Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.

Protein dynamics have been suggested to have a crucial role in biomolecular recognition, but the precise molecular mechanisms remain unclear. Herein, we performed single-molecule fluorescence resonance energy transfer measurements for wild-type maltose-binding protein (MBP) and its variants to demonstrate the interplay of conformational dynamics and molecular recognition. Kinetic analysis provided direct evidence that MBP recognizes a ligand through an 'induced-fit' mechanism, not through the generally proposed selection mechanism for proteins with conformational dynamics such as MBP. Our results indicated that the mere presence of intrinsic dynamics is insufficient for a 'selection' mechanism. An energetic analysis of ligand binding implicated the critical role of conformational dynamics in facilitating a structural change that occurs upon ligand binding.

Evolution of supramolecular chirality from self-assembly of achiral compounds and control over its handedness is closely related to the evolution of life and development of supramolecular materials with desired handedness. Here we report a system where the entire process of induction, control and locking of supramolecular chirality can be manipulated by light. Combination of triphenylamine and diacetylene moieties in the molecular structure allows photoinduced self-assembly of the molecule into helical aggregates in a chlorinated solvent by visible light and covalent fixation of the aggregate via photopolymerization by ultraviolet light, respectively. By using visible circularly polarized light, the supramolecular chirality of the resulting aggregates is selectively and reversibly controlled by its rotational direction, and the desired supramolecular chirality can be arrested by irradiation with ultraviolet circularly polarized light. This methodology opens a route to ward the formation of supramolecular chiral conducting nanostructures from the self-assembly of achiral molecules.

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy.

The dendritic building blocks with a focal pyrene unit self-organize into vesicles in aqueous phase. The in situ inclusion of the focal pyrene units into the cavity of ␤-or ␥-cyclodextrin (CD) induces self-assembled organic nanotubes with an average outer diameter of Ϸ45 nm and inner diameter of 22 nm. The surface of the nanotube is covered with CD. Therefore, the functional group on the surface of the nanotube is controlled simply by modifying the functionality of CD. The removal of CD from the nanotube with poly(propylene glycol) reversibly generates vesicles. This work provides an efficient methodology not only to create an additional class of CD-covered organic nanotubes but also to exhibit reversible transformation of nanotubes and vesicles triggered by the motifs of dendron self-assembly, CD inclusion, and pseudorotaxane formation.vesicle ͉ amphiphile S elf-assembly and transformation of biological or synthetic macromolecules in a wide range of scientific fields are crucial subjects for the achievement of well defined nanostructures and the precise control of the function of supramolecules at the molecular level (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). A multitude of biological or chemical assemblies including vesicle, tubule, fibril, and viral helical coats perform numerous biochemical operations in nature. In particular, vesicular and tubular assemblies are of much interest because of their unique characteristics as a biomimetic system, carrier for drug or gene, biochemical sensor, electronic or photonic material, nanoreactor, and template for hybrid structure (9-18). Therefore, in these viewpoints, self-assembly of synthetic building blocks by noncovalent interactions is expected to provide a unique methodology for creating supramolecular functional materials (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).Self-organization of dendrons (6) into supramolecular assemblies has been demonstrated in a thermotropic fashion (19,27), in aqueous phase (21-25), in organic media (20,22,23,26), and at solid-liquid interface (26). Recently, we reported that the amide dendrons can self-organize in various conditions to exhibit a multiplicity of architectures and functions (22-26). For the preparation of self-assembling nanomaterials, we designed the amide dendritic building blocks consisting of amide branches for hydrogen bonding, carboxyl functionality at the focal point, and alkyl tails for the stabilization of assembled structures by van der Waals interaction (22). Particularly, it was suggested that the dimeric form of the amide dendron, induced by secondary interactions such as hydrogen bonding and -interaction at the focal functional units, is the primary building block in the self-aggregation process in organic media (22,23,26). In addition, the amphiphilic nature of these amide dendritic building blocks provides an opportunity for the formation of various self-assembled nanostructures in aqueous phase. For example, we reported that the transition of the self-organiz...