Raillard S A, Joyce G F
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037, USA.
Biochemistry. 1996 Sep 10;35(36):11693-701. doi: 10.1021/bi960845g.
A variant of the Tetrahymena ribozyme that efficiently cleaves single-stranded DNA under simulated physiological conditions [Tsang, J., & Joyce, G. F. (1994) Biochemistry 33, 5966-5973] was evaluated as a potential therapeutic agent on the basis of its ability to cleave synthetic oligonucleotide substrates corresponding to conserved target sites within HIV-I cDNA. In order to increase the sequence selectivity of the ribozyme, its substrate recognition domain was extended from 6 to 12 nucleotides, allowing base pairing with substrate nucleotides that lie both upstream and downstream of the cleavage site. The sequence of the extended recognition domain could be changed to allow cleavage of a variety of different DNA targets. The ribozyme exhibited a high degree of sequence specificity, discriminating by a factor of 10(2) to more than 10(4) against substrates that form a single-base mismatch with the ribozyme's recognition domain. Mismatches that occurred close to the cleavage site led to a greater decrease in activity compared to those that occurred farther away.
在模拟生理条件下能有效切割单链DNA的四膜虫核酶变体[曾杰和乔伊斯，《生物化学》，1994年，第33卷，第5966 - 5973页]，基于其切割与HIV - I cDNA内保守靶位点对应的合成寡核苷酸底物的能力，被评估为一种潜在的治疗剂。为了提高核酶的序列选择性，其底物识别结构域从6个核苷酸扩展到12个核苷酸，允许与切割位点上下游的底物核苷酸进行碱基配对。扩展识别结构域的序列可以改变，以允许切割多种不同的DNA靶标。该核酶表现出高度的序列特异性，对与核酶识别结构域形成单碱基错配的底物的区分度达10²至超过10⁴倍。与远离切割位点的错配相比，靠近切割位点的错配导致活性下降幅度更大。
