Wee Keng Boon, Pramono Zacharias Aloysius Dwi, Wang Jian Li, MacDorman Karl F, Lai Poh San, Yee Woon Chee
Bioinformatics Institute, Singapore, Singapore.
PLoS One. 2008 Mar 26;3(3):e1844. doi: 10.1371/journal.pone.0001844.
Antisense oligonucleotides (AONs) mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency) of 94% of 176 previously reported AONs. Four novel insights are inferred: (1) the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2) engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3) the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4) engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.
反义寡核苷酸（AONs）介导的外显子跳跃为杜氏肌营养不良症提供了潜在的治疗方法。然而，由于缺乏预测其结合可及性的精确方法，有效AON靶位点的鉴定仍不尽人意。本研究证明了共转录前体mRNA折叠在确定AON靶位点可及性方面的重要性，该可及性对于AON诱导DMD中的选择性外显子跳跃至关重要。由于转录和剪接是串联发生的，AON必须在剪接因子之前与其靶位点结合。此外，共转录前体mRNA折叠形成瞬时二级结构，这会重新分布可及的结合位点。在我们的分析中，为了近似转录延伸，将包含整个靶向外显子的“分析窗口”沿着前体mRNA一次移动一个核苷酸。使用转录分析每个步骤中的序列预测可能的共转录二级结构。如果一个核苷酸在特定步骤的所有预测二级结构中形成互补碱基对，则该核苷酸被认为是“参与的”。AON靶位点中参与核苷酸的频率和定位的相关性解释了先前报道的176个AON中94%的性能（有效性和效率）。由此推断出四个新见解：（1）参与核苷酸频率最低的与最有效的AON相关；（2）靶位点3'或5'端的参与核苷酸比其他位点更能削弱AON性能；（3）与较短的AON相比，较长AON的性能受靶位点3'或5'端参与核苷酸的影响较小；（4）短靶位点3'端的参与核苷酸比5'端更能削弱AON效率。
