Inhibition of gene expression inside cells by peptide nucleic acids: Effect of mRNA target sequence, mismatched bases, and PNA length
Inhibition of gene expression inside cells by peptide nucleic acids: Effect of mRNA target sequence, mismatched bases, and PNA length
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DOI：
10.1021/bi0020630
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发表时间：
2001-01-09
期刊：
影响因子：
2.9
通讯作者：
Corey, DR
中科院分区：
文献类型：
--
作者：
Doyle, DF;Braasch, DA;Corey, DR
基因组测序已经揭示了数以千计的新基因，重新强调控制基因表达的化学方法。直接从测序产生的信息设计的反义寡聚体是实现这种控制的一种选择。在这里，我们探索的规则，由肽核酸（PNA）细胞内的基因表达的抑制。PNA是一种DNA/RNA模拟物，其中磷酸脱氧核糖骨架已被不带电荷的键取代。与互补序列的结合不受静电排斥的阻碍，其特征在于高缔合速率和高亲和力。在这里，我们测试的假设，PNA的有利性质提供了优势，在体内的mRNA识别和基因表达的反义抑制。我们已经将27个PNA靶向至遍及荧光素酶mRNA的5 ' -非翻译区（5 ' -UTR）、起始位点和编码区的18个不同位点。将PNA作为PNA-DNA-脂质复合物引入培养中的活细胞中，为细胞递送提供了方便的高通量方法。我们发现靶向5 ' -UTR末端的PNA是有效的和序列特异性的反义试剂。长度为15 ~ 18个碱基的PNA是最佳的抑制剂。一个或两个错配的引入消除了抑制，并且针对有义链的互补PNA也是无活性的。与针对末端区域的PNA的有效抑制形成鲜明对比的是，与5 ' -UTR内的其它位点互补的PNA不抑制基因表达。我们还观察到与起始位点或编码区的其余部分互补的PNA没有抑制作用，我们也没有检测到高度C/G富集且对其靶序列具有极高亲和力的PNA的抑制作用。我们的研究结果表明，PNA可以阻断翻译机制的结合，但不太能够阻断核糖体沿着mRNA的进展。PNA的反义抑制的高度特异性强调了PNA作为反义试剂的前景和挑战，并为使用PNA探测细胞内生物靶标的分子识别提供了一般指导。
Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5 ' -untranslated region (5 ' -UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5 ' -UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5 ' -UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells.