A Bump-Hole Strategy for Increased Stringency of Cell-Specific Metabolic Labeling of RNA

Nguyen, Kim; Kubota, Miles; Arco, Jon Del; Feng, Chao; Singha, Monika; Beasley, Samantha; Sakr, Jasmine; Gandhi, Sunil; Blurton-Jones, Matthew; Fernández‐Lucas, Jesús; Spitale, Robert C.

doi:10.1021/acschembio.0c00755

Cited by 10 publications

(16 citation statements)

References 30 publications

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“…The potential to use this novel catalyst in advanced organic synthesis is highlighted by the fact that compounds 2r and 2s have been successfully formed from important drug molecules without affecting other functionalities. Here, 2s is a particularly interesting product as its equivalent alkyne ( 1s ) was in the top 200 highest grossing drugs in 2020 under the name Mirena, , while 1r is a component of the urology drugs Yaz and Lo Loestren. , Finally, product 2t , which is a component of an RNA biomarker as well as antiviral , and anticancer agents, was prepared in a straightforward manner. The starting material 1t was not soluble in acetonitrile, but performing the reaction in water allowed the formation of 2t in an 81% yield.…”

Section: Resultsmentioning

confidence: 99%

Designing a Green Replacement for the Lindlar Catalyst for Alkyne Semi-hydrogenation Using Silica-Supported Nickel Nanoparticles Modified by N-Doped Carbon

McNeice

Müller²,

Medlock³

et al. 2022

ACS Sustainable Chem. Eng.

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A lead-and palladium-free alternative to the Lindlar catalyst has been developed for industrially relevant alkyne semihydrogenation using silica-supported nickel nanoparticles modified by nitrogen-doped carbon. The optimal nickel catalyst acts under mild conditions (5 bar H 2 , 100 °C, and 4 h) for the selective semihydrogenation of a range of alkynes, including vitamin precursors that are important food supplements. Notably, this novel catalyst is selective for alkynes in the presence of easily reducible groups such as carbonyl and nitrile, which also makes it attractive for other synthetic applications.

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Section: Resultsmentioning

confidence: 99%

Designing a Green Replacement for the Lindlar Catalyst for Alkyne Semi-hydrogenation Using Silica-Supported Nickel Nanoparticles Modified by N-Doped Carbon

McNeice

Müller²,

Medlock³

et al. 2022

ACS Sustainable Chem. Eng.

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“…58 However, human PRTs have very low tolerance for substrate variation and cannot be used to incorporate unnatural nucleobases. This was shown by Pfefferkorn and Pfefferkorn in an early study with tritium-labeled uridine and uracil, where it was demonstrated that human cells could incorporate 3 H-uridine via nucleotide salvage pathways, but not 3 H-uracil. 59 In contrast, the parasite Toxoplasma gondii 4B), whereas unmodified HeLa cells did not.…”

Section: Cell-type-specific Rna Metabolic Labelingmentioning

confidence: 78%

“…The compatibility of the cells expressing the mutant TgUPRT and treatment with 5VU was studied by the MTT cell metabolic activity assay, and no significant difference between treated and untreated cells was observed. 3 With these results, we demonstrated cell-specific RNA metabolic labeling via the use of 5-VU and a triple-mutant TgUPRT, overcoming the nonspecific incorporation challenges faced with wild-type TgUPRT.…”

Section: Nonspecific Incorporation Of Uracil Nucleobasesmentioning

confidence: 82%

“…Therefore, we created a "hole" in the active site by replacing certain nearby amino acids with amino acids that had smaller side chains (i.e., alanine and/or glycine). 3 Single, double, and triple mutants were designed and transiently transfected into HEK293T cells. After transfection, the cells were treated with modified nucleobases (5-EU, 5-VU, 5-azidomethyluracil, or 5-azidouracil; Figure 7A), and the cellular RNA was extracted and biotinylated with appropriate "click" chemistry to analyze the incorporation of uracil analogues by dot-blot assay (Figure 7B).…”

Section: Nonspecific Incorporation Of Uracil Nucleobasesmentioning

confidence: 99%

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Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

2022

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Conspectus The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling. To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels–Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological...

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“…A major limitation with chemical approaches to track RNA expression is the inability to tag RNAs from cell types of interest. Expression of exogenous enzymes (Figure a) that, when paired with modified nucleosides/nucleobases, can enable cell-specific metabolic labeling of RNA. − However, each of these methods relies on expression of an exogenous enzyme, which requires laborious procedures to produce expression constructs; further, the utilization in living animals takes extensive time to implement. Overall, these limitations suggest additional methods need to be explored for cell-specific metabolic labeling of RNA, but may also present an opportunity for more sophisticated chemical approaches toward this problem.…”

mentioning

confidence: 99%

Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo

et al. 2022

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Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.

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A Bump-Hole Strategy for Increased Stringency of Cell-Specific Metabolic Labeling of RNA

Cited by 10 publications

References 30 publications

Designing a Green Replacement for the Lindlar Catalyst for Alkyne Semi-hydrogenation Using Silica-Supported Nickel Nanoparticles Modified by N-Doped Carbon

Designing a Green Replacement for the Lindlar Catalyst for Alkyne Semi-hydrogenation Using Silica-Supported Nickel Nanoparticles Modified by N-Doped Carbon

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo

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