Mutually Orthogonal Bioconjugation of Vinyl Nucleosides for RNA Metabolic Labeling

Gupta, Mrityunjay; Singha, Monika; Rasale, Dnyaneshwar B.; Zhou, Zehao; Bhandari, Srijana; Beasley, Samantha; Sakr, Jasmine; Parker, Shane M.; Spitale, Robert C.

doi:10.1021/acs.orglett.1c02584

Cited by 8 publications

(14 citation statements)

References 14 publications

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“…The chemistry remained orthogonal with RNA, where 5-VUrd-containing RNA reacted specifically with maleimide−biotin and 2-VAdo-containing RNA reacted specifically with TCEP (Figure 9C). 4 We demonstrated the use of these reactions for fixed-cell imaging, where metabolically labeled cells were tagged with TCEP-and maleimidelinked fluorophores (Figure 9D). These reactions provide not only metal-free, RNA-compatible conjugation conditions but also a potential method for the simultaneous use of two vinyl nucleosides for RNA metabolic labeling.…”

Section: Orthogonal Conjugation Of Vinyl Nucleosidesmentioning

confidence: 97%

“…1 While both nucleosides contain a terminal alkene, the electronics of these alkenes are quite different. 4 The alkene in 2-VAdo is highly delocalized with extended conjugation to the adenine ring and is a part of an electrondeficient α,β-unsaturated imine. In contrast, the terminal alkene of 5-VUrd has limited delocalization with the internal alkene, forming a diene.…”

Section: Orthogonal Conjugation Of Vinyl Nucleosidesmentioning

confidence: 99%

“…We reasoned that 5-VUrd could react as a diene in a [4 + 2] cycloaddition reaction with a dienophile such as maleimide, while the 2-VAdo could react as a Michael acceptor with a phosphine nucleophile such as tris(2carboxyethyl)phosphine (TCEP) (Figure 9A,B). 4 We performed these reactions in D 2 O and observed high conversion and rapid reactions in both cases via 1 H NMR spectroscopy. These reactions were also mutually orthogonal: 2-VAdo did not react as a diene with maleimide, and 5-VUrd did not react as a Michael acceptor with TCEP.…”

Section: Orthogonal Conjugation Of Vinyl Nucleosidesmentioning

confidence: 99%

“…C. Gupta, M. Singha, M. Rasale, D. B. Zhou, Z. Bhandari, S. Beasley, S. Sakr, J. Parker, S. M. Spitale, R. C. Org. Lett.20212371837187 …”

Section: Key Referencesmentioning

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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Section: Orthogonal Conjugation Of Vinyl Nucleosidesmentioning

confidence: 97%

Section: Orthogonal Conjugation Of Vinyl Nucleosidesmentioning

confidence: 99%

Section: Orthogonal Conjugation Of Vinyl Nucleosidesmentioning

confidence: 99%

“…C. Gupta, M. Singha, M. Rasale, D. B. Zhou, Z. Bhandari, S. Beasley, S. Sakr, J. Parker, S. M. Spitale, R. C. Org. Lett.20212371837187 …”

Section: Key Referencesmentioning

confidence: 99%

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Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

2022

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“…Isolating and profiling RNA molecules from specified cells is a long-standing challenge in molecular biology. Metabolic labeling using nucleosides endowed with several types of chemical functional groups such as alkyne, azide, and more recently vinyl groups enable tracking and analysis by fluorescence or enrichment (Figure a). − …”

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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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Cyclopropene als Chemische Reporter für die Duale Bioorthogonale und Orthogonale Metabolische Markierung von DNA

Seul,

Lamade,

Stoychev

et al. 2024

Angewandte Chemie

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Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross‐reactivity. We developed a pair of two differently modified 2’‑deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse‐electron demand Diels‐Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1‐ and 3‐methylcyclopropenyl‐modified 2’‑deoxynucleosides, two 2’‐deoxyuridines were identified to be the fastest‐reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2‘‑deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2’‐deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine‐styryl tetrazine and a fluorogenic pyrene‐tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments.

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Mutually Orthogonal Bioconjugation of Vinyl Nucleosides for RNA Metabolic Labeling

Cited by 8 publications

References 14 publications

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

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

Cyclopropene als Chemische Reporter für die Duale Bioorthogonale und Orthogonale Metabolische Markierung von DNA

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