Exploring the dynamic nature of divalent metal ions involved in DNA cleavage by CRISPR-Cas12a.

CRISPR-Cas12a has been widely used in genome editing and nucleic acid detection. In both of these applications, Cas12a cleaves target DNA in a divalent metal ion-dependent manner. However, when and how metal ions contribute to the cleavage reaction is unclear. Here, using a single-molecule FRET assay, we reveal that these metal ions are necessary for stabilising cleavage-competent conformations and that they are easily exchangeable, suggesting that they are dynamically coordinated.

Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage.

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

A thermodynamic understanding of the salt-induced B-to-Z transition of DNA containing BZ junctions.

Z-DNA has attracted interest due to its distinctive left-handed helical structure. This non-canonical DNA structure is able to form transiently and plays an important role in cellular processes such as transcriptional regulation and DNA recombination. Alternating purine-pyrimidine sequences are well known to form Z-DNA under high-salt conditions, but the detailed mechanism of B-to-Z transition of DNA containing BZ junctions under these conditions is not well understood. Here, using single-molecule FRET and circular dichroism experiments, we studied the effect of BZ junctions on Z-DNA formation under high-salt conditions. Further thermodynamic analysis revealed that a discrepancy of different DNA substrates in the presence and absence of BZ junctions in Z-DNA formation can be attributed mainly to the competition between enthalpy and entropy. Salt-induced B-to-Z transition is entropically favored in the presence of BZ junctions and is enthalpically favored in their absence. This thermodynamic information provides a deeper understanding of Z-DNA formation of DNA containing BZ junctions.

Single-Molecule FRET Detection of Sub-Nanometer Distance Changes in the Range below a 3-Nanometer Scale.

Single-molecule fluorescence energy transfer (FRET) detection has become a key technique to monitor intra- and intermolecular distance changes in biological processes. As the sensitive detection range of conventional FRET pairs is limited to 3-8 nm, complement probes are necessary for extending this typical working range. Here, we realized a single-molecule FRET assay for a short distance range of below 3 nm by using a Cy2-Cy7 pair having extremely small spectral overlap. Using two DNA duplexes with a small difference in the labeling position, we demonstrated that our assay can observe subtle changes at a short distance range. High sensitivity in the range of 1-3 nm and compatibility with the conventional FRET assay make this approach useful for understanding dynamics at a short distance.

Munc18-1 induces conformational changes of syntaxin-1 in multiple intermediates for SNARE assembly.

In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex.

Selection of DNA Cleavage Sites by Topoisomerase II Results from Enzyme-Induced Flexibility of DNA.

Topoisomerase II cleaves DNA at preferred sequences with different efficiencies; however, the mechanism of cleavage site selection is not known. Here we used single-molecule fluorescence assays that monitor several critical steps of DNA-topoisomerase II interactions, including binding/dissociation, bending/straightening, and cleavage/religation, and reveal that the cleavage site is selected mainly during the bending step. Furthermore, despite the sensitivity of the bending rate to the DNA sequence, it is not an intrinsic property of the DNA itself. Rather, it is determined by protein-DNA interactions.

Improved thickness estimation of liquid water using Kramers-Kronig relations for determination of precise optical parameters in terahertz transmission spectroscopy.

In terahertz transmission spectroscopy, there is a typical problem of thickness uncertainty, which hampers to determine precise optical parameters of samples. In order to resolve this experimental problem, a method optimizing sample thickness using singly subtractive Kramers-Kronig relations is proposed. For tens of micrometers thick water samples, we improved the accuracy of sample thickness by an order of magnitude (up to sub-micrometer) using the algorithm leading to obtain precise optical parameters of water. The broad applicability of the method is demonstrated for measuring various materials in addition to highly absorbing liquid water in the spectral range from 0.3 to 1.6 THz.

Stiffness measurement using terahertz and acoustic waves for biological samples.

A method is proposed to measure sample stiffness using terahertz wave and acoustic stimulation. The stiffness-dependent vibration is measured using terahertz wave (T-ray) during an acoustic stimulation. To quantify the vibration, time of the peak amplitude of the reflected T-ray is measured. In our experiment, the T-ray is asynchronously applied during the period of the acoustic stimulation, and multiple measurements are taken to use the standard deviation and the maximum difference in the peak times to estimate the amplitude of the vibration. Some preliminary results are shown using biological samples.

Z-DNA stabilization is dominated by the Hofmeister effect.

The manner in which Z-DNA is stabilized at high salt concentrations remains unclear. Here, we systematically examine the Z-DNA-stabilizing capabilities of different salts. The strong correlation between the double-stranded DNA denaturation and B-to-Z transition efficiencies indicates that Z-DNA is mainly stabilized by the Hofmeister effect.

Dielectric relaxation change of water upon phase transition of a lipid bilayer probed by terahertz time domain spectroscopy.

We investigate the influence of the 1, 2-ditetradecanoyl-sn-glycero-3-phosphocholine lipid bilayer phases on the water reorientation dynamics with terahertz time domain spectroscopy. The phase of the lipids was controlled by the temperature in the range of 14-35 °C. During the gel-to-fluid phase transition, the hydration water ratio drastically changed from 0.3 to 0.6. The absorption coefficient of the hydration water increased with the temperature in the gel phase and then decreased in the fluid phase. The dielectric relaxation time of the lipid solution decreased initially but then increased after the phase transition. This indicates that the hydration water reorientation dynamics are restricted by lipids and that this phenomenon is pronounced in a biologically relevant fluid phase.