Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.
Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.
复制标题
核靶向肽-反义寡脱氧核苷酸缀合物的合成和评估。
DOI：
10.1021/bc00031a012
复制
发表时间：
1995
影响因子：
4.7
通讯作者：
Hinrichsen,RD
中科院分区：
文献类型：
--
作者：
Reed,MW;Fraga,D;Schwartz,DE;Scholler,J;Hinrichsen,RD
内源性核酶RNase H是决定反义寡脱氧核苷酸（ODN）功效的重要组分。为了提高反义ODN的效力，制备了具有三种不同核靶向信号肽的缀合物。这些短肽序列已显示出促进大分子转运到细胞核中。描述了用于合成ODN-肽缀合物的有效化学。5 ′-氨基己基修饰的ODNs与碘乙酸酐反应，以良好的产率得到纯的碘乙酰胺ODNs（IA-ODNs）。这些亲电中间体与含巯基的肽反应，以优异的产率和纯度得到ODN-肽。ODN-肽通过用胰蛋白酶的蛋白水解进一步表征。ssDNA靶的热变性研究表明，5 '-肽修饰对ODN的杂交性质几乎没有影响。在淡水纤毛虫草履虫中评价了核信号肽对反义效力的影响。与钙调素mRNA互补的3 '-己醇修饰的24-mer反义ODN改变了这些细胞的膜离子通道和游泳行为的调节。该ODN的2 '-0-甲基类似物是无活性的，因此提供了草履虫中该活性由RNA酶H介导的证据。采用电穿孔法将反义寡核苷酸-核信号肽偶联物转染入细胞。令人惊讶的是，这些缀合物与5 '-未修饰的对照ODN相比没有显示出反义作用。与5 '端偶联的随机肽或氨基酸不降低反义活性。
An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates isdescribed. Reaction of 5'-aminohexylmodified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA—ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN—peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-0-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN—nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did notdecrease antisense activity.