Genetic and Genomics Medicine Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, London, UK.
NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK.
Methods Mol Biol. 2022;2434:53-62. doi: 10.1007/978-1-0716-2010-6_3.
Bifunctional antisense oligonucleotide (AON) is a specially designed AON to regulate pre-messenger RNA (pre-mRNA) splicing of a target gene. It is composed of two domains. The antisense domain contains sequences complementary to the target gene. The tail domain includes RNA sequences that recruit RNA binding proteins which may act positively or negatively in pre-mRNA splicing. This approach can be designed as targeted oligonucleotide enhancers of splicing, named TOES, for exon inclusion; or as targeted oligonucleotide silencers of splicing, named TOSS, for exon skipping. Here, we provide detailed methods for the design of TOES for exon inclusion, using SMN2 exon 7 splicing as an example. A number of annealing sites and the tail sequences previously published are listed. We also present methodology of assessing the effects of TOES on exon inclusion in fibroblasts cultured from a SMA patient. The effects of TOES on SMN2 exon 7 splicing were validated at RNA level by PCR and quantitative real-time PCR, and at protein level by western blotting.
双功能反义寡核苷酸 (AON) 是一种专门设计的 AON，用于调节靶基因的前信使 RNA (pre-mRNA) 剪接。它由两个结构域组成。反义结构域包含与靶基因互补的序列。尾结构域包含招募 RNA 结合蛋白的 RNA 序列，这些蛋白可能在 pre-mRNA 剪接中发挥正或负作用。这种方法可以设计为靶向剪接的寡核苷酸增强子，称为 TOES，用于外显子包含；或设计为靶向剪接的寡核苷酸沉默子，称为 TOSS，用于外显子跳过。在这里，我们提供了设计用于外显子包含的 TOES 的详细方法，以 SMN2 外显子 7 剪接为例。列出了以前发表的一些退火位点和尾序列。我们还介绍了在从 SMA 患者培养的成纤维细胞中评估 TOES 对外显子包含的影响的方法。通过 PCR 和定量实时 PCR 在 RNA 水平，通过 Western blot 在蛋白质水平验证了 TOES 对 SMN2 外显子 7 剪接的影响。
