The observation of biological processes at the molecular scale in real time requires high spatial and temporal resolution. Magnetic tweezers are straightforward to implement, free of radiation or photodamage, and provide ample multiplexing capability, but their spatiotemporal resolution has lagged behind that of other single-molecule manipulation techniques, notably optical tweezers and AFM. Here, we present, to our knowledge, a new high-resolution magnetic tweezers apparatus. We systematically characterize the achievable spatiotemporal resolution for both incoherent and coherent light sources, different types and sizes of beads, and different types and lengths of tethered molecules. Using a bright coherent laser source for illumination and tracking at 6 kHz, we resolve 3 Å steps with a 1 s period for surface-melted beads and 5 Å steps with a 0.5 s period for double-stranded-dsDNA-tethered beads, in good agreement with a model of stochastic bead motion in the magnetic tweezers. We demonstrate how this instrument can be used to monitor the opening and closing of a DNA hairpin on millisecond timescales in real time, together with attendant changes in the hairpin dynamics upon the addition of deoxythymidine triphosphate. Our approach opens up the possibility of observing biological events at submillisecond timescales with subnanometer resolution using camera-based detection.

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

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In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNAbinding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps-DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps-DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.DNA condensation | Dps | cooperativity | hysteresis | Ising model P urified DNA behaves as an entropic spring with a radius of gyration that scales as a function of the contour length (1). In contrast, DNA in vivo is highly organized and condensed. In bacteria, this condensation is caused by nucleoid-associated proteins (NAPs) that collectively shape the chromosome (2, 3). NAPs are capable of binding genomic DNA and in doing so alter its shape, control the transcriptional expression of genes, and remodel the structure of the nucleoid in response to external stimuli (2, 3).DNA-binding protein from starved cells (Dps) is an NAP structurally related to ferritins and associated with the response to stress. Dps is highly expressed in stationary phase (4-7) and is also involved in the cellular response to oxidative (4, 8-10), UV (8, 11), thermal (8), and pH shocks (8). In addition, Dps has been implicated in biofilm formation and tolerance to bacteriophage attacks (12). Dps monomers have a molecular mass of 19 kDa and assemble into a dodecameric shell (Fig. S1A) (13). The resulting complex binds to both supercoiled and linear DNA to form a dense biocrystal structure (4,7,9,14).Although the crystal structure of the Dps dodecamer has been solved (13), no atomic-scale structure of Dps-DNA assemblies currently exists and little is known about complex formation. The affinity of Dps for DNA is very sensitive to buffer conditions. Like many DNA-binding proteins, Dps binds DNA more weakly in the presence of higher salt concentrations. Less typically, divalent cations such as Mg 2+ can substantially weaken the affinity of Dps for DNA (9, 15). It has been proposed that fluctuations in divalent cation concentrations act as a trigger for biocrystal as...

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.