Player M R, Maitra R K, Silverman R H, Torrence P F
Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0805, USA.
Antivir Chem Chemother. 1998 May;9(3):225-31. doi: 10.1177/095632029800900303.
In an attempt to develop a lead for the application of 2-5A-antisense to the targeted destruction of human immunodeficiency virus (HIV) RNA, specific target sequences within the HIV mRNAs were identified by analysis of the theoretical secondary structure. 2-5A-antisense chimeras were chosen against a total of 11 different sequences: three in the gag mRNA, three in the rev mRNA and five in the tat mRNA. 2-5A-antisense chimera synthesis was accomplished using solid-phase phosphoramidite chemistry. These chimeras were evaluated for their activity in a cell-free assay system using purified recombinant human RNase L to effect cleavage of 32P-labelled RNA transcripts of plasmids derived from HIV NL4-3. This screening revealed that of the three 2-5A-antisense chimeras targeted against gag mRNA, only one had significant HIV RNA cleavage activity, approximately 10-fold-reduced compared to the parent 2-5A tetramer and comparable to that reported for the prototypical 2-5A-anti-PKR chimera, targeted against PKR mRNA. The cleavage activity of this chimera was specific, since a scrambled antisense domain chimera and a chimera without the key 5'-monophosphate moiety were both inactive. The 10 other 2-5A-antisense chimeras against tat and rev had significantly less activity. These results imply that HIV gag RNA, like PKR RNA and a model HIV tat-oligoA-vif RNA, can be cleaved using the 2-5A-antisense approach. The results further imply that not all regions of a potential RNA target are accessible to the 2-5A-antisense approach.
为了开发一种将2-5A反义技术用于靶向破坏人类免疫缺陷病毒（HIV）RNA的先导物，通过分析理论二级结构确定了HIV mRNA内的特定靶序列。针对总共11个不同序列选择了2-5A反义嵌合体：gag mRNA中有3个，rev mRNA中有3个，tat mRNA中有5个。使用固相亚磷酰胺化学方法完成了2-5A反义嵌合体的合成。使用纯化的重组人RNase L在无细胞检测系统中评估这些嵌合体的活性，以实现对源自HIV NL4-3质粒的32P标记RNA转录本的切割。该筛选显示，针对gag mRNA的三个2-5A反义嵌合体中，只有一个具有显著的HIV RNA切割活性，与亲本2-5A四聚体相比活性降低了约10倍，与针对PKR mRNA的原型2-5A-抗PKR嵌合体报道的活性相当。该嵌合体的切割活性具有特异性，因为一个乱序的反义结构域嵌合体和一个没有关键5'-单磷酸部分的嵌合体均无活性。针对tat和rev的其他10个2-5A反义嵌合体活性明显较低。这些结果表明，HIV gag RNA与PKR RNA和模型HIV tat-oligoA-vif RNA一样，可以使用2-5A反义方法进行切割。结果还进一步表明，并非潜在RNA靶标的所有区域都可被2-5A反义方法所作用。
