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.
C-rich DNAh as the capacity to form at etrastranded structure knowna sa ni -motif.T he i-motifs within genomic DNAh ave been proposed to contribute to the regulation of DNAtranscription. 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 stateof-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.O ur 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 DNAi -motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNAbiosensors for intracellular applications.
Studies on DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA–ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.
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,n amely Xenopus laevis oocytes and in human HeLa cells.T he riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly,w e 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:T hus far,i n-cell NMR studies on RNAinmammalian cells have been limited to investigations of short (< 15 nt) RNAfragments that were extensively modified by protecting groups to limit their degradation in the intracellular space.Here,weshow that the in-cell NMR setup can be adjusted for characterization of muchl arger (% 70 nt) functional and chemically non-modified RNA.
Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe’s discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex–ligand interactions in the complex environment of living cells.
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.
High-resolution studies of DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this issue, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of high-resolution NMR data of cells electroporated with pre-formed DNA-ligand complex. The impact of the intracellular environment on the integrity of the complex is assessed on the basis of in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. By using this technique, we studied complexes of model DNA fragments and four ligands, representative of DNA minor-groove binders (netropsin) or ligands binding to DNA pairing defects (naphthalenophanes). We demonstrate that some of the <i>in vitro</i> validated ligands retain their ability to form stable on-target DNA interactions <i>in situ</i>, while other<i> </i>lose this ability due to off-target interactions with genomic DNA as well as cellular metabolic components. Collectively, our data suggest that direct evaluation of behavior of drug-like molecules in the intracellular environment provides important insights for the design and development of DNA-binding ligands with the desired biological action and minimal side effects resulting from off-target binding.<br><div><br></div>
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