Lin S B, Blake K R, Miller P S, Ts'o P O
Division of Biophysics, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205.
Biochemistry. 1989 Feb 7;28(3):1054-61. doi: 10.1021/bi00429a020.
EDTA-derivatized oligonucleoside methylphosphonates were prepared and used to characterize hybridization between the oligomers and single-stranded DNA or RNA. The melting temperatures of duplexes formed between an oligodeoxyribonucleotide 35-mer and complementary methylphosphonate 12-mers were 4-12 degrees C higher than those of duplexes formed by oligodeoxyribonucleotide 12-mers as determined by spectrophotometric measurements. Derivatization of the methylphosphonate oligomers with EDTA reduced the melting temperature by 5 degrees C. Methylphosphonate oligomer-nucleic acid complexes were stabilized by base stacking interactions between the terminal bases of the two oligomers binding to adjacent binding sites on the target. In the presence of Fe2+ and DTT, the EDTA-derivatized oligomers produce hydroxyl radicals that cause degradation of the sugar-phosphate backbone of both targeted DNA and RNA. Degradation occurs specifically in the region of the oligomer binding site and is approximately 20-fold more efficient for single-stranded DNA than for RNA. In comparison to the presence of one oligomer, the extent of target degradation was increased considerably by additions of two oligomers that bind at adjacent sites on the target. For example, the extent of degradation of a single-stranded DNA 35-mer caused by two contiguously binding oligomers, one of which was derivatized by EDTA, was approximately 2 times greater than that caused by the EDTA-derivatized oligomer alone. Although EDTA-derivatized oligomers are stable for long periods of time in aqueous solution, they undergo rapid autodegradation in the presence of Fe2+ and DTT with half-lives of approximately 30 min. This autodegradation reaction renders the EDTA-derivatized oligomers unable to cause degradation of their complementary target nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)
制备了乙二胺四乙酸（EDTA）衍生的寡核苷酸甲基膦酸酯，并用于表征寡聚物与单链DNA或RNA之间的杂交。通过分光光度法测量发现，由35聚体寡脱氧核糖核苷酸与互补的12聚体甲基膦酸酯形成的双链体的解链温度比由12聚体寡脱氧核糖核苷酸形成的双链体高4-12摄氏度。用EDTA对甲基膦酸酯寡聚物进行衍生化使解链温度降低了5摄氏度。甲基膦酸酯寡聚物-核酸复合物通过两个寡聚物的末端碱基与靶标上相邻结合位点结合之间的碱基堆积相互作用而稳定。在Fe2+和二硫苏糖醇（DTT）存在下，EDTA衍生的寡聚物产生羟基自由基，导致靶标DNA和RNA的糖-磷酸主链降解。降解特异性发生在寡聚物结合位点区域，对单链DNA的效率比对RNA高约20倍。与存在一种寡聚物相比，添加两个在靶标上相邻位点结合的寡聚物可显著增加靶标降解程度。例如，由两个相邻结合的寡聚物（其中一个用EDTA衍生化）引起的35聚体单链DNA的降解程度比单独由EDTA衍生化的寡聚物引起的降解程度大约高2倍。尽管EDTA衍生的寡聚物在水溶液中长时间稳定，但在Fe2+和DTT存在下会迅速自降解，半衰期约为30分钟。这种自降解反应使EDTA衍生的寡聚物无法导致其互补靶标核酸的降解。（摘要截短于250字）
