Ochi Akinori, Umekage So, Kikuchi Yo
Division of Life Science, Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
Nucleic Acids Symp Ser (Oxf). 2009(53):275-6. doi: 10.1093/nass/nrp138.
Circular RNA is more stable than linear RNA both in vitro and in vivo because of its inaccessibility to exoribonucleases. Therefore, circularization of functional RNAs is a potentially useful methodology for designing therapeutic RNA reagents. We designed a circular hammerhead ribozyme that can cleave the template region of human telomerase RNA. This circular hammerhead ribozyme was generated by in vitro transcription followed by spontaneous self-circularization activity using the permuted intron-exon (PIE) method. Two-dimensional gel electrophoresis and alkaline digestion of the in vitro transcription products revealed that the circular hammerhead ribozyme could be produced by the PIE method. The purified circular hammerhead ribozyme cleaved the template region of human telomerase RNA in a magnesium-dependent manner. These results indicated that the circular hammerhead ribozyme generated by the PIE method maintained the specific feature of canonical hammerhead catalytic activity.
由于环状RNA对外切核糖核酸酶具有不可接近性，因此在体外和体内都比线性RNA更稳定。因此，功能性RNA的环化是设计治疗性RNA试剂的一种潜在有用方法。我们设计了一种环状锤头状核酶，它可以切割人端粒酶RNA的模板区域。这种环状锤头状核酶是通过体外转录，然后使用置换内含子-外显子（PIE）方法进行自发自我环化活性而产生的。体外转录产物的二维凝胶电泳和碱性消化表明，PIE方法可以产生环状锤头状核酶。纯化的环状锤头状核酶以镁依赖的方式切割人端粒酶RNA的模板区域。这些结果表明，通过PIE方法产生的环状锤头状核酶保持了经典锤头状催化活性的特定特征。
