Pascolo E, Hudrisier D, Sproat B, Nguyen T T, Toulmé J J
Laboratoire de Biophysique Moléculaire, INSERM U386, Université de Bordeaux II, France.
Biochim Biophys Acta. 1994 Sep 13;1219(1):98-106. doi: 10.1016/0167-4781(94)90251-8.
We investigated the properties of two antisense oligonucleotides, 11 alpha Pso and 14TMP, 11 and 14 nucleotides long, respectively, and conjugated to psoralen derivatives. These oligonucleotides were complementary to the mini-exon sequence of Leishmania amazonensis. Upon ultraviolet (UV) irradiation these oligomers were selectively cross-linked to DNA or RNA target sequences, either 14 or 35 nucleotides long. The yield of photo-addition was much lower on the longer targets than on the shorter ones, due to the presence of a hairpin structure. The co-addition of a helper oligonucleotide, whose binding site, on the 35-mer, was adjacent to that of the psoralen-derivatized antisense oligomer, improved the cross-linking efficiency. We then determined the effect of 14TMP on in vitro translation of Leishmania mRNA in cell-free extracts. Non-irradiated antisense oligonucleotide/mRNA complexes reduced the protein synthesis in wheat germ extract but not in rabbit reticulocyte lysate. Conversely, UV irradiation induced a 14TMP-dependent reduction of translation in reticulocyte lysate whereas the inhibition was not improved in the wheat germ extract. These results are discussed with respect to the involvement of RNase-H in the oligonucleotide-mediated effect on protein synthesis.
我们研究了两种反义寡核苷酸的特性，分别为11αPso和14TMP，长度分别为11和14个核苷酸，并与补骨脂素衍生物偶联。这些寡核苷酸与亚马逊利什曼原虫的小外显子序列互补。经紫外线（UV）照射后，这些寡聚物可选择性地与长度为14或35个核苷酸的DNA或RNA靶序列交联。由于存在发夹结构，较长靶序列上的光加成产率比较短靶序列上的低得多。辅助寡核苷酸的共加成提高了交联效率，该辅助寡核苷酸在35聚体上的结合位点与补骨脂素衍生的反义寡核苷酸的结合位点相邻。然后，我们测定了14TMP对无细胞提取物中亚马逊利什曼原虫mRNA体外翻译的影响。未照射的反义寡核苷酸/mRNA复合物降低了小麦胚芽提取物中的蛋白质合成，但未降低兔网织红细胞裂解物中的蛋白质合成。相反，紫外线照射导致网织红细胞裂解物中翻译的14TMP依赖性降低，而小麦胚芽提取物中的抑制作用未得到改善。就核糖核酸酶H在寡核苷酸介导的蛋白质合成效应中的作用对这些结果进行了讨论。
