Journal: Nature Communications

Article Title: Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection

doi: 10.1038/s41467-025-60265-8

Figure Lengend Snippet: A Schematic representation of spatial hindrance introduced by streptavidin binding to scaffold-biotin via biotin-SA binding. [Created in BioRender. Hu, H. (2025) https://BioRender.com/gzhemn8 ]. B Fluorescence curves after adding different concentrations of SA to the system. The final concentrations of the system were Cas12a, 20 nM; scaffold, scaffold-biotin and spacer, 20 nM; activator dsDNA, 60 nM; ssDNA reporter, 100 nM. Reaction condition: 37 °C, 50 μL, 50 mM NaCl, 10 mM Tris-HCl, 14 mM MgCl2, 100 µg/ml recombinant albumin. The incubation time was 30 min. 1× represents the concentration of 20 nM. The final concentrations of SA were 0×, 0.25×, 0.5×, 1×, 2×, and 4×. C Schematic representation of biotin competitively binding SA to promote Cas12a activation. [Created in BioRender. Hu, H. (2025) https://BioRender.com/gzhemn8 ]. D Rise in fluorescence of the system after the addition of free biotin. The basic reaction conditions were consistent with B , and the final concentration of free biotin was 1 μM. E Optimization of incubation time for competitive binding of free biotin and scaffold-biotin to SA. The incubation times were set to 0.5 h, 0.75 h, 1 h, and 1.5 h. The experiments were conducted in three technical replicates and error bars represent mean ± SD ( n = 3). F The sensitivity analysis for different concentrations of biotin. G The selectivity analysis among GSH, acrylamide, m6A, glycine, glucose,biotin. The final concentration of all small molecules was 1 μM. The experiments were conducted in three technical replicates and error bars represent mean ± SD ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Streptavidin was purchased from Yeasen Biotechnology (Shanghai, China). m6A antibody (68055-1-Ig) was purchased from Proteintech Group (Wuhan, China).

Techniques: Binding Assay, Fluorescence, Recombinant, Incubation, Concentration Assay, Activation Assay

Journal: Nature Communications

Article Title: Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection

doi: 10.1038/s41467-025-60265-8

Figure Lengend Snippet: A Schematic of SBS-Cas approach based on antigen-antibody binding. [Created in BioRender. Hu, H. (2025) https://BioRender.com/gzhemn8 ]. B Structure of scaffold-m6A1 and inhibition of Cas12a activity by anti-m6A antibody under 1× PBS buffer conditions (pH 7.4). The concentrations of the system were Cas12a, 20 nM; scaffold-m6A1 and spacer, 20 nM; activator dsDNA, 60 nM; antibody 20 nM, ssDNA reporter, 100 nM. Reaction condition: 37 °C, 50 μL, 50 mM NaCl, 10 mM Tris-HCl, 14 mM MgCl2, 100 µg/ml recombinant albumin. The incubation time was 30 min. C The m6A structure and the position of m6A in place of rA on the scaffold. D Optimization of m6A modification positions on scaffold. The experimental conditions were set as in B . The experiments were conducted in three technical replicates and error bars represent mean ± SD ( n = 3). E Heatmap for optimization of antibody concentration (1× represented 20 nM) and incubation time. The antibody concentration were set to 0.5×, 1×, 1.5× and 2×. The incubation times were set to 0.5 h, 1 h, and 2 h. F The sensitivity analysis for different concentrations of m6A. G The selectivity analysis among GSH, acrylamide, biotin, A, G, C, T and m6A. The final concentration of all small molecules was 4 μM. The experiments were conducted in three technical replicates and error bars represent mean ± SD ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Streptavidin was purchased from Yeasen Biotechnology (Shanghai, China). m6A antibody (68055-1-Ig) was purchased from Proteintech Group (Wuhan, China).

Techniques: Binding Assay, Inhibition, Activity Assay, Recombinant, Incubation, Modification, Concentration Assay