The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs‐CRISPR). Gibbs free energy analysis suggests that the hairpin‐spacer crRNAs (hs‐crRNAs) suppress Cas13a's affinity to off‐target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high‐specificity of hs‐CRISPR/Cas13a system. Compared to ordinary crRNA, hs‐crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs‐crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system.

The potential carrier role of β-lactoglobulin (β-LG) and its interactions with oxaliplatin were studied using various spectroscopic techniques (fluorescence, UV-visible, and circular dichroism (CD)) in an aqueous medium at two temperatures of 25 and 37 °C in combination with a molecular docking study. Fluorescence measurements have shown that the observed quenching is a combination of static and dynamic quenching with a predominant contribution of static mode. The presence of a single binding site located in the internal cavity of the β-barrel of β-LG was confirmed by molecular docking calculations. Thermodynamic data as well as molecular docking indicated that the hydrophobic interactions dominate in the binding site. Results of fluorescence resonance energy transfer (FRET) measurements in combination with docking results imply that resonance energy transfer occurs between β-LG and its ligand oxaliplatin. Additionally, CD results revealed that oxaliplatin binding has no influence on the β-LG structure. The molecular docking results indicate that docking may be an appropriate method for the prediction and confirmation of experimental results. Complementary molecular docking results may be useful for the determination of the binding mechanism of proteins such as β-LG in pharmaceutical and biophysical studies providing new insight in the novel pharmacology and new solutions in the formulation of advanced oral drug delivery systems.

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In recent decades, considerable efforts have been made to understand the mechanism of memory, cognition, and relevant neurodegenerative diseases in the human brain. Several studies have shown the importance of microtubule proteins in the memory mechanism and memory dysfunction. Microtubules possess dynamicity, which is essential for functions of neuronal networks. Microtubule-associated proteins, i.e., tau, play vital roles in microtubule stability. On the other hand, the ferromagnetic mineral magnetite (Fe(3)O(4)) has been detected in the normal human brain, and elevated levels of magnetite are also observed in the brains of Alzheimer's disease patients. Therefore, we propose that a relationship between microtubule organization in axons and brain magnetite nanoparticles is possible. In this study we found alterations of microtubule polymerization in the presence of increasing concentrations of magnetite through transmission electron microscopy images and a turbidimetry method. Structural changes of microtubule and tau protein, as an essential microtubule-associated protein for tubulin assembly, were detected via circular dichroism spectroscopy, intrinsic fluorescence, and 8-anilino-1-naphthalenesulfonic acid fluorometry. We predicted three possible binding sites on tau protein and one possible binding site on tubulin dimer for magnetite nanoparticles. Magnetite also causes the morphology of PC12 cells to change abnormally and cell viability to decrease. Finally, we suggest that magnetite changes microtubule dynamics and polymerization through two paths: (1) changing the secondary and tertiary structure of tubulin and (2) binding to either tubulin dimer or tau protein and preventing tau-tubulin interaction.