Direct Immunodetection of Global A‐to‐I RNA Editing Activity with a Chemiluminescent Bioassay

Knutson, Steve D.; Arthur, Robert A.; Johnston, H. Richard; Heemstra, Jennifer M.

doi:10.1002/anie.202102762

Cited by 11 publications

(19 citation statements)

References 73 publications

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“…Thus, we developed a plate-based assay termed the EndoV-linked immunosorbent assay (EndoVLISA) which is closely related to typical ELISAs with only a few modifications to the workflow. 4 In this bioassay format, cellular mRNA is glyoxalated and biotinylated and then conjugated to a streptavidin-coated multiwell plate (Figure 6a). The immunodetection of inosine is then possible using EndoV and a secondary antibody having a reporter enzyme which provides a chemiluminescent signal to quantify the inosine content.…”

Section: Proteins and Enzymesmentioning

confidence: 99%

“…This application was of particularly high interest because global A-to-I editing levels can be indicative of a disease state, yet convenient and high-throughput methods to quantify overall inosine content were lacking. Thus, we developed a plate-based assay termed the EndoV-linked immunosorbent assay (EndoVLISA) which is closely related to typical ELISAs with only a few modifications to the workflow . In this bioassay format, cellular mRNA is glyoxalated and biotinylated and then conjugated to a streptavidin-coated multiwell plate (Figure a).…”

Section: Repurposing Naturally Occurring Proteins and Enzymesmentioning

confidence: 99%

Section: Conclusion and Future Outlookmentioning

confidence: 99%

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Harnessing Nature’s Molecular Recognition Capabilities to Map and Study RNA Modifications

et al. 2022

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Conspectus RNA editing or “epitranscriptomic modification” refers to the processing of RNA that occurs after transcription to alter the sequence or structure of the nucleic acid. These chemical alterations can be found on either the ribose sugar or the nucleobase, and although many are “silent” and do not change the Watson–Crick–Franklin code of the RNA, others result in recoding events. More than 170 RNA modifications have been identified so far, each having a specific biological purpose. Additionally, dysregulated RNA editing has been linked to several types of diseases and disorders. As new modifications are discovered and our understanding of their functional impact grows, so does the need for selective methods of identifying and mapping editing sites in the transcriptome. The most common methods for studying RNA modifications rely on antibodies as affinity reagents; however, antibodies can be difficult to generate and often have undesirable off-target binding. More recently, selective chemical labeling has advanced the field by offering techniques that can be used for the detection, enrichment, and quantification of RNA modifications. In our method using acrylamide for inosine labeling, we demonstrated the versatility with which this approach enables pull-down or downstream functionalization with other tags or affinity handles. Although this method did enable the quantitative analysis of A-to-I editing levels, we found that selectivity posed a significant limitation, likely because of the similar reactivity profiles of inosine and pseudouridine or other nucleobases. Seeking to overcome the inherent limitations of antibodies and chemical labeling methods, a more recent approach to studying the epitranscriptome is through the repurposing of proteins and enzymes that recognize modified RNA. Our laboratory has used Endonuclease V, a repair enzyme that cleaves inosine-containing RNAs, and reprogrammed it to instead bind inosine. We first harnessed EndoV to develop a preparative technique for RNA sequencing that we termed EndoVIPER-seq. This method uses EndoV to enrich inosine-edited RNAs, providing better coverage in RNA sequencing and leading to the discovery of previously undetected A-to-I editing sites. We also leveraged EndoV to create a plate-based immunoassay (EndoVLISA) to quantify inosine in cellular RNA. This approach can detect differential A-to-I editing levels across tissue types or disease states while being independent of RNA sequencing, making it cost-effective and high-throughput. By harnessing the molecular recognition capabilities of this enzyme, we show that EndoV can be repurposed as an “anti-inosine antibody” to develop new methods of detecting and enriching inosine from cellular RNA. Nature has evolved a plethora of proteins and enzymes that selectively recognize and act on RNA modifications, and exploiting the affinity of these biomolecules offers a promising new direction for the field of epitranscriptomics.

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Section: Proteins and Enzymesmentioning

confidence: 99%

Section: Repurposing Naturally Occurring Proteins and Enzymesmentioning

confidence: 99%

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Harnessing Nature’s Molecular Recognition Capabilities to Map and Study RNA Modifications

et al. 2022

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“…Chemiluminescent (CL) immunoassays have been extensively employed in many areas including disease diagnosis, environmental monitoring, and drug safety owing to their high sensitivity, ideal selectivity, wide dynamic range, and simple instrument. − Easy denaturation and unsatisfactory cost-effectiveness of natural enzymes such as alkaline phosphatase, horseradish peroxidase, and laccase enormously restrain their application in CL immunoassays. − Alternatively, as substitutes of natural enzymes, many nanomaterial-based mimicking enzymes possess intrinsic catalytic activity toward CL reactions. − Unfortunately, insufficient catalytic efficiency of these nanozymes resulting from bulk active centers cannot meet the acquirement of practicability in CL immunoassays. , Thus, it is necessary to noticeably improve the catalytic efficiency of nanozymes for highly sensitive CL platforms.…”

