In Cell NMR Spectroscopy: Investigation of G-Quadruplex Structures Inside Living Xenopus laevis Oocytes
G-quadruplexes are inherently polymorphic nucleic acid structures. Their folding topology depends on the nucleic acid primary sequence and on physical–chemical environmental factors. Hence, it remains unclear if a G-quadruplex topology determined in the t
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Introduction In principle, in-cell NMR spectroscopy can be considered as a direct analogy of conventional solution NMR spectroscopy with a nature of the sample being a single, but a key difference. While the conventional NMR sample comprises target molecule (G-quadruplex, for example) dissolved in a low-complexity buffered solution, the in-cell sample constitutes of the target “dissolved” in the interior of living cells. However, the “dissolution” of the target in the interior of a cell, without compromising its viability and metabolic status, at concentration amiable to notoriously insensitive NMR detection represents a major technical challenge. As first demonstrated for proteins [1, 2] and later also for nucleic acids [3], this challenge can be resolved by microinjecting bio-macromolecules into large (~1 mm in diameter) oocytes from the African clawed frog (Xenopus laevis)—Fig. 1. When microinjected material is labeled, e.g., with non-native nuclei such as 19F [4] or enriched in isotopes natively occurring at low abundance such as the 13C
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_25, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Fig. 1 Top: Schematic representation of in-cell NMR experimental setup. Bottom: Comparison of the imino regions of 1D 1H NMR spectra of the G-quadruplex forming telomeric DNA, d(G3(TTAG3)3T), acquired in buffer (in vitro), in X. laevis oocytes (in-cell), and in cleared cellular lysate (ex vivo). Note that in-cell NMR spectrum has notably lower resolution compared to its in vitro counterpart. Additionally, note the evident difference between spectral fingerprint in buffer and in cleared lysate. Figure was adapted with permission from ref. [3]
and/or 15N [3, 5], the NMR signals specific to the introduced target of interest can be readily observed even in the complex environment of living cells. Unfortunately, in-cell NMR spectra of nucleic acids exhibit a lower resolution compared to NMR spectra acquired under simplistic conditions in vitro [3, 6, 7] (Fig. 1). The resolution of in-cell NMR spectra generally does not permit their use for de novo structure determination. Although the low spectral resolution notably limits applicability of in-cell NMR for inherently polymorphic G-quadruplex structures, the resolution and informational content of in-cell NMR spectra was shown to be still sufficient to address number of biological relevant questions. For example, in-cell NMR was successfully used to address roles of low molecular weight compounds (metabolites) [3] as well as of native intracellular molecular crowding in promoting G-quadruplex polymorphism [8]. In-cell NMR was also used to assess the role of an intracellular environment in modulating interactions between a DNA G-quadruplex and a G-quadruplex stabilizing ligand (drug-like molecule) [5]. Most recently, the in-cell NMR study in X. laevis
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