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A technique called condense-seq has been developed to measure nucleosome condensability and used to show that mononucleosomes contain sufficient information to condense into large-scale compartments without requiring any external factors.

Here the authors by applying a single-molecule detection platform enabling FRET measurement on a long plasmid DNA construct, provide detailed insights on how negatively supercoiled DNA impacts co-transcriptional R-loop and G4 formation.

Yellow fluorescent proteins (YFPs) photobleach rapidly, restricting microscopy experiments. Here, the authors report mGold2s and mGold2t, YFPs that extend imaging durations up to 25 times longer than standard probes without sacrificing brightness.

Cas9 remains bound to DNA after cleavage and its removal is required for DNA double-strand break repair. Here, the authors show that the HLTF translocase disrupts the Cas9- DNA post-cleavage complexes in a process that requires the HLTF HIRAN ___domain and ATPase activity.

SNARE protein-mediated vesicle fusion is usually monitored by indirect lipid mixing. Diaoet al. have developed a FRET-based single-vesicle content mixing assay, which elucidates fusion pore formation, and shows that the yeast SNARE complex mediates pore expansion in the absence of accessory proteins.

Theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another in the absence of proteins. Here, the authors show that GC-rich DNA with methylated cytosine and AT-rich DNA duplexes associate more strongly than GC-rich duplexes regardless of the sequence homology.

Integrins come in subtypes that bind distinct ligands. This work reveals and quantifies subtype-specific mechanical force transmission and assembly of cytoskeleton architectures.

Sequence recognition through base pairing is essential for DNA repair and gene regulation, but the basic rules underlying this process have been unclear. Data from single-molecule fluorescence studies, used to visualize annealing and melting reactions of two untethered strands containing a single mismatch, suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA.

During DNA metabolism, single-stranded DNA intermediates are often generated that are protected from degradation by binding of ssDNA-binding (SSB) proteins. Bacterial SSB protein forms a tetramer that wraps ssDNA using its four subunits. Here it is shown that tetrameric SSB protein can spontaneously migrate along ssDNA; this diffusional movement introducing a new model for the redistribution of the SSB protein, while remaining bound to ssDNA during recombination and repair processes.

Voltage-gated sodium channels are expressed in excitable tissues and mutations have been linked to cardiac arrhythmias and channelopathies. Here the authors show that the sodium channel α-subunits interact to form a dimer and gate as dimer and that this functional dimerisation is conserved.

Many RNAs become functional before their synthesis completes. Here the authors employ a single-molecule vectorial folding assay mimicking RNA transcription and show that the ZTP riboswitch is kinetically controlled and activated by slower unwinding and strategic pausing.

DISCOVER-Seq+ is a high-sensitivity method for the detection of off-target effects of CRISPR genome editing.

Phoenix Fluor 555 (PF555) is a bright dye with an exceptional, order-of-magnitude longer photobleaching lifetime than conventional organic dyes that enables extended live-cell single-molecule imaging without anti-photobleaching additives.

Kim et al. used directed evolution methods to identify a high-fidelity SpCas9 variant, Sniper2L, which exhibits high general activity but maintains high specificity at a large number of target sites.

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs) but only few specific interactions are known. Using TIRF microscopy based assays, functional studies and an experimentally validated prediction algorithm, the authors show that specific PIP binding is widespread among human PH domains.

Single-molecule analysis reveals a novel binding state of Holliday junction (HJ) resolving enzymes where the enzymes partially dissociate from the HJ and allowing nearly unencumbered HJ dynamics, suggesting coupled branch migration and HJ resolution.

Signaling via the mTOR complex 1 (mTORC1) maintains cellular and organismal homeostasis. Here the authors show that the AAA + ATPase Thorase binds mTOR to promote disassembly and inactivation of mTORC1 to fine tune TOR signaling according to amino acid availability.

A DNA-based device can act as a pH sensor inside living cells.

Semiconducting nanocrystals emit light in many different colours, but they blink on and off at random. The latest nanocrystals emit photons steadily, thanks to the blending of their cores into their outer shells.

The growth factor TGF-ß1 is located inside a protein cage, and is thought to be released by force applied through integrin proteins. A structure of TGF-ß1 in complex with integrin α_Vß₆ sheds light on the uncaging process. See Article p.55

The photoluminescence properties of carbon nanotubes are sensitive to molecular adsorption. By studying the response of a pair of single-wall carbon nanotubes, researchers have now shown that analytes of biological interest can be identified and measured in real-time within living cells.

DNA is replicated by a replisome containing two DNA polymerase molecules, one of which copies the leading-strand template in a continuous manner while the second copies the lagging-strand template in a discontinuous manner; however, the two strands are synthesized at the same net rate. RNA primers are now shown to be made as DNA is being synthesized and then passed on to the polymerase; to allow for this transfer, the lagging-strand polymerase has a faster rate.

