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oai:figshare.com:article/34027270 2026-09-29T18:19:23Z category_1 category_4 category_146 category_8 category_12 category_14 category_46 category_61 category_135 portal_63 item_type_6 month_year_09_2026

Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex Gabriel Lamothe (9921765) Felix Veillette (25138525) Idorenyin A. Iwe (25138528) Ariel Corsano (19639903) Suman Shrestha (20107207) Serena Singh (2658559) Camille Bouchard (25138531) Kelly Godbout (25138534) Yaoyao Lu (2839361) Joel Rousseau (25138537) Keith Pardee (603874) Jacques P. Tremblay (9921768) Biophysics Biochemistry Space Science Microbiology Cell Biology Molecular Biology Immunology Developmental Biology Computational Biology less target sequence improved structural selectivity functional assays indicate electrophoretic mobility shift constrains target selection duplex architecture provides less dna strand proximal duplex crispr dna activator chimeric dna pd duplex weakening hybridization ultraspecific platform spatially separating rna increased resulting split programmable rna nucleotide specificity nucleotide polymorphisms mediated stabilization fluorescence polarization facilitate binding either fragment diagnostic applications competent complex adjacent protospacer adjacent motif CRISPR-Cas12a is a programmable RNA-guided endonuclease whose activation by double-stranded DNA normally depends on the recognition of an adjacent protospacer-adjacent motif (PAM), which constrains target selection in diagnostic applications. Here, we divide the DNA activator into PAM-proximal (Pp) and PAM-distal (Pd) duplexes, spatially separating the PAM requirement from the interrogation of a PAM-less target sequence. FRET, electrophoretic mobility shift, fluorescence polarization, and functional assays indicate that either fragment can associate with Cas12a, but that robust productive activation requires the PAM-containing Pp duplex together with the Pd duplex. Our data further support a model in which PAM-mediated protein–DNA contacts stabilize an assembly-competent complex and facilitate binding of the PAM-less Pd target capture. Weakening hybridization between the guide and Pp with a chimeric DNA/RNA increased the dependence on PAM-mediated stabilization and improved structural selectivity in the Pp region and single-nucleotide specificity in the Pd region. The resulting split-duplex architecture provides an ultraspecific platform for resolving single-nucleotide polymorphisms in the PAM-less DNA strand. 2026-09-29T00:00:00Z Text Journal contribution 10.1021/acs.analchem.6c03855.s001 https://figshare.com/articles/journal_contribution/Split_Cas12a_Protospacer_Engineering_Enables_Ultraspecific_Recognition_of_a_PAM-Less_DNA_Strand_via_an_External_PAM-Proximal_Duplex/34027270 CC BY-NC 4.0