Evaluation of Parameters Critical for Observing Nucleic Acids Inside Living <i>Xenopus laevis</i> Oocytes by In-Cell NMR Spectroscopy

Hänsel, Robert; Foldynová-Trantírková, Silvie; Löhr, Frank; Buck, Janina; Bongartz, Eva; Bamberg, Ernst; Schwalbe, Harald; Dötsch, Volker; Trantı́rek, Lukáš

doi:10.1021/ja9052027

Cited by 100 publications

(112 citation statements)

References 64 publications

(102 reference statements)

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“…To evaluate the stability of i‐motifs formed from naturally occurring C‐rich sequences from the human genome in a complex intracellular environment, we employed in‐cell NMR spectroscopy6 of DNA in living human cells. Four oligonucleotide constructs, corresponding to the i‐motif forming sequences from DAP, HIF‐1α, PDGF‐A, and JAZF1 promoter regions7, 8 from the human genome (see Table 1) and displaying the capacity to form i‐motif structures in vitro under near‐physiological conditions including ionic strength and near‐neutral pH,7, 8 were selected for the in‐cell NMR study.…”

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confidence: 99%

Evaluation of the Stability of DNA i‐Motifs in the Nuclei of Living Mammalian Cells

et al. 2018

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C‐rich DNA has the capacity to form a tetra‐stranded structure known as an i‐motif. The i‐motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i‐motif structures form in complex environment of living cells is not currently known. Herein, using state‐of‐the‐art in‐cell NMR spectroscopy, we evaluate the stabilities of i‐motif structures in the complex cellular environment. We show that i‐motifs formed from naturally occurring C‐rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i‐motif stabilities in vivo are generally distinct from those in vitro. Our results are the first to interlink the stability of DNA i‐motifs in vitro with their stability in vivo and provide essential information for the design and development of i‐motif‐based DNA biosensors for intracellular applications.

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confidence: 99%

Evaluation of the Stability of DNA i‐Motifs in the Nuclei of Living Mammalian Cells

et al. 2018

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“…However, the stronger background in the cellular environment often leads to complicated or poor-quality in-cell NMR spectra. For example, 1 H imino proton NMR analysis of a telomere DNA G-quadruplex in the cellular environment yields a low-resolution spectrum, thus distinguishing the resonance signals for molecules of interest from the background noise due to the other molecules present in cells remains a challenge (40). …”

Section: Resultsmentioning

confidence: 99%

Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy

Bao

Ishizuka

Shimoyama

et al. 2017

Nucleic Acids Research

131

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Human telomeric RNA has been identified as a key component of the telomere machinery. Recently, the growing evidence suggests that the telomeric RNA forms G-quadruplex structures to play an important role in telomere protection and regulation. In the present studies, we developed a 19F NMR spectroscopy method to investigate the telomeric RNA G-quadruplex structures in vitro and in living cells. We demonstrated that the simplicity and sensitivity of 19F NMR approach can be used to directly observe the dimeric and two-subunits stacked G-quadruplexes in vitro and in living cells and quantitatively characterize the thermodynamic properties of the G-quadruplexes. By employing the 19F NMR in living cell experiment, we confirmed for the first time that the higher-order G-quadruplex exists in cells. We further demonstrated that telomere RNA G-quadruplexes are converted to the higher-order G-quadruplex under molecular crowding condition, a cell-like environment. We also show that the higher-order G-quadruplex has high thermal stability in crowded solutions. The finding provides new insight into the structural behavior of telomere RNA G-quadruplex in living cells. These results open new avenues for the investigation of G-quadruplex structures in vitro and in living cells.

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“…Under such conditions, the characteristics like molecular crowding or composition of endogenous compounds are maintained, whereas the line-width of the spectra is significantly decreased (26). This set-up allowed us to prepare and detect RNA concentrations between 75 and 150 μM and metabolite concentrations up to 600 μM.…”

Section: Discussionmentioning

confidence: 99%

Ligand binding to 2′-deoxyguanosine sensing riboswitch in metabolic context

et al. 2017

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The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit β of ribonucleotide reductase in Mesoplasma florum upon 2΄-deoxyguanosine binding. We characterized binding of 2΄-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and ‘in-cell-like’ conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. ‘In-cell-like’ environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under ‘in-cell-like’-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2΄-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2΄-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2΄-deoxyguanosine, guanosine plays a role in riboswitch function in vivo. Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2΄-deoxyguanosine was not able to regulate gene expression.

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Evaluation of Parameters Critical for Observing Nucleic Acids Inside Living Xenopus laevis Oocytes by In-Cell NMR Spectroscopy

Cited by 100 publications

References 64 publications

Evaluation of the Stability of DNA i‐Motifs in the Nuclei of Living Mammalian Cells

Evaluation of the Stability of DNA i‐Motifs in the Nuclei of Living Mammalian Cells

Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy

Ligand binding to 2′-deoxyguanosine sensing riboswitch in metabolic context

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