Deshpande et al show that MRN nuclease-dependent processing of DNA ends in human cells occurs at sites bound by DNA-PK. Chromatin immunoprecipitation analysis of DNA-PK, MRN, and CtIP supports a sequential model of pathway choice.

Analysing single-molecule fluorescence resonance energy transfer (smFRET) enables an unprecedented view of the dynamics and kinetics of biomolecular conformational changes and interactions. In this Primer, Ha et al. discuss technological advances that have led to smFRET and how the method can be adapted to address various research questions in structural and molecular biology.

A high-throughput, chromosome-wide analysis of DNA looping reveals its contribution to the organization of chromatin, and provides insight into how nucleosomes are deposited and organised de novo.

Basu and colleagues comprehensively characterize how sequence and epigenetic modifications impact the local mechanical properties of DNA. The results suggest that DNA mechanics may have evolved to aid diverse DNA-deforming biological processes.

Living cells change their behaviour in response to the viscosity of the medium surrounding them. An in vitro study shows that cells spread wider and move faster in a highly viscous medium, provided they have an actively ruffling lamellipodium.

PCR is an essential method for the amplification and manipulation of nucleic acids, but the requirement for a thermocycler limits access. Here, authors engineer a helicase to replace the heating step of PCR with enzymatic unwinding, allowing the isothermal amplification of fragments up to 6 kb.

Visualisation of point mutations in situ is informative for studying genetic diseases. Here the authors report single guide genome oligopaint via local denaturation fluorescence in situ hybridisation, sgGOLDFISH, a direct hybridisation genome imaging method with single-nucleotide sensitivity.

Light-up aptamers are widely used for fluorescence visualization of non-coding RNA in vivo. Here the authors employ single-molecule fluorescence-force spectroscopy to characterize the mechanical responses of the G-Quadruplex based light-up aptamers Spinach2, iMangoIII and MangoIV, which is of interest for the development of improved fluorogenic modules for imaging applications.

Much of our knowledge about biological systems has been obtained by examining ensembles of molecules. However, this has begun to change because of the unprecedented precision and clarity afforded by single-molecule measurements. The last decade has seen amazing advances in the resolution and complexity of these methods, making it possible to ask and answer entirely new types of biological questions.

CRISPR-Cas9 has enabled an unprecedented ability to manipulate the genome yet it is still poorly understood how target recognition functions at a molecular level. Here the authors use single-molecule FRET to probe Cas9-target interaction and identify distinct search and proofreading states.

In viruses, multi-subunit ring-ATPases are involved in genome packaging. Here, using single-molecule techniques, the authors determine that the active bacteriophage T4 DNA packaging motor is a pentamer and show that the motor can tolerate inactive subunits, suggesting that strict coordination between the subunits is not crucial.

Analyses of hippocampal AMPA receptor–auxiliary subunit complexes provide insights into the predominant assemblies and organization of the AMPA receptor, TARP-γ8 and CNIH2/SynDIG4 and explain the mechanism of inhibition of a clinically relevant, brain-region-specific allosteric inhibitor.

The authors describe the mechanism of exo-endocytosis coupling at synapses. They find that actin forms a ring around the region of exocytosis. This ring conserves membrane area, allowing induction of inward membrane buckling following exocytosis.

Pif1 plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA. Here the authors solve the structure of Bacteroides sp Pif1 (BaPif1) in complex with a symmetrical double forked dsDNA and provide unprecedented insights into forked dsDNA unwinding by BaPif1.

The single stranded DNA binding protein RPA coordinates DNA metabolism using multiple protein and DNA interaction domains. Here, the authors show that the chaperone-like protein Rtt105 staples RPA domains to prevent untimely protein interactions.

Cytosine modifications are important epigenetic markers yet their physical influence on DNA is not well understood. Here, Ngo et al. show that different alterations affect DNA flexibility, suggesting a mechanism where modifications change accessibility of nucleosome bound DNA.

Tracking single molecules on long stretches of single-stranded DNA poses technical challenges due to its propensity to form hairpin structures. To solve this problem, the authors combine TIRF microscopy with optical tweezers to stretch the DNA and capture the dynamics of DNA unwinding by UvrD DNA helicase.

The combination of a genetically encoded aldehyde tag and optimized labeling method allows high-efficiency, site-specific labeling of tagged proteins after purification or in cell extracts. The authors use the high labeling efficiency for single-molecule measurements of the dynamic interactions between two DNA polymerases and polymerase processivity factor bound to DNA.

The combination of an ultrahigh-resolution dual optical trap with a confocal microscope allowed single-fluorophore detection of labeled oligonucleotide binding and simultaneous measurement of angstrom-scale changes in DNA tether extension.

Bacterial small RNA SgrS binds and regulates its primary target, ptsG mRNA. Here the authors employ Sort-Seq and super resolution imaging to investigate in vivo target recognition and rejection kinetics of SgrS.