Development of a new generation of 2-5A-antisense and its property for the regulation of gene expression.

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1.8-Methyladenosine-substituted 2-5A tetramers with hydroxyalkyl groups at the 5'-phosphates and the corresponding chimeras were synthesized by the phosphoramidite method with a DNA/RNA synthesizer. Incorporation of the hydroxyethyl group into 2-5A tetramer and 2-5A-antisense chimera slightly reduced the abilities of their analogs to activate recombinant human RNase L, but the abilities of the 2-5A tetramer and the 2-5A-antisense chimera both with hydroxyethyl group and 8-methyladenosine returned to 80 and 50% relative to those of oligonucleotides without the hydroxyethyl group and 8-methyladenosine, respectively.2.A novel 2-5A-antisense chimera having two molecules of a 2-5A tetramer at the 5'-terminal of the antisense moiety with a 2-(hydroxymethyl)-1,3-propanediol linker was synthesized. The double-headed 2-5A-antisense chimeras more efficiently cleaved the target RNA.3.We examined the properties of RNA analogs containing 2'-deoxy-2'-α-fluorouridine or 2'-O-methyluridine as inhibitors against human RNase L, that cleaved a single-stranded RNA in the presence of 2',5'-linked oligoadenylate (2-5A).4.Among the single amino acid mutants examined, Y712A and F716A resulted in a significant decrease of RNase activity with a reduced RNA binding activity.5.It was found the analogs containing an acyclonucleoside at the second position and at the third position from the 5'-end were only 9-and 1.7-fold less potent than the parent 2-5A tetramer.6.We presented the crystal structure of the N-terminal ankyrin repeat domain of human RNase L complexed with activator 2-5A containing 2',5' internucleotide linkages. The structure basis for 2-5A recognition by ANK was essential for designing stable 2-5As with high likelihood of activating RNase L.7.The ankyrin-repeat domain of RNase L constricted its structure by binding of 2-5A.8.The synthesis of 2-5A-antisense chimera targeting a viral mRNA and its antiviral activity are now in progress.