open field arena  (Data Sciences International)

Journal: RSC Chemical Biology

Article Title: Guanine base modifications in antisense oligonucleotides mitigate acute central nervous system toxicity

doi: 10.1039/d5cb00316d

Figure Lengend Snippet: Comparative analysis of hypoxanthine substitutions at different bases reveals guanine-specific mitigation of acute neurotoxicity. (A) Sequences and T m values of LNA-gapmer ASO1 (16-mer) targeting mouse Mapt mRNA. “n.d.” indicates values that were not determined. The terms “a to i,” “t to i,” and “c to i” refer to substitutions in which adenine, thymine, cytosine are respectively replaced by hypoxanthine. Capital letters indicate DNA, bold and italic letters indicate LNA-modified nucleoside, and G, A, T, C, and I represent DNA with guanine, adenine, thymine, cytosine, and hypoxanthine, respectively. (B) Acute tolerability scores in mice assessed 1–4 h after i.c.v. injection of ASO1, ASO1 a to i, t to i, and c to i (9.4 nmol, 50 µg per mouse). (C) Locomotor activity parameters (maximum speed) at 3 h post-injection in the open-field tests. Data are shown as mean ± SEM ( n = 4), except for the ASO1 a to i group, in which one animal died 3 h post-injection ( n = 3) and ASO1 c to i group, in which one animal died 2 h post-injection ( n = 3). (D) Quantitative real-time PCR analysis of relative Mapt mRNA expression (% of vehicle) in the hippocampus. Data are presented as mean ± SEM ( n = 4 per group), except for ASO1 a to i ( n = 2, due to two animal deaths at 3–5 h post-i.c.v. injection) and ASO1 c to i ( n = 3, due to one death at 2 h post-injection). Statistical differences were performed using one-way ANOVA followed by Tukey's post hoc test. (B): vs. ASO1 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (C), (D): vs. vehicle and ASO1 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). No significant differences were observed where p > 0.05.

Article Snippet: Each mouse was placed at the center of a square open-field arena (50 cm × 50 cm × 40 cm; Muromachi Kikai Co., Tokyo, Japan) consisting of a floor and surrounding walls, and allowed to explore freely for 5 min.

Techniques: Modification, Injection, Activity Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: RSC Chemical Biology

Article Title: Guanine base modifications in antisense oligonucleotides mitigate acute central nervous system toxicity

doi: 10.1039/d5cb00316d

Figure Lengend Snippet: Guanine base modifications, particularly 7-deazaguanine, mitigate acute CNS toxicity of gapmer ASO1 targeting Mapt mRNA while maintaining silencing activity. (A) Chemical structures of N 2 -methylguanine, N 2 -isobutylguanine, and 7-deazaguanine. (B) Sequences and T m values of LNA-gapmer ASO1 (16-mer) targeting mouse Mapt mRNA. Capital letters indicate DNA, bold and italic letters indicate LNA-modified nucleoside. All internucleoside linkages are phosphorothioate. G, A, T, C, and I represent DNA with guanine, adenine, thymine, cytosine, and hypoxanthine. ASO1 methyl g, ASO1 isobutyl g, and ASO1 deaza g represent guanine modified to N 2 -methylguanine, N 2 -isobutylguanine, or 7-deazaguanine, respectively. M, N, and D correspond to 2′-deoxyribonucleosides containing these modifications and are highlighted in red. (C) Acute tolerability scores in mice assessed 1–4 h after i.c.v. injection of ASO1, ASO1 g to i, ASO1 methyl g, ASO1 isobutyl g, and ASO1 deaza g (9.4 nmol, 50 µg per mouse). (D) Representative track plots from open-field tests conducted 3 h post-injection. (E) Locomotor activity parameters (maximum speed) assessed 3 h post-injection. (F) Quantitative real-time PCR analysis of relative Mapt mRNA expression in the hippocampus. (G) RNase H–mediated RNA cleavage activity. ASO1/RNA and ASO1 methyl g/RNA duplexes (1.25 µM) were incubated with E. coli RNase H (2.5 mU µL −1 or 10 mU µL −1 ) at 37 °C for 30 min. The reaction was stopped with stop solution and electrophoresed on a 15% denaturing PAGE gel. Gels were stained with GelRed and imaged using the ChemiDoc system. Data are presented as mean ± SEM ( n = 4 per group for panels C, E, and F). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. (C): vs. ASO-treated groups (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (E) and (F): vs. vehicle and ASO1 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Each mouse was placed at the center of a square open-field arena (50 cm × 50 cm × 40 cm; Muromachi Kikai Co., Tokyo, Japan) consisting of a floor and surrounding walls, and allowed to explore freely for 5 min.

Techniques: Activity Assay, Modification, Injection, Real-time Polymerase Chain Reaction, Expressing, Incubation, Staining