Cell-Selective Bioorthogonal Metabolic Labeling of RNA

Nguyen, Kim; Fazio, Michael; Kubota, Miles; Nainar, Sarah; Feng, Chao; Li, Xiang; Atwood, Scott X.; Bredy, Timothy W.; Spitale, Robert C.

doi:10.1021/jacs.6b11401

Cited by 42 publications

(70 citation statements)

References 27 publications

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“…The time of incubation of un-“caged” 3 are similar to the results we have observed with UPRT-dependent RNA labelling with 5EU. 12 These results further demonstrate the ability of 5EU to be caged and efficiently liberated by light exposure. Further, they support the notion that once 5EU is liberated it is incorporated into cellular RNA with similar kinetics of just 5EU.…”

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confidence: 60%

“…After 5EU incubation, the majority of signal comes from the nucleolus, consistent with our previous reports. 12 Incubation of UPRT expressing cells with 3 alone resulted in no cellular imaging signals (Fig. 3E).…”

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confidence: 96%

“…One example is the liberation of a protected 2′-azidoadenosine by the enzyme penicillin G amidase (PGA). 12 The key observation from these studies is that modified nucleoside intermediates can be chemically controlled by functional group placement. These intermediates can remain inert until being transformed into a metabolic-active form by functional group removal.…”

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confidence: 99%

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Photo-controlled cell-specific metabolic labeling of RNA

Feng

Spitale

2017

Org. Biomol. Chem.

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Elucidating gene expression programs within a cell-specific manner is a grand challenge for biologists. Harder still is the abilty to have kinetic control over such experiments. Metabolic labeling with bioorthogonally-functionalized metabolic intermediates provides a means to profile RNA expression in a cell-specific manner, but there is still lack of kinetic resolution. Herein we present the synthesis and evaluation of photocaged metabolic uracil intermediates. We compare the photo-decaging properties and demonstrate their utility in metabolic labeling experiments in a cell-specific manner. We anticipate our approach will have far-reaching impact as they provide control over tagging of nascent RNA.

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confidence: 60%

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confidence: 96%

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confidence: 99%

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Photo-controlled cell-specific metabolic labeling of RNA

Feng

Spitale

2017

Org. Biomol. Chem.

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“…Bioorthogonal nucleotide precursors with RNA polymerase selectivity or different chemical reactivities could expand the types of information obtained by biosynthetic RNA tagging (Nainar et al, ). Promising alternative means of achieving cell type specificity include targeted expression of penicillin G amidase (this enzyme “uncages” a 2′‐azidoadenosine nucleoside that is subsequently added to poly(A) tails via cytoplasmic poly(A) polymerase) and photoregulated uncaging of 5‐ethynyluracil (Feng, Li, & Spitale, ; Nguyen et al, ). Additional specificity may be achieved through reconstitution of functional CD from independently expressed fragments.…”

Section: Discussionmentioning

confidence: 99%

Uncovering cell type‐specific complexities of gene expression and RNA metabolism by TU‐tagging and EC‐tagging

Cleary

2018

WIREs Developmental Biology

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Cell type-specific transcription is a key determinant of cell fate and function. An ongoing challenge in biology is to develop robust and stringent biochemical methods to explore gene expression with cell type specificity. This challenge has become even greater as researchers attempt to apply high-throughput RNA analysis methods under in vivo conditions. TU-tagging and EC-tagging are in vivo biosynthetic RNA tagging techniques that allow spatial and temporal specificity in RNA purification. Spatial specificity is achieved through targeted expression of pyrimidine salvage enzymes (uracil phosphoribosyltransferase and cytosine deaminase) and temporal specificity is achieved by controlling exposure to bioorthogonal substrates of these enzymes (4-thiouracil and 5-ethynylcytosine). Tagged RNAs can be purified from total RNA extracted from an animal or tissue and used in transcriptome profiling analyses. In addition to identifying cell type-specific mRNA profiles, these techniques are applicable to noncoding RNAs and can be used to measure RNA transcription and decay. Potential applications of TU-tagging and EC-tagging also include fluorescent RNA imaging and selective definition of RNA-protein interactions. TU-tagging and EC-tagging hold great promise for supporting research at the intersection of RNA biology and developmental biology. This article is categorized under: Technologies > Analysis of the Transcriptome.

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“…Other uses of click-chemistry have been to develop better labelling of RNA in vitro and in vivo to better profile RNA populations in distinct cellular populations. 12–13 Furthermore, click chemistry has also been shown to be an effective tool to analyze the transport of stem cells to different locations 14–15 and has been used to understand the molecular dynamics and potential heterogeneity in drug activity in vivo 16 .…”

Section: Emerging Targets In Gene Therapy and Use Of Synthetic Biomentioning

confidence: 99%

“Clicking” Gene Therapeutics: A Successful Union of Chemistry and Biomedicine for New Solutions

et al. 2018

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The use of nucleic acid, DNA and RNA, based strategies to disrupt gene expression as a therapeutic is quickly emerging. Indeed, synthetic oligonucleotides represent a major component of emerging gene therapeutics. However, the efficiency and specificity of intracellular uptake for non-modified oligonucleotides is rather poor. Utilizing RNA based oligonucleotides as therapeutics is even more challenging to deliver, due to extremely fast enzymatic degradation of the RNAs. Like unmodified oligonucleotides, RNAs also get rapidly degraded in vivo and demonstrate large off-target binding events when they can reach and enter the desired target cells. One approach that holds much promise is the utilization of “click chemistry” to conjugate receptor or cell specific targeting molecules directly to the effector oligonucleotides. We discuss here the applications of the breakthrough technology of CuAAC “click-chemistry” and the immense potential in utilizing “click chemistry” in the development of new age targeted oligonucleotide therapeutics.

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Cell-Selective Bioorthogonal Metabolic Labeling of RNA

Cited by 42 publications

References 27 publications

Photo-controlled cell-specific metabolic labeling of RNA

Photo-controlled cell-specific metabolic labeling of RNA

Uncovering cell type‐specific complexities of gene expression and RNA metabolism by TU‐tagging and EC‐tagging

“Clicking” Gene Therapeutics: A Successful Union of Chemistry and Biomedicine for New Solutions

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