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.

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

Transport of proteins across membranes is a fundamental process, achieved in every cell by the ‘Sec’ translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.

C-rich DNAh as the capacity to form at etrastranded structure knowna sa ni -motif.T he i-motifs within genomic DNAh ave been proposed to contribute to the regulation of DNAtranscription. 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 stateof-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.O ur 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 DNAi -motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNAbiosensors for intracellular applications.