SUNY at Albany
June 19-23, 2001
Translation inhibition by RNA Padlocks: Kinetics, specificity, and efficacy in vector-based delivery to cultured cells
We have developed novel agents for gene inhibition based on the principle of antisense inhibition, but with certain advantages over "traditional" antisense oligonucleotides and cleaving ribozymes. These molecules, called RNA Padlocksª, hybridize to and become topologically linked with target RNA molecules, creating complexes that are stable under highly denaturing conditions. They strongly inhibit the initiation and elongation of translation at target sites where ordinary antisense RNAs are completely ineffective.
Active RNA Padlock molecules form strong complexes with target RNAs following first order kinetics (0.02/min) and with an affinity of ~ 30 nM. Padlocks modified so that their circular forms were less stable bound with lower affinity. Control Padlocks whose antisense sequences were scrambled or truncated, or contained even a single mismatch (out of 20 nucleotides of complementarity to the target), failed to bind even under nondenaturing conditions. Pulse-chase experiments indicated that strong binding was practically irreversible.
A replication-deficient alphavirus vector was shown to infect and express a marker RNA in 60Ð90% of cells of the mouse macrophage-like line RAW 264.7. Under conditions of 60% infectivity, virally-expressed anti-TNFa Padlocks inhibited by 60% the secretion of TNFa protein in response to stimulation by lipopolysaccharide, suggesting that inhibition is nearly complete in cells that express the Padlocks.
A Padlock complexed with cationic lipids showed substantial stability in 50% human serum. The stability and efficacy of RNA Padlocks suggest that they may be useful for the analysis of gene function and perhaps as human therapeutics.
Brian H. Johnston (1, 2), Babak Alizadeh (1), Ivo Austin (1), Sergei A. Kazakov (1) and Attila Seyhan (1)
Somagenics, Inc.(1) , 325 E. Middlefield Rd., Mountain View, CA 94043;
Department of Pediatrics(2) , Stanford University School of Medicine, Stanford, CA 94305