Hovig E, Maelandsmo G, Mellingsaeter T, Fodstad O, Mielewczyk S S, Wolfe J, Goodchild J
The Norwegian Radium Hospital, Oslo.
Antisense Nucleic Acid Drug Dev. 2001 Apr;11(2):67-75. doi: 10.1089/108729001750171272.
Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6+8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6+6) and (5+5) nt. Stimulation was particularly strong in the case of the (5+5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.
此前研究表明，骨肉瘤细胞中内源性表达的核酶对S100A4 mRNA的抑制作用可抑制其在大鼠体内的转移。作为使用外源性合成核酶进行类似研究的前奏，我们比较了一系列针对mRNA不同位点的锤头状核酶。这些核酶仅在与底物互补的7个碱基侧翼序列上有所不同，并通过化学修饰来抵抗核酸酶。切割效率差异很大，且与靶RNA的预测二级结构没有明显关联。该系列中活性最高的核酶被选用于进一步优化。延长其侧翼序列会适得其反，即使使用过量核酶也会降低切割效率。使用过量底物（多周转动力学）时，具有6个核苷酸（nt）的茎III和8个nt的茎I的（6 + 8）核酶切割速度最快。尽管这些茎对核酶性能有很大影响，但其优化仍基于经验。通过向具有较短茎（6 + 6）和（5 + 5）nt的核酶中添加促进寡核苷酸可实现更快的切割。在（5 + 5）核酶的情况下，刺激作用尤为强烈，该核酶本身活性较差。不同促进寡核苷酸引起的增强作用与其预期的与RNA杂交的能力呈平行关系，该能力是长度和化学修饰的函数。
