Siwkowski A, Humphrey M, De-Young M B, Hampel A
Northern Illinois University, DeKalb, USA.
Biotechniques. 1998 Feb;24(2):278-84. doi: 10.2144/98242st05.
Random mutagenesis followed by an in vitro selection procedure was shown to be capable of identifying important bases of the hairpin ribozyme for cleavage of an RNA target sequence. The selection scheme enriched the RNA population for those molecules capable of efficient site-specific self-cleavage in the absence of ligation. Cleavable mutants were selected for all positions in loop 4 except for position A38, supporting the notion that A38 is an important base in the hairpin ribozyme. This has been confirmed by direct mutagenesis, validating the utility of this procedure. Thus, the method developed and reported here has utility for the selection of efficient hairpin ribozymes capable of highly efficient cleavage of a substrate RNA without a requirement for ribozyme-catalyzed ligation, conditions desired for many applications of catalytic RNA such as gene therapy.
随机诱变随后进行体外选择程序，结果表明该方法能够识别发夹状核酶切割RNA靶序列的重要碱基。该选择方案在不进行连接的情况下，富集了能够进行高效位点特异性自我切割的RNA群体。除了A38位点外，对环4中的所有位置都选择了可切割突变体，这支持了A38是发夹状核酶中重要碱基的观点。这已通过直接诱变得到证实，验证了该程序的实用性。因此，本文开发并报道的方法可用于选择能够高效切割底物RNA的高效发夹状核酶，而无需核酶催化连接，这是催化RNA的许多应用（如基因治疗）所期望的条件。
