Drift correction in localization microscopy using entropy minimization.

Localization microscopy offers resolutions down to a single nanometer but currently requires additional dedicated hardware or fiducial markers to reduce resolution loss from the drift of the sample. Drift estimation without fiducial markers is typically implemented using redundant cross correlation (RCC). We show that RCC has sub-optimal precision and bias, which leaves room for improvement. Here, we minimize a bound on the entropy of the obtained localizations to efficiently compute a precise drift estimate. Within practical compute-time constraints, simulations show a 5x improvement in drift estimation precision over the widely used RCC algorithm. The algorithm operates directly on fluorophore localizations and is tested on simulated and experimental datasets in 2D and 3D. An open source implementation is provided, implemented in Python and C++, and can utilize a GPU if available.

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute.

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT.

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.

Facilitated diffusion of Argonaute-mediated target search.

Argonaute (Ago) proteins are of key importance in many cellular processes. In eukaryotes, Ago can induce translational repression followed by deadenylation and degradation of mRNA molecules through base pairing of microRNAs (miRNAs) with a complementary target on a mRNA sequence. In bacteria, Ago eliminates foreign DNA through base pairing of siDNA (small interfering DNA) with a target on a DNA sequence. Effective targeting activities of Ago require fast recognition of the cognate target sequence among numerous off-target sites. Other target search proteins such as transcription factors (TFs) are known to rely on facilitated diffusion for this goal, but it is undetermined to what extent these small nucleic acid-guided proteins utilize this mechanism. Here, we review recent single-molecule studies on Ago target search. We discuss the consequences of the recent findings on the search mechanism. Furthermore, we discuss the open standing research questions that need to be addressed for a complete picture of facilitated target search by small nucleic acids.

High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics.

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.

mRNA structural dynamics shape Argonaute-target interactions.

Small interfering RNAs (siRNAs) promote RNA degradation in a variety of processes and have important clinical applications. siRNAs direct cleavage of target RNAs by guiding Argonaute2 (AGO2) to its target site. Target site accessibility is critical for AGO2-target interactions, but how target site accessibility is controlled in vivo is poorly understood. Here, we use live-cell single-molecule imaging in human cells to determine rate constants of the AGO2 cleavage cycle in vivo. We find that the rate-limiting step in mRNA cleavage frequently involves unmasking of target sites by translating ribosomes. Target site masking is caused by heterogeneous intramolecular RNA-RNA interactions, which can conceal target sites for many minutes in the absence of translation. Our results uncover how dynamic changes in mRNA structure shape AGO2-target recognition, provide estimates of mRNA folding and unfolding rates in vivo, and provide experimental evidence for the role of mRNA structural dynamics in control of mRNA-protein interactions.

Argonaute bypasses cellular obstacles without hindrance during target search.

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.

RecA filament maintains structural integrity using ATP-driven internal dynamics.

At the core of homologous DNA repair, RecA catalyzes the strand exchange reaction. This process is initiated by a RecA loading protein, which nucleates clusters of RecA proteins on single-stranded DNA. Each cluster grows to cover the single-stranded DNA but may leave 1- to 2-nucleotide (nt) gaps between the clusters due to three different structural phases of the nucleoprotein filaments. It remains to be revealed how RecA proteins eliminate the gaps to make a seamless kilobase-long filament. We develop a single-molecule fluorescence assay to observe the novel internal dynamics of the RecA filament. We directly observe the structural phases of individual RecA filaments and find that RecA proteins move their positions along the substrate DNA to change the phase of the filament. This reorganization process, which is a prerequisite step for interjoining of two adjacent clusters, requires adenosine triphosphate hydrolysis and is tightly regulated by the recombination hotspot, Chi. Furthermore, RecA proteins recognize and self-align to a 3-nt-period sequence pattern of TGG. This sequence-dependent phase bias may help the RecA filament to maintain structural integrity within the kilobase-long filament for accurate homology search and strand exchange reaction.