Syntheses of alternating oligro-2 '-O-methylribonucleoside methylphosphonates and their interactions with HIV TAR RNA

Oligonucleotide analogues 15-20 nucleotides in length have been prepared, w hose sequences are complementary to nucleotides in the upper hairpin of HIV TAR RNA. These alternating oligonucleoside methylphosphonates, mr-AOMPs, c ontain 2'-O-methylribonucleosides and alternating methylphosphonate and pho sphodiester internucleotide linkages. The methylphosphonate and phosphodies ter linkages of these oligomers are highly resistant to hydrolysis by exonu clease activity found in mammalian serum and to endonucleases, such as S1 n uclease. The oligomers were prepared using automated phosphoramidite chemis try and terminate with a 5'-phosphate group, which provides an affinity han dle for purification by strong anion exchange HPLC. A 15-mer mr-AOMP, 1676, that is complementary to the 5'-side of the TAR RNA hairpin, including the 3-base bulge and 6-base loop region, forms a 1:1 duplex with a complementar y RNA 18-mer, mini-TAR RNA. The T-m of this duplex is 71 degrees C, which i s similar to that of the duplex formed by the corresponding all phosphodies ter 15-mer. Introduction of two mismatched bases reduces the T-m by 17 degr ees C. The apparent dissociation constant, K-d, for the 1676/mini-TAR RNA d uplex as determined by an electrophoretic mobility shift assay at 37 degree s C is 0.3 nM. Oligomer 1676 also binds tightly to the full length TAR RNA target under physiological conditions (K-d = 20 nM), whereas no-binding was observed by the mismatched oligomer. A 19-mer that is complementary to the entire upper hairpin also binds to TAR RNA with a K-d that is similar to t hat of 1676, a result that suggests only part of the oligomer binds. When t wo of the methylphosphonate linkages in the region complementary to the 6-b ase loop are replaced with phosphodiester linkages, the Kd is reduced by ap proximately a factor of 10. This result suggests that interactions between TAR RNA and the oligomer occur initially with nucleotides in the 6-base loo p, and that these interactions are sensitive to presence and possibly the c hirality of the methylphosphonate linkages in the oligomer. The high affini ties of mr-AOMPs for TAR RNA and their resistance to nuclease hydrolysis su ggests their potential;utility as antisense agents in cell culture.