Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver BC, V6T 1Z1, Canada.
ACS Comb Sci. 2013 Apr 8;15(4):174-82. doi: 10.1021/co3001378. Epub 2013 Mar 13.
The convenient use of SELEX and related combinatorial methods of in vitro selection provides a formidable gateway for the generation of DNA enzymes, especially in the context of improving their potential as gene therapeutic agents. Here, we report on the selection of DNAzyme 12-91, a modified nucleic acid catalyst adorned with imidazole, ammonium, and guanidinium groups that provide for efficient M(2+)-independent cleavage of an all-RNA target sequence (kobs = 0.06 min(-1)). While Dz12-91 was selected for intramolecular cleavage of an all-RNA target, it surprisingly cleaves a target containing a lone ribocytosine unit with even greater efficiency (kobs = 0.27 min(-1)) than Dz9-86 (kobs = 0.13 min(-1)). The sequence composition of Dz12-91 bears a marked resemblance to that of Dz9-86 (kobs = 0.0014 min(-1) with an all-RNA substrate) that was selected from the same library to cleave a target containing a single ribonucleotide. However, small alterations in the sequence composition have a profound impact on the substrate preference and catalytic properties. Indeed, Dz12-91 displays the highest known rate enhancement for the M(2+)-independent cleavage of all-RNA targets. Hence, Dz12-91 represents a step toward the generation of potentially therapeutically active DNAzymes and further underscores the usefulness of modified triphosphates in selection experiments.
SELEX 及其相关的体外选择组合方法的便捷使用为 DNA 酶的生成提供了一个强大的途径，特别是在提高其作为基因治疗剂的潜力方面。在这里，我们报告了 DNAzyme 12-91 的选择，这是一种经过修饰的核酸催化剂，带有咪唑、铵和胍基，可有效实现对全 RNA 靶序列的 M(2+)-非依赖性切割（kobs = 0.06 min(-1)）。虽然 Dz12-91 是为了切割全 RNA 靶分子的分子内切割而被选择的，但它令人惊讶地以比 Dz9-86（kobs = 0.13 min(-1)）更高的效率切割含有单个核糖胞嘧啶单元的靶分子（kobs = 0.27 min(-1)）。Dz12-91 的序列组成与从同一文库中选择切割含有单个核苷酸的靶分子的 Dz9-86（kobs = 0.0014 min(-1)，与全 RNA 底物）有明显的相似之处。然而，序列组成的微小改变对底物偏好和催化特性有深远的影响。事实上，Dz12-91 显示了对全 RNA 靶分子的 M(2+)-非依赖性切割的已知最高的速率增强。因此，Dz12-91 代表了朝着生成潜在治疗活性 DNA 酶的方向迈出的一步，进一步强调了修饰三磷酸在选择实验中的有用性。
