CLEAVAGES OF TRANSCRIPTS OF TOBACCO MOSAIC VIRUS BY SYNTHETIC RIBOZYMES in vitro

Based upon computer-assisted predictions on the secondary structures of tobacco mosaicvirus (TMV) genomic RNA (both polarities), hammerhead type ribozymes were synthesizedin vitro, which all shared a conserved domain adapted from satellite tobacco ringspot virus(sTobRV)RNA. Ribozymes RZ1, RZ2 and RZ3 were designed to cleave the phosphodiester bondsimmediate to the 3'--end of GUC between the residues 5384-5385 and 6312--6313 on the plusstrand and 1214-1215 on the minus strand, respectively. The in vitro data indicated that RZ1 wasable to cleave completely its substrates BT1(+ ) and BT2(+ ), representing partial sequencesof the plus strand of the TMV MP region at 50, 37 and 30℃ with a molar ratio of ribozymeto the target as low as 1:1. Its two iso-ribozymes RZ1A and RZ1B which were respectivelymodified to contain a CUUCGG sequence in the conserved region and in an additional 3'-ter-minal stem-loop of UUUUUCUUCGGAAAAA were able to cleave BT1(+) and BT2(+) asefficiently as RZ1. Ribozyme RZ3 cleaved, with l     

核酶的体外合成及其对烟草叶病毒RNA靶的作用

通过微机对烟草花叶病毒(TMV)RNA二级结构的分析并参照烟草环斑卫星病毒(sTobRV)的锤头型(hammer head)催化性RNA(即ribozyme或核酶)序列,我们在体外分别合成了以TMV移动蛋白(MP)基因区域正链和负链及正链3’末端序列为靶的锤头型核酶RZ1,RZ3和RZ2。体外结果表明,RZ1在50,37和30℃都能切割含有其靶序列的体外转录TMV RNA BT1(+)和BT2(+);与之相比,RZ3在50,37和30℃都只能部分切割靶RNA BT2(-)。至于RZ2,它对TMV正链3’末端内的靶序列的任何切割作用则未能观察到。我们在核酶RZ1的催化序列的茎环内或其3’末端引入了一个起稳定作用的序列CUUCGG以观察该序列对核酶的影响,实验表明这种修饰并不改变核酶的作用效率。