In-cell NMR of a DNA G-quadruplex and ligand
A parallel d(TG4T)4 G-quadruplex remains NMR-visible and long-lived in Xenopus oocytes, but ligand 360A can hide the complex on the NMR timescale.
Source
G-quadruplex DNA and ligand interaction in living cells using NMR spectroscopy
Study at a glance
- Design
- Animal / in-vitro — In-cell NMR of 15N/13C-labeled d(TG4T)4 microinjected into Xenopus oocytes ± 360A ligand
- N
- Oocyte in-cell NMR — no single reported oocyte analytic N in stored summary
- Population
- Living Xenopus laevis oocytes
- Outcome
- Intracellular G-quadruplex persistence and ligand-bound NMR visibility
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Inspect source passages
In-cell NMR of a DNA G-quadruplex and ligand
These passages occur verbatim in the stored paper text. They support the points listed below; they do not verify every statement in the explanation.
NMR experiments were run at approximately 16 °C on an 800 MHz spectrometer.
All NMR experiments were executed at approximately 16 °C in order to minimize damage to the cells and at same time improve the observation of the imino signals.
Oocyte robustness allowed acquisitions longer than 48 h.
laevis oocytes’ robustness, when compared with other cells, allowed us to perform acquisitions for periods longer than 48 h.
What they did
Authors micro-injected 15N/13C-labeled d(TG4T)4 into living Xenopus laevis oocytes and recorded 800 MHz SOFAST-HMQC imino spectra with and without the G-quadruplex ligand 360A, comparing in-cell and in vitro fingerprints.
What they found
The all-parallel G-quadruplex forms in cells and survives enzymatic attack for many hours. Ligand-bound oligonucleotide appears NMR-invisible in cells, implying extra binding partners or precipitation, so in vitro ligand studies may not transfer directly.
The limits
What it doesn't show
The study uses oocytes, not human tumor cells, and does not assign the cellular partners that silence the ligand complex.
Key terms
- G-quadruplex
- Stacked guanine quartets stabilized by Hoogsteen hydrogen bonds and cations such as K+.
- Imino signature
- Downfield 1H NMR peaks of guanine imino protons that report G-quadruplex fold.
- SOFAST-HMQC
- Fast 2D 1H–15N correlation experiment used here for in-cell imino detection.
- 360A
- A G-quadruplex-binding ligand titrated against d(TG4T)4.
- In-cell NMR
- NMR of labeled molecules inside living cells rather than in dilute buffer.
Flashcards
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Quiz yourself
In-cell NMR here shows d(TG4T)4 is:
Common questions
Does the G-quadruplex fold in cells?
Yes—imino NMR shows the parallel d(TG4T)4 fold under physiological conditions.
What happens with ligand 360A in cells?
The complex can become NMR-invisible, unlike typical in vitro spectra.
What cells were used?
Living Xenopus laevis oocytes.
Why record at ~16 °C?
To limit cell damage while still seeing imino signals.
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