Shedding light on the base-pair opening dynamics of nucleic acids in living human cells.

Base-pair opening is a fundamental property of nucleic acids that plays important roles in biological functions. However, studying the base-pair opening dynamics inside living cells has remained challenging. Here, to determine the base-pair opening kinetics inside living human cells, the exchange rate constant ([Formula: see text]) of the imino proton with the proton of solvent water involved in hairpin and G-quadruplex (GQ) structures is determined by the in-cell NMR technique. It is deduced on determination of [Formula: see text] values that at least some G-C base pairs of the hairpin structure and all G-G base-pairs of the GQ structure open more frequently in living human cells than in vitro. It is suggested that interactions with endogenous proteins could be responsible for the increase in frequency of base-pair opening. Our studies demonstrate a difference in dynamics of nucleic acids between in-cell and in vitro conditions.

Detection of parallel and antiparallel DNA triplex structures in living human cells using in-cell NMR.

We introduced oligodeoxynucleotides (ODNs) that form parallel and antiparallel triplex structures in vitro into living human cells and recorded their in-cell NMR spectra. Observation of landmark signals for triplex structures proved for the first time that parallel and antiparallel triplex structures are formed in living human cells.

Observation of nucleic acids inside living human cells by in-cell NMR spectroscopy.

The intracellular environment is highly crowded with biomacromolecules such as proteins and nucleic acids. Under such conditions, the structural and biophysical features of nucleic acids have been thought to be different from those in vitro. To obtain high-resolution structural information on nucleic acids in living cells, the in-cell NMR method is a unique tool. Following the first in-cell NMR measurement of nucleic acids in 2009, several interesting insights were obtained using Xenopus laevis oocytes. However, the in-cell NMR spectrum of nucleic acids in living human cells was not reported until two years ago due to the technical challenges of delivering exogenous nucleic acids. We reported the first in-cell NMR spectra of nucleic acids in living human cells in 2018, where we applied a pore-forming toxic protein, streptolysin O. The in-cell NMR measurements demonstrated that the hairpin structures of nucleic acids can be detected in living human cells. In this review article, we summarize our recent work and discuss the future prospects of the in-cell NMR technique for nucleic acids.

Recent progress of in-cell NMR of nucleic acids in living human cells.

The inside of living cells is highly crowded with biological macromolecules. It has long been considered that the properties of nucleic acids and proteins, such as their structures, dynamics, interactions, and enzymatic activities, in intracellular environments are different from those under in vitro dilute conditions. In-cell NMR is a robust and powerful method used in the direct measurement of those properties in living cells. However, until 2 years ago, in-cell NMR was limited to Xenopus laevis oocytes due to technical challenges of incorporating exogenous nucleic acids. In the last 2 years, in-cell NMR spectra of nucleic acid introduced into living human cells have been reported. By use of the in-cell NMR spectra of nucleic acids in living human cells, the formation of hairpin structures with Watson-Crick base pairs, and i-motif and G-quadruplex structures with non-Watson-Crick base pairs was demonstrated. Others investigated the mRNA-antisense drug interactions and DNA-small compound interactions. In this article, we review these studies to underscore the potential of in-cell NMR for addressing the structures, dynamics, and interactions of nucleic acids in living human cells.