chemical Profiling of A‐to‐I RNA Editing Using a Click‐Compatible Phenylacrylamide

Knutson

Korn

Johnson

et al. 2020

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

Straightforwardm ethods for detecting adenosine-to-inosine (A-to-I) RNA editinga re key to ab etter understanding of its regulation, function, and connection with disease. Wea ddress this need by developing an ovel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in as ingle step, reacts rapidlyw ith inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probesa te diting sites. We first validate EPhAA reactivity and selectivityf or inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinantA DAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides ac ost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings. RNA is chemically modified by anumber of enzymes after transcription, in turn influencing RNA stability, localizationa nd activity within the cell. Adenosine-to-inosine (A-to-I) RNA editing is one of the most widespread modifications, and is performed by adenosine deaminases acting on RNA (ADARs) (Scheme 1a). [1] Adenosine deaminationc hanges the molecular structurea nd hydrogen-bonding pattern of the nucleobase, and resulting inosines insteadb ase pairw ith cytidinet oe ffectively recode these sites as guanosine. Editings ites within protein-coding mRNAsd irectly alter amino acid sequences and produce different protein isoforms. Non-coding RNAs also undergo extensive editing, including microRNAs and small-interfering RNAs, significantly alteringt heir biosynthesis, localization, and gene regulation properties. [2-3] A-to-I editing is essential for an umber of biological processes including tissue development, [4-5] neurologicalf unction, [6] and immune system activation. [7] Dysfunctional editing is also directlyl inked with autoimmune diseases, [8-9] neurological disorders, [10] and several types of cancer. [11-12] Despite this importance,o ur overall understanding of A-to-I editingr egulation is limited. In particular,w hile many sites have been identified (> 5million), [13-14] it is unclear why certain sites are edited at higherf requency than others and what precise function they each serve. [15] Efforts to map A-to-I locations and ADAR binding sites have revealed that editingp atterns are highly complex and variable in humans, [7, 16-18] and the precise mechanismsb yw hich ADAR enzymes bind to and edit specific RNA sequences remain unclear.T his gap is also significant for therapeutic site-directed RNA editing strategies, [19] as both the design and precise implementation of this machinery is reliant on at horough understanding of ADAR regulation. Detecting inosine formation in RNA is of central importance for characterizing editingm echanisms. While high-throughput RNA sequencin...

show abstract

Section: Methodssupporting

confidence: 70%

Section: Methodsmentioning

confidence: 90%

Chemical Profiling of A‐to‐I RNA Editing Using a Click‐Compatible Phenylacrylamide

Knutson

Korn

Johnson

et al. 2020

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

show abstract

“…Another inosine labeling approach uses bulky acrylamidofluorescein for selective N1-inosine derivatization. Such labeling does not require subsequent reactions for affinity capture with specific antibodies and it can be used for the comprehensive transcriptome-wide analysis of A-to-I editing [ 43 ].…”

Section: Analysis Of Rna Modifications By Ngsmentioning

confidence: 99%

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

Motorin

Marchand

2021

Genes

View full text Add to dashboard Cite

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.

show abstract

“…It has been known for several decades that Michael acceptors, in particular acrylonitrile, can react with N 1 on inosine to form a stable adduct on the Watson‐Crick‐Franklin face (Yoshida & Ukita, 1965). We and others have applied this chemistry toward the design of inosine‐enrichment reagents; however, these compounds also reacted with pseudouridine and uridine, which overall limited their enrichment potential (Knutson, Ayele, & Heemstra, 2018; Li, Göhl, Ke, Vanderwal, & Spitale, 2019). In search of improved methods to address this enrichment problem we were inspired by Endonuclease V (EndoV), an evolutionary conserved repair enzyme that recognizes and cleaves inosine in nucleic acids.…”

Section: Commentarymentioning

confidence: 99%

EndoVIPER‐seq for Improved Detection of A‐to‐I Editing Sites in Cellular RNA

Knutson

Heemstra

2020

CP Chemical Biology

Self Cite

View full text Add to dashboard Cite

Adenosine to-inosine (A-to-I) RNA editing is a conserved post-transcriptional modification that is critical for a variety of cellular processes. A-to-I editing is widespread in nearly all types of RNA, directly imparting significant global changes in cellular function and behavior. Dysfunctional RNA editing is also implicated in a number of diseases, and A-to-I editing activity is rapidly becoming an important biomarker for early detection of cancer, immune disorders, and neurodegeneration. While millions of sites have been identified, the biological function of the majority of these sites is unknown, and the regulatory mechanisms for controlling editing activity at individual sites is not well understood. Robust detection and mapping of A-to-I editing activity throughout the transcriptome is vital for understanding these properties and how editing affects cellular behavior. However, accurately identifying A-to-I editing sites is challenging because of inherent sampling errors present in RNA-seq. We recently developed Endonuclease V immunoprecipitation enrichment sequencing (EndoVIPER-seq) to directly address this challenge by enrichment of Ato-I edited RNAs prior to sequencing. This protocol outlines how to process cellular RNA, enrich for A-to-I edited transcripts with EndoVIPER pulldown, and prepare libraries suitable for generating RNA-seq data.

show abstract

Chemical Labeling and Affinity Capture of Inosine-Containing RNAs Using Acrylamidofluorescein

Cited by 29 publications

References 25 publications

Chemical Profiling of A‐to‐I RNA Editing Using a Click‐Compatible Phenylacrylamide

Chemical Profiling of A‐to‐I RNA Editing Using a Click‐Compatible Phenylacrylamide

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

EndoVIPER‐seq for Improved Detection of A‐to‐I Editing Sites in Cellular RNA

Contact Info

Product

Resources

About