Section: Introductionmentioning

confidence: 99%

Emitter–Quencher Pair of Single Atomic Co Sites and Monolayer Titanium Carbide MXenes for Luminol Chemiluminescent Reactions

Ouyang

Xian

Luo

et al. 2021

ACS Appl. Mater. Interfaces

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A facile, one-step doping protocol was adopted to synthesize Co single atomic site catalysts (SASCs) in UiO-66 metal–organic frameworks. In view of highly uniform active sites of Co–O6 moieties, the SASCs specifically contribute to catalyzing the generation of a large amount of singlet oxygen instead of superoxide or hydroxyl radicals, which endows Co SASCs with a the remarkable enhancement effect (∼3775 times) on luminol chemiluminescent (CL) emission. Interestingly, monolayer titanium carbide MXenes can drastically quench the CL signal of the Co SASC-boosted luminol reaction by ∼94.6% as highly efficient luminescent absorbents. Furthermore, the emitter–quencher pair of Co SASCs and titanium carbide MXenes was successfully adopted to develop an immunoassay method for cardiac troponin I (cTnI) on an immunochromatographic test strip platform. With a sandwich immunoreaction mode, a titanium carbide MXene-labeled cTnI tracer antibody was captured on the test line of a test strip, which significantly inhibited the CL response of the Co SACs-boosted luminol system. The dynamic range for quantitating cTnI is 1.0–100 pg mL–1, with a detection limit of 0.33 pg mL–1 (3σ). The test strip was successfully used to detect cTnI in human serum samples collected from cardiopathy patients. This proof-of-principle work manifests both the CL enhancement of SASCs and the quenching behavior of MXenes, which shows the thrilling prospects of combinational usage of the two functionalized nanomaterials for tracking biological recognition events.

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“…Chemiluminescent immunoassay (CLIA) exhibited many superiorities including facile operations, simple instruments, wide dynamic range, high sensitivity, and ideal specificity. Thus, it has been employed for a variety of purposes including food quality control, clinic diagnosis, environmental monitoring, etc. − Since natural enzymes possess thrilling catalysis efficiency on diverse CL reactions, they have long been widely employed as powerful signal tracers for establishing various CLIA protocols. − Unfortunately, CL reactions that involved enzymes inevitably suffer from the intrinsic shortcomings including limited sources, high cost, and poor stability, which dramatically hamper their practicability on CLIA techniques. − …”

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confidence: 99%

Highly Sensitive Chemiluminescent Immunoassay of Mycotoxins Using ZIF-8-Derived Yolk-Shell Co Single-Atom Site Catalysts as Superior Fenton-like Probes

et al. 2022

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Superior to traditional nanoscale catalysts, single-atom site catalysts (SASCs) show such merits as maximal catalysis efficiency and outstanding catalytic activity for the construction of analytical methodological platforms. Hereby, an in situ etching strategy was designed to prepare yolk-shell Co SASCs derived from ZIF-8@SiO2 nanoparticles. On the basis of direct chemical interactions between precursors and supports, the Co element with isolated atomic dispersion was anchored on ZIF-8@SiO2 nanoparticles. The Co SASCs possess high Fenton-like activity and thus can catalyze the decomposition of H2O2 to produce massive superoxide radical anions instead of singlet oxygen and hydroxyl radicals. With the activity for producing superoxide radical anion, Co SASCs can greatly improve the chemiluminescent (CL) response of a luminol system by 3133.7 times. Furthermore, the SASCs with active sites of Co–O5 moieties were utilized as the CL probes for establishment of an immunoassay method for sensitive detection of mycotoxins by adopting aflatoxin B1 as a mode analyte. The quantitation range is 10–1000 pg/mL, and the limit of detection is 0.44 pg/mL (3σ) for aflatoxin B1. The proof-of-principle work elucidates the practicability of direct chemical interactions between precursors and supports for forming SASCs with ultrahigh CL response, which can be extended to the exploitation of more sorts of SASCs for tracing biological binding events.

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Direct Immunodetection of Global A‐to‐I RNA Editing Activity with a Chemiluminescent Bioassay

Cited by 11 publications

References 73 publications

Harnessing Nature’s Molecular Recognition Capabilities to Map and Study RNA Modifications

Harnessing Nature’s Molecular Recognition Capabilities to Map and Study RNA Modifications

Emitter–Quencher Pair of Single Atomic Co Sites and Monolayer Titanium Carbide MXenes for Luminol Chemiluminescent Reactions

Highly Sensitive Chemiluminescent Immunoassay of Mycotoxins Using ZIF-8-Derived Yolk-Shell Co Single-Atom Site Catalysts as Superior Fenton-like Probes

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