Antisense-induced ribosomal frameshifting

Henderson, Clark M.; Anderson, Christine B.; Howard, Michael T.

AZ1A, AZ1B and AZ1C were designed to complement RNA sequences encoded by the originating vector. To determine if duplexes formed between the antisense oligonucleotide and 3 0 adjacent antizyme sequences would result in more efficient frameshift stimulation, reporter vectors were designed to contain a portion of the antizyme 3 0 stimulator. Construct p2luc-AZ1PKm1 contains sequences from the 5 0 half of the axis formed by the stacking of stem 1 and stem 2 of the pseudoknot ( Figure 1A) . Two complementary 2 0 -O-Methyl antisense oligonucleotides were designed. First, PKm1 has perfect complimentarity to the region starting 3 nt and ending 21 nt downstream of the UGA shift site codon. Second, PKm2 is the same except that a mispaired C and bulged A were located at positions 9 and 16, respectively. These two alterations were included to more closely mimic the natural pseudoknot which also contains a mispaired C and bulged A at equivalent positions along the extended stem formed by the stacking of pseudoknot stems 1 and 2 ( Figure 1 ; compare p2luc-AZ1wt with the duplex formed between p2luc-PKm1 and antisense oligonucleotide PKm2). PKm1 and PKm2 induced 30 and 22% frameshifting, respectively, when added to coupled transcription and translation reactions of p2luc-AZ1PKm1 in the presence spermidine ( Figure 4A and B, and Supplementary Table 4 ). Neither PKm1 nor Pkm2 induced frameshifting to the same levels seen with AZ1B, suggesting that the sequence content of the duplex region can affect the efficiency of frameshift stimulation and that native antizyme sequences are not required.