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Angew Chem Int Ed Engl
2021 Jan 11;602:865-872. doi: 10.1002/anie.202007184.
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In-Cell NMR Spectroscopy of Functional Riboswitch Aptamers in Eukaryotic Cells.
Broft P
,
Dzatko S
,
Krafcikova M
,
Wacker A
,
Hänsel-Hertsch R
,
Dötsch V
,
Trantirek L
,
Schwalbe H
.
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We report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2'-deoxyguanosine to the aptamer domain of the bacterial 2'-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, we show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Our data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intracellular space. Here, we show that the in-cell NMR setup can be adjusted for characterization of much larger (≈70 nt) functional and chemically non-modified RNA.
CRC902 Deutsche Forschungsgemeinschaft, 871037 Horizon 2020, 19-26041X Czech Science Foundation, CZ.02.1.01/0.0/0.0/15_003/0000477 Ministry of Education, Youth, and Sports of the Czech Republic
Figure 1 A–D) Secondary structures of 2′‐dG aptamer 70mer (A), sv‐2′‐dG aptamer (B), RNA 14mer employed as a reference for in‐cell NMR measurements (C), and 2′‐dG aptamer 72mer (D). The RNAs are delivered into the cells either by injection into oocytes (A+B) or by electroporation in HeLa cells (C+D). Ligand binding of the aptamers is mainly stabilized by Watson–Crick‐type hydrogen bonding of 2′‐dG to C74 in the three‐way‐junction of the RNA aptamer. Upon ligand binding, the closing base‐pairs A21‐U75 and G25‐U45 are stabilized and give rise to reporter imino proton signals.
Figure 2 A) Watson–Crick base pair formed between C74 of the RNA aptamer domain and the ligand 2′‐deoxyguanosine. The 15N‐isotope of the ligand imino nitrogen is highlighted in green, the imino proton giving rise to the signal in the spectra shown in panels (B) and (C) is highlighted in blue. B+C) Comparison of 15N‐edited imino proton spectra of the RNA aptamer–70mer‐2′‐deoxyguanosine complex in‐cell (B) and in intraoocyte buffer (C). 60 nL of a 1.62 mm stock solution were injected into each cell, yielding ≈100 μm RNA–ligand complex per cell. The imino proton giving rise to the single signal at ≈13 ppm is marked with blue circle. The in‐cell spectrum (B) was recorded with 8192 scans and the intraoocyte buffer spectrum (C) was recorded with 2048 scans, both at T=291 K. For this experiment, unlabeled RNA and 15N‐labeled ligand has been used. D–G) Series of 2D‐spectra of the RNA aptamer–70mer‐2′‐deoxyguanosine complex in oocyte extract. The reporter signal for ligand binding, U75, is annotated in spectrum (D) and is still observed after 18 h (E). For this experiment, 15N‐labeled RNA and unlabeled ligand has been used.
Figure 3 (In‐cell) 15N‐HMQC spectra of G, U‐15N‐labeled sv‐2′‐dG aptamer. The resonances of the G57 and the U69 imino protons are annotated in blue and also highlighted in blue in the secondary structure inset. The resonances of the imino protons of G25, G32, U45, and U75, which are reporter signals for ligand binding,8 are marked in red in the spectra and highlighted in red in the secondary structure inset. A) In vitro spectrum of the RNA–ligand complex (≈200 μm) in intraoocyte buffer. B) Spectrum of the RNA–ligand complex (≈200 μm) in oocyte extract. C) In‐cell spectrum of the RNA (≈120 μm) in the absence of ligand. The 15N‐HMQC was recorded at 700 MHz as a SOFAST‐HMQC20 employing a 2.25 ms PC9‐pulse21 centered at 12 ppm for imino proton excitation (corresponding to an excitation bandwidth of ≈1.4 kHz) and a 1.5 ms Reburp refocusing pulse[22](covering ≈1.4 kHz). 1024×32 complex points were acquired in the direct and the indirect dimension with 256 scans per point and an inter‐scan delay of 0.7 s. The temperature was 291 K.
Figure 4. A) Confocal microscopy images of cells transfected with RNA 14mer (FAM). The green color indicates the localization of RNA 14mer (FAM). The blue color corresponds to a cell nucleus stained by Hoechst 33342. B) Double‐staining (PI/FAM) FCM analysis of transfected HeLa cells with the RNA 14mer (FAM). Percentages of a viable non‐transfected cells, viable RNA‐containing cells, non‐transfected dead/compromised cells, and transfected dead/compromised cells with RNA are indicated in left‐bottom, right‐bottom (red), left‐top, and right‐top quadrants, respectively. C) Imino region of 1D 1H NMR spectra of RNA 14mer in vitro (TOP) in EB‐buffer (140 mm sodium phosphate, 5 mm KCl, 10 mm MgCl2, pH 7.2) and corresponding spectrum of HeLa cells transfected with RNA 14mer (FAM) (MIDDLE). Imino region of 1D 1H NMR spectrum of extracellular fluid (supernatant) taken from the in‐cell NMR samples after completion of the spectra acquisition (BOTTOM). The (in‐cell) NMR spectra were acquired at 20 °C.
A) Confocal microscopy images of cells transfected with aptamer–ligand complex. The green color indicates the localization of (FAM)‐aptamer/(FAM)‐aptamer–ligand complex. The blue color corresponds to a cell nucleus stained by Hoechst 33342. B) Double‐staining (PI/FAM) FCM analysis of transfected HeLa cells with the aptamer–ligand complex. Percentages of a viable non‐transfected cells, viable aptamer–ligand complex containing cells, non‐transfected dead/compromised cells, and transfected dead/compromised cells with aptamer–ligand complex are indicated in left‐bottom, right‐bottom (red), left‐top, and right‐top quadrants, respectively. C) Imino region of 1D 1H NMR spectra of aptamer–ligand complex in vitro (TOP) in EB‐buffer (140 mm sodium phosphate, 5 mm KCl, 10 mm MgCl2, pH 7.2) and corresponding spectrum of HeLa cells transfected with aptamer–ligand complex (MIDDLE). Imino region of 1D 1H NMR spectrum of extracellular fluid (supernatant) taken from the in‐cell NMR samples after completion of the spectra acquisition (BOTTOM). D) 1D 13C‐edited NMR spectra of the aptamer–ligand complex in vitro in EB‐buffer (TOP) and corresponding spectra of HeLa cells transfected with aptamer–ligand complex. Note: The NMR spectra from two independent experiments are provided (MIDDLE). 1D 13C‐edited NMR spectra of extracellular fluid (supernatant) taken from the respective in‐cell NMR samples after completion of the spectra acquisition (BOTTOM). E) Zoomed region of 1D 13C‐edited NMR spectra from D). Note: For confocal microscopy images and FCM analysis from the second in‐cell NMR experiment, see Supporting Figure S4. The (in‐cell) NMR spectra were acquired at 20 °C.
Figure 6. 1D 13C‐edited in‐cell NMR spectra of cells transfected with aptamer–ligand complex (400 μm aptamer/2 mm 2′‐deoxyguanosine), cells transfected with 400 μm and 2 mm ligand, and non‐transfected cells. 1D 13C‐edited in vitro NMR spectra of extracellular fluid taken from the 2 mm 2′‐deoxyguanosine in‐cell NMR sample after completion of the spectra acquisition (leakage), and Leibovitz L15 medium. The (in‐cell) NMR spectra were acquired at 20 °C.
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