Solid-Phase Synthesis, Thermal Denaturation Studies, Nuclease Resistance, and Cellular Uptake of (Oligodeoxyribonucleoside)methylborane Phosphine鈥揇NA Chimeras
详细信息 查看全文
- 作者：Heera Krishna ; Marvin H. Caruthers
- 刊名：Journal of the American Chemical Society
- 出版年：2011
- 出版时间：June 29, 2011
- 年：2011
- 卷：133
- 期：25
- 页码：9844-9854
- 全文大小：1061K
- 年卷期：v.133,no.25(June 29, 2011)
- ISSN：1520-5126
文摘
The major hurdle associated with utilizing oligodeoxyribonucleotides for therapeutic purposes is their poor delivery into cells coupled with high nuclease susceptibility. In an attempt to combine the nonionic nature and high nuclease stability of the P鈥揅 bond of methylphosphonates with the high membrane permeability, low toxicity, and improved gene silencing ability of borane phosphonates, we have focused our research on the relatively unexplored methylborane phosphine (Me鈥揚鈥揃H3) modification. This Article describes the automated solid-phase synthesis of mixed-backbone oligodeoxynucleotides (ODNs) consisting of methylborane phosphine and phosphate or thiophosphate linkages (16-mers). Nuclease stability assays show that methylborane phosphine ODNs are highly resistant to 5鈥?and 3鈥?exonucleases. When hybridized to a complementary strand, the ODN:RNA duplex was more stable than its corresponding ODN:DNA duplex. The binding affinity of ODN:RNA duplex increased at lower salt concentration and approached that of a native DNA:RNA duplex under conditions close to physiological saline, indicating that the Me鈥揚鈥揃H3 linkage is positively charged. Cellular uptake measurements indicate that these ODNs are efficiently taken up by cells even when the strand is 13% modified. Treatment of HeLa cells and WM-239A cells with fluorescently labeled ODNs shows significant cytoplasmic fluorescence when viewed under a microscope. Our results suggest that methylborane phosphine ODNs may prove very valuable as potential candidates in antisense research and RNAi.