When Small becomes Too Big: Expanding the Use of In‐Cell Solid‐State NMR Spectroscopy

Abstract

state NMR spectroscopy has become a powerful tool to study soluble proteins in cells, provided that they tumble sufficiently fast. In addition, cryo-electron tomography (cryo-ET) has recently displayed a tremendous potential to probe structures of large proteins and assemblies in their native cellular environments. However, challenges remain to obtain atomic-level information in native cell settings for proteins that are small, disordered, or are strongly engaged in intermolecular interactions. In this Minireview, we discuss recent progress in using sensitivity enhanced solid-state NMR spectroscopy methods in the context of cellular structural biology.[a] S.

In‐Cell NMR Spectroscopy of Functional Riboswitch Aptamers in Eukaryotic Cells

“…Additionally spectra of sv-2'-dG aptamer in intraoocyte buffer ( Figure 3A) and in oocyte extract ( Figure 3B), both in the presence of ligand, were recorded. Thea ptamer in-cell adopts the same global fold as in vitro,a se videnced by the annotated imino signal pattern in the 2D 1 H, 15 Nc orrelation spectrum (Figure 3C).…”

“…Thes v-2'-dG aptamer ( Figure 1B), which is as trongly sequence-modified RNAa ptamer with high stability,c an be prepared at higher concentration and contains more stable GC-rich stems as well as more stable tertiary interactions than the 2'-dG aptamer.T os tabilize the 2'-dG aptamer 70mer ( Figure 1A), the sequence is modified by 3G C-closing base pairs.T he 2'-dG aptamer 72mer ( Figure 1D)c ontains less GC-rich stems than the sv-aptamer but comes closer to the natural riboswitch in its sequence because it was only modified at the 5'-end with an additional G, which has no stabilizing effect on the RNAs tructure in order to generate higher transcription yields.A ll three aptamers bind 2'deoxyguanosine in vitro and adopt ac haracteristic tertiary fold upon ligand binding,which is well illustrated in the imino proton region of 1 H, 15 N-correlated 2D NMR spectra. [8]…”

“…In-Cell NMR of 2' '-dG Aptamer 72mer in HeLa Cells Using the electroporation procedure described above,we introduced the preformed aptamer-ligand complex, consisting of 2'-dG aptamer 72mer and its 13 C, 15 Nlabeled ligand 2'deoxyguanosine into HeLa cells.F CM analysis showed that after electroporation more than 90 %ofthe cells were viable RNAc ontaining cells and less than 6% of the cells were either dead or had compromised cell membrane integrity ( Figure 5B). Confocal microscopy indicated that the RNA…”

“…[11][12][13][14] An umber of previous reports have focused on the NMR investigation of proteins under in-cell conditions. [15,16] However,r eports for nucleic acids in general and of RNAinparticular are rare. [17] We investigate stability,structure and ligand binding capacity of an aptamer domain derived from the natural riboswitch (2'-dG aptamer 70mer) and aG switch-dGswitch chimera (sv-2'-dG aptamer) [18] both in living oocytes and oocyte extract ( Figures 1A and B).…”

In‐Cell NMR Spectroscopy of Functional Riboswitch Aptamers in Eukaryotic Cells

“…Here, we seize the unique opportunity to directly test ligand binding in living eukaryotic cells, namely in Xenopus laevis oocytes and in HeLa cells, using state‐of‐the‐art approach of in‐cell NMR spectroscopy [11–14] . A number of previous reports have focused on the NMR investigation of proteins under in‐cell conditions [15, 16] . However, reports for nucleic acids in general and of RNA in particular are rare [17] .…”