The synthesis and application of a diazirine-modified uridine analogue for investigating RNA–protein interactions

Smith, Christine Catherine; Hollenstein, Marcel; Leumann, Christian J.

doi:10.1039/c4ra08682a

Cited by 21 publications

(20 citation statements)

References 101 publications

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“…6‐Iodouridine was reported as a potent inhibitor of the malaria parasite . Nucleoside derivatives are also applied in molecular probes , fluorescent surrogates , duplex stabilizing component , glucose uptake , anti‐epileptic, and identification of RNA binding proteins . Furthermore, many nucleoside analogues possessing antiviral and antitumor activities are already used clinically.…”

Section: Methodsmentioning

confidence: 99%

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Conjugation of Uridine with Oleanolic Acid Derivatives as Potential Antitumor Agents

Cheng

Huang

et al. 2016

Chem Biol Drug Des

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According to fused two bioactive moieties together by bonds covalently and available as a new single hybrid entity known as pharmacophore hybridization, a total of 10 targeted uridine-oleanolic acid hybrids were synthesized. Most of these hybrids showed excellent proliferation inhibition against tested Hep-G2, A549, BGC-823, MCF-7, and PC-3 tumor cell lines (IC50 < 8 μm), even with some IC50 values under 0.1 μm. The detection of cytotoxicity selectivity revealed that hybrids 5 and 18 exhibited low cytotoxicity toward normal human liver cell HL-7702. Further studies revealed that selected hybrid 5 could induce apoptosis in Hep-G2 cells through the investigation of acridine orange/ethidium bromide, Hoechst 33258 fluorescence stainings, and annexin V/propidium iodide assay. It was also found that hybrid 5 could induce mitochondrial membrane potential disruption, arrest Hep-G2 cell line at G1 phase, and activate effector caspase-3/9 to trigger cell apoptosis.

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Section: Methodsmentioning

confidence: 99%

“…or acyl chloride (4 equiv.) to get acyl oleanolic acid compounds (9)(10)(11)(12)(13)(14) at moderate yields of 61-89%. These acyl oleanolic acid 9-14 (1 equiv.)…”

Section: Synthesismentioning

confidence: 99%

Conjugation of Uridine with Oleanolic Acid Derivatives as Potential Antitumor Agents

Cheng

Huang

et al. 2016

Chem Biol Drug Des

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“…In this sense, tC O TP behaves better or on par with a majority of previously modified nucleotides, 35,36,[55][56][57] including bulkier triphosphate analogues. 58,59 It has been reported that the T7 RNA polymerase binds the incoming nucleotide substrate in an open conformation, 60 which could enable it to accommodate our modified base. Cell-free enzymatic incorporation of tC O into mRNA transcript…”

Section: Cell-free Enzymatic Incorporation Of Tc O Into Rnamentioning

confidence: 99%

Stealth fluorescence labeling for live microscopy imaging of mRNA delivery

Baladi

Nilsson

Gallud

et al. 2020

Preprint

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AbstractMethods for tracking of RNA molecules inside living cells are critical to probe their dynamics and biological functions, but also to monitor delivery of therapeutic RNA. We here describe a method for fluorescence labeling of RNAs of any length, via the enzymatic incorporation of the minimally perturbing and intrinsically fluorescent tricyclic cytosine analogue tCO. Using this approach, we demonstrate incorporation of tCO in up to 100% of all natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). The resulting transcript is fully compatible with both in vitro transcription and subsequent in cell translation. Spectroscopic characterization of the in vitro transcribed mRNA, shows that the incorporation rate of tCO is on par with cytosine, facilitating efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently and correctly translated into H2B:GFP upon electroporation as well as lipid-mediated transfection of human Huh-7 cells; correct translation was further confirmed in cell-free systems. Importantly, the spectral properties of the tCO-modified transcripts and their translation product, in this case H2B:GFP, allow for their straightforward and simultaneous visualization in live cells.

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“…Aryl ketones, aryl azides and diazirines are the most popular photoreactive groups used for the photo cross-linking 30 , 31 . In fact, these have been used to study the cross-linking of DNA-DNA 32 or protein-DNA/RNA complexes 33 – 36 , to probe protein-protein interactions 37 , 38 or to attach a variety of biomolecules to polymer surfaces 39 , 40 . In addition, novel click chemistry-based photo-crosslinkers (e.g., tetrazole) have proved to be very promising as alternative photo-labels 41 .…”

Section: Introductionmentioning

confidence: 99%

A versatile method for the UVA-induced cross-linking of acetophenone- or benzophenone-functionalized DNA

Jakubovska

Tauraitė

Meškys

2018

Sci Rep

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Bioconjugation, biosensing, bioimaging, bionanomaterials, etc., are only a few examples of application of functionalized DNA. Since base-modified nucleic acids contribute not only to a broad range of biotechnological fields but also to the understanding of various cellular processes, it is crucial to design novel modifications with unique properties. Here, we demonstrate the utilization of N4-cytidine modified oligonucleotides, which contain reactive acetophenone (AP) or benzophenone (BP) groups, for the UV-induced cross-linking. We find that terminal deoxynucleotidyl transferase-mediated 3′-tailing using AP/BP-containing modified nucleotides generates photoactive DNA, suitable for a straightforward covalent cross-linking with both interacting proteins and a variety of well-known solid polymeric supports. Moreover, we show that AP/BP-functionalization of nucleic acid molecules induces an efficient cross-linking upon exposure to UVA light. Our findings reveal that 3′-tailed single-stranded DNA bearing AP/BP-moieties is easily photoimmobilized onto untreated polystyrene, polypropylene, polylactate, polydimethylsiloxane, sol-gel and borosilicate glass substrates. Furthermore, we demonstrate that such immobilized DNA probes can be further used for successful hybridization of complementary DNA targets. Our results establish novel N4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids.

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The synthesis and application of a diazirine-modified uridine analogue for investigating RNA–protein interactions

Abstract: A uridine analogue equipped with a photoactive diazirine unit was generated and incorporated into RNA either syntheticallyviaphosphoramidite chemistry or by enzymatic polymerization. The new analogue was developed to identify and investigate RNA–protein interactions.

Cited by 21 publications

References 101 publications

Conjugation of Uridine with Oleanolic Acid Derivatives as Potential Antitumor Agents

Conjugation of Uridine with Oleanolic Acid Derivatives as Potential Antitumor Agents

Stealth fluorescence labeling for live microscopy imaging of mRNA delivery

A versatile method for the UVA-induced cross-linking of acetophenone- or benzophenone-functionalized DNA

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