Scherr M, Rossi J J
Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3011, USA.
Nucleic Acids Res. 1998 Nov 15;26(22):5079-85. doi: 10.1093/nar/26.22.5079.
A major concern for antisense experiments is the prediction of effective oligonucleotide binding sites. We have developed a system to carry out oligodeoxyribonucleotide-RNA and ribozyme-RNA binding experiments in cell extracts to create a protein environment known to directly influence the structure of the mRNA. In these experiments the native, endogenous mRNA is probed using oligodeoxyribonucleotides (ODNs) to identify RNase H-accessible sites. The resulting RNase H-mediated cleavages in the cell extracts were quantified using RT-PCR with fluorescein and rhodaminetagged primers to generate fluorescent products that are analyzed and quantified on an automated DNA sequencer. As a model substrate for testing this system, we have targeted the murine DNA methyltransferase (MTase) mRNA. An ODN binding site in native MTase mRNA was identified that was cleaved by endogenous RNase H with an efficiency of 85% in the extracts. The ODN that was most effective in the cell extracts was also found to provide the best activity in vivo , resulting in a 75-85% reduction of the MTase mRNA. These data support the use of cell extracts and native transcripts to identify antisense and perhaps ribozyme target sites.
反义实验的一个主要问题是有效寡核苷酸结合位点的预测。我们开发了一个系统，用于在细胞提取物中进行寡脱氧核糖核苷酸-RNA和核酶-RNA结合实验，以营造一种已知会直接影响mRNA结构的蛋白质环境。在这些实验中，使用寡脱氧核糖核苷酸（ODN）探测天然的内源性mRNA，以识别可被核糖核酸酶H作用的位点。细胞提取物中核糖核酸酶H介导的切割产物，通过使用荧光素和罗丹明标记的引物进行逆转录聚合酶链反应（RT-PCR）进行定量，以生成荧光产物，并在自动DNA测序仪上进行分析和定量。作为测试该系统的模型底物，我们选择了小鼠DNA甲基转移酶（MTase）mRNA。在天然MTase mRNA中鉴定出一个ODN结合位点，该位点在提取物中被内源性核糖核酸酶H切割，切割效率为85%。在细胞提取物中最有效的ODN在体内也具有最佳活性，导致MTase mRNA减少75-85%。这些数据支持使用细胞提取物和天然转录本来鉴定反义以及可能的核酶靶位点。
