The effect of structure in a long target RNA on ribozyme cleavage efficiency
The effect of structure in a long target RNA on ribozyme cleavage efficiency
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DOI：
10.1093/nar/25.24.4985
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发表时间：
1997-12-15
影响因子：
14.9
通讯作者：
Hagen, M
中科院分区：
文献类型：
--
作者：
Campbell, TB;McDonald, CK;Hagen, M
通过催化RNA(核酶)抑制基因表达需要核酶有效地切割大靶RNA中的特定位置。然而，核酶对长靶RNA的切割效率远低于对短寡核苷酸底物的切割效率，这是因为长靶RNA中存在较高的有序结构。为了进一步研究长靶RNA结构对核酶切割效率的影响，我们确定了含有人类免疫缺陷病毒1型(HIV-1)vif-vpr的靶RNA中7个锤头状核酶切割位点的可及性。然后通过化学修饰图谱评估每个切割位点上单个核苷酸的碱基配对有效性，锤头状核酶切割长目标RNA的能力与切割位点附近与核酶进行碱基配对的核苷酸的可用性最相关，而且，长目标RNA中七个锤头状核酶切割位点的可及性变化高达400倍，但直接取决于与核酶进行碱基配对的切割位点的可用性。因此，立体干扰不太可能影响锤头状核酶的切割，切割位点结构的化学修饰作图因此可能提供一种手段来鉴定长靶RNA中有效的锤头状核酶切割位点。
Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif-vpr. The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping, The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme, Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. it is therefore unlikely that steric interference affected hammerhead ribozyme cleavage, Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.