PHOTOCHEMICAL CROSS-LINKING OF PSORALEN-DERIVATIZED OLIGONUCLEOSIDE METHYLPHOSPHONATES TO RABBIT GLOBIN MESSENGER-RNA
PHOTOCHEMICAL CROSS-LINKING OF PSORALEN-DERIVATIZED OLIGONUCLEOSIDE METHYLPHOSPHONATES TO RABBIT GLOBIN MESSENGER-RNA
复制标题
DOI：
10.1021/bi00426a008
复制
发表时间：
1988-12-27
期刊：
影响因子：
2.9
通讯作者：
MILLER, PS
中科院分区：
文献类型：
--
作者：
KEAN, JM;MURAKAMI, A;MILLER, PS
针对 mRNA 或前体 mRNA 各个区域的反义寡脱氧核糖核苷甲基磷酸盐是无细胞系统和培养细胞中 mRNA 表达的选择性抑制剂。通过引入能够与mRNA交联的可光激活的补骨脂素衍生物，可以显着提高甲基膦酸酯寡聚物与mRNA相互作用并由此抑制翻译的效率。与兔α-或β-球蛋白mRNA的编码区互补的寡核苷甲基膦酸酯通过将补骨脂素基团经由核酸酶抗性氨基磷酸酯键连接至寡聚物的5''端而用4''-(氨基烷基)-4,5'',8-三甲基补骨脂素衍生化。补骨脂素基团和寡聚物5''末端之间的距离可以通过改变氨基烷基连接臂中的亚甲基的数量来调节。补骨脂素衍生的寡聚物与目标 mRNA 上的互补序列特异性交联。例如，与α-珠蛋白mRNA的核苷酸56-67互补的寡聚物在4℃照射寡聚物和兔珠蛋白mRNA的溶液后与α-珠蛋白mRNA特异性交联。 C.用4''-[[N-2(2-氨基乙基)氨基]甲基]-4,5'',8-三甲基补骨脂素衍生的低聚物与mRNA的交联程度最高。交联程度还取决于寡聚物的链长和寡聚物结合位点的结构。与对单链特异性核酸酶水解敏感的mRNA区域互补的寡聚物的交联程度比与对核酸酶水解不敏感的区域互补的寡聚物大约10-30倍。其中补骨脂素基团与 mRNA 中的尿嘧啶残基相对的寡聚物的交联程度比补骨脂素基团与胞嘧啶残基相对的寡聚物高约 10 倍。 5-20μM补骨脂素衍生的寡聚物与珠蛋白mRNA的交联导致靶mRNA翻译的特异性抑制达到40-60％的程度，这与寡聚物的交联程度一致。补骨脂素衍生的寡核苷甲基膦酸酯与 mRNA 单链区域特异性交联的能力、交联寡聚物对 mRNA 翻译的抑制作用以及这些寡聚物在含血清培养基中相对较长的半衰期表明这些核酸类似物可用于特异性控制活细胞中的 mRNA 表达。
Antisense oligodeoxyribonucleoside methylphosphonates targeted against various regions of mRNA or precursor mRNA are selective inhibitors of mRNA expression both in cell-free systems and in cells in culture. The efficiency with which methylphosphonate oligomers interact with mRNA, and thus inhibit translation, can be considerably increased by introducing photoactivatable psoralen derivatives capable of cross-linking with the mRNA. Oligonucleoside methylphosphonates complementary to coding regions of rabbit .alpha.- or .beta.-globin mRNA were derivatized with 4''-(aminoalkyl)-4,5'',8-trimethylpsoralens by attaching the psoralen group to the 5'' end of the oligomer via a nuclease-resistant phosphoramidate linkage. The distance between the psoralen group and the 5'' end of the oligomer can be adjusted by changing the number of methylene groups in the aminoalkyl linker arm. The psoralen-derivatized oligomers specifically cross-link to their complementary sequences on the targeted mRNA. For example, an oligomer complementary to nucleotides 56-67 of .alpha.-globin mRNA specifically cross-linked to .alpha.-globin mRNA upon irradiation of a solution of the oligomer and rabbit globin mRNA at 4.degree. C. Oligomers derivatized with 4''-[[N-2(2-amino-ethyl)amino]methyl]-4,5'',8-trimethylpsoralen gave the highest extent of cross-linking to mRNA. The extent of cross-linking was also determined by the chain length of the oligomer and the structure of the oligomer binding site. Oligomers complementary to regions of mRNA that are sensitive to hydrolysis by single-strand-specific nucleases cross-linked to an approximately 10-30-fold greater extent than oligomers complementary to regions that are insensitive to nuclease hydrolysis. Oligomers in which the psoralen group is opposite a uracil residue in the mRNA cross-linked to approximately a 10-fold higher extent than those with the psoralen group opposite a cytosine residue. Cross-linking of 5-20 .mu.M psoralen-derivatized oligomers to globin mRNA results in specific inhibition of translation of the targeted mRNA to the extent of 40-60%, which is consistent with the extent of cross-linking of the oligomer. The ability of psoralen-derivatized oligonucleoside methylphosphonates to specifically cross-link with single-stranded regions of mRNA, the inhibitory effect of cross-linked oligomers on mRNA translation, and the relatively long half-lives of these oligomers in serum-containing media suggest that these nucleic acid analogues could be used to specifically control mRNA expression in living cells.