Synthesis and characterization of dumbbell oligonucleotides containing loops of phosphodiester , two/ three trimethylene, C3 moieties in each loop and phosphorothioate linkages is described. Incubation of dumbbell oligonucleotides containing loops of phosphorothioate with S-1 did not result in significant cleavage. Binding of dumbbell oligonucleotides to MoMuL Virus reverse transcriptase was evaluated by PAGE band mobility shift assays. Dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. Investigation of inhibition of RNase H activity of reverse transcriptase showed that the dumbbell oligonucleotides containing loops of phosphorothioate are better inhibitor of the retroviral RNase H activity than the dumbbell oligonucleotides containing loops of phosphodiester , two/ three trimethylene, C3 moieties in each loop. Our studies demonstrated that the oligonucleotides containing loops of phosphorothioate displays an IC50 of 18- 25 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.