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Conventional biophysical and chemical biology approaches for delineating relationships between the structure and biological function of nucleic acids (NAs) abstract NAs from their native biological context. However, cumulative experimental observations have revealed that the structure, dynamics and interactions of NAs might be strongly influenced by a broad spectrum of specific and nonspecific physical-chemical environmental factors. This consideration has recently sparked interest in the development of novel tools for structural characterization of NAs in the native cellular context. Here, we review the individual methods currently being employed for structural characterization of NA structure in a native cellular environment with a focus on recent advances and developments in the emerging fields of in-cell NMR and electron paramagnetic resonance spectroscopy and in-cell single-molecule FRET of NAs.

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.

The Sec translocon is a transmembrane assembly highly conserved among all forms of life as the principal route for transport of polypeptides across or into lipid bilayers. In bacteria translocation involves allosteric communication between the membrane pore SecYEG and the associated SecA ATPase. Using singlemolecule fluorescence we reveal that slow conformational changes associated with the ATPase SecA modulate fast opening and closure of the SecY lateral gate. Such a mismatch of timescales is not compatible with direct coupling between SecA and SecYEG. A dynamic allosteric model is proposed in which the SecA ATPase cycle ‘steers’ the energy landscape for SecY pore opening. We map the experimental traces onto reduced reaction coordinates derived from molecular dynamics trajectories, providing a model for the energy landscape and a structural interpretation of the associated dynamics. Dynamic allostery may be common among motor ATPases that drive conformational changes in molecular machines.Graphical TOC Entry

Chromatin-remodeling ATPases modulate histones-DNA interactions within nucleosomes and regulate transcription. At the heart of remodeling, ATPase is a helicase-like motor flanked by a variety of conserved targeting domains. CHD4 is the core subunit of the nucleosome remodeling and deacetylase complex NuRD and harbors tandem plant homeo finger (tPHD) and chromo (tCHD) domains. We describe a multifaceted approach to link the domain structure with function, using quantitative assays for DNA and histone binding, ATPase activity, shape reconstruction from solution scattering data, and single molecule translocation assays. These approaches are complementary to high-resolution structure determination.

The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form.