Optimising in-cell NMR acquisition for nucleic acids.
J Biomol NMR
; 2024 Aug 20.
Article
em En
| MEDLINE
| ID: mdl-39162911
ABSTRACT
Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT1), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppression for in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.
Texto completo:
1
Base de dados:
MEDLINE
Idioma:
En
Revista:
J Biomol NMR
/
J. biomol. NMR
/
Journal of biomolecular NMR
Assunto da revista:
BIOLOGIA MOLECULAR
/
DIAGNOSTICO POR IMAGEM
/
MEDICINA NUCLEAR
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Suécia