Pierce M L, Ruffner D E
Department of Pharmaceutics and Pharmaceutical Chemistry and Department of Bioengineering, University of Utah, 421 Wakara Way, Suite 318, Salt Lake City, UT 84108, USA.
Nucleic Acids Res. 1998 Nov 15;26(22):5093-101. doi: 10.1093/nar/26.22.5093.
Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention.
反义介导的基因抑制利用短的互补DNA或RNA寡核苷酸来阻断任何感兴趣的mRNA的表达。反义疗法成败的一个关键参数是在所选mRNA上鉴定合适的靶位点。最终，靶标对反义剂的可及性决定了靶标的适用性。由于可及性是许多复杂因素的函数，目前我们还无法预测。因此，鉴定最有效的靶标需要对每个位点进行检查。为了实现这一目标，我们描述了一种构建针对任何所选mRNA的定向核酶文库的方法。该文库包含几乎等量的靶向所选转录本上每个位点的核酶，并且该文库仅包含能够与该转录本结合的核酶。在培养细胞中表达核酶文库应该能够在自然条件下鉴定最佳靶位点，但要受制于功能完全正常的细胞的复杂性。以这种方式鉴定的最佳靶位点应该是治疗干预的最有效位点。
