smai restriction enzyme  (New England Biolabs)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Orthogonal characterization of rAAV reveals vector attributes that drive ITR repair, self-complementary genome formation, and transgene expression

doi: 10.1016/j.omtn.2026.102899

Figure Lengend Snippet: Recombinant AAV genome element and heterogeneity analysis by native and alkaline electrophoresis (A) Graphic representation of the main recombinant AAV vector genome elements evaluated: wtITR (143 nt, black) versus dITR/wtITR (11-nt deletion in the 5′ C arm, 130 nt, red), EAlbAAT (albumin enhancer-alpha1-antitrypsin promoter, blue), Egfp (enhanced green fluorescent protein, green), pA (bovine/human growth hormone polyadenylation signal, yellow), and CBA (CMV (cytomegalovirus) enhancer-chicken beta actin promoter, orange). Vector genome sizes are shown next to each representation. (B) Native automated electrophoresis of extracted rAAV genomes from dITR/wtITR and wtITR. EalbAAT . Egfp vectors by TapeStation. From left to right: AAV2, AAV3B, AAV5, AAV8, AAV9, and AAVLK03 serotypes. As a run reference, the upper marker (10,000 bp) and lower marker (15 bp) of the ladder are highlighted in purple and green, respectively. (C) Native automated electrophoresis of extracted rAAV genomes from AAV5 and AAV8 serotypes with dITR vs. wtITR and CBA versus EalbAAT promoters. As a run reference, the upper marker (10,000 bp) and lower marker (15 bp) of the ladder are highlighted in purple and green, respectively. (D) Native automated electrophoresis of extracted rAAV genomes from the AAVLK03 serotype with dITR and CBA vs. EalbAAT promoters. As a run reference, the upper marker (10,000 bp) and lower marker (15 bp) of the ladder are highlighted in purple and green, respectively. (E) Native electrophoresis of extracted rAAV genomes from AAV9 and AAVLK03 serotypes with dITR and CBA versus EalbAAT promoters, before and after digestion at 37°C for 1 h with SmaI. (F) Graphic representation of the dITR/tITR design (11-nt deletion in the 5′ C arm and 22-nt deletion of the 3′ B arm, 121 nt, purple), scITR (C arm deletion, 106 nt, green), and the dITR. AAT . Egfp expression cassette including an hypoxanthine phosphoribosyltransferase 1 ( HPRT 1 ) DNA stuffer sequence (GenBank: NG_012329.2 ) (bottom). Vector genome sizes are shown next to each design. (G) Alkaline electrophoresis of AAV8 vectors with different ITR configurations and CBA versus EalbAAT promoters. (H) Alkaline electrophoresis of AAV8 and AAV3B vectors with dITR/wtITR, dITR/tITR, and scITR configurations. (I) Alkaline electrophoresis of AAV8 and AAVLK03 vectors with the dITR/tITR configuration. (J) Alkaline electrophoresis of AAV3B, AAV5, and AAVLK03 vectors with dITR/wtITR configuration and CBA versus EalbAAT promoters. Combinations are displayed on top of each lane. Agarose gel ladder: FastGene 1 kb DNA marker (10,000–100 bp).

Article Snippet: SmaI restriction enzyme (20,000 units/mL; New England Biolabs) was used to digest rAAV genomes for 1 h at 37°C.

Techniques: Recombinant, Electrophoresis, Plasmid Preparation, Marker, Expressing, Sequencing, Agarose Gel Electrophoresis