The major hurdle associated with utilizing oligo-deoxyribonucleotides 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-C 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-P-BH(3)) modification. This Article describes the automated solid-phase synthesis of mixed-backbone oligodeoxynu-cleotides (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' and 3' exonucleases. When hybridized to a complementary strand, the ODN:RNA duplex was more stable than its corresponding ODN:DNA duplex. The binding affinity of ODNANA 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-P-BH(3) 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.