Functional Aptamers In Vivo

Some RNA aptamers that were isolated in vitro have also been expressed in vivo to study their biological function within the context of a living pro- or eucaryotic cell. Among them is an aptamer which binds to the reverse transcriptase (Rev) protein of the human immunodeficiency virus type 1 (HIV-1) [42,43,70],This anti-HIV-l-Rev aptamer was cloned into an expression cassette based on the U6 snRNA promoter, in which aptamer transcripts are protected against nuclease degradation to some extent. Transient expression in the nucleus of cultured cells led to 107-109 full-length aptamer transcripts per cell. When anti-HIV-l-Rev aptamer-expressing cells were co-transfected with the HIV-1 provirus, viral replication was efficiently inhibited in these cells, as shown by an assay in which the production of HIV-1 reverse transcriptase was measured [71], 

Another study also investigated the effect of various anti-Rev aptamers in vivo, but from a somewhat different angle. Symensma et al. substituted the wild-type Rev-binding element for the Rev-binding aptamer, and tested whether the synthetic Rev-binding RNA would still be able to substitute the biological function of the wild-type, i.e.Ao facilitate Rev-dependent mRNA transport [72]. Various classes of anti-HIV-1-Rev-binding aptamers were assayed One class closely resembled the wild-type sequence, whereas the 

As stated by Conrad et al. [75], the two studies discussed above suggest that in some instances wild-type sequences are functionally optimal, whereas in other cases wild-type sequences can be readily replaced by alternative sequences. Some RNA-binding sequences, such as the Rev-binding element, obviously are able to function in a structure-independent manner, whereas other RNA-binding sequences, such as the SelB-binding mRNA-SECIS element, function in a structure-dependent manner. 

Growth of cells expressing Reduced growth of cells negative control RNA expressing aptamer RNA 

Tire complementary DNA of these aptamers was inserted in tandem into the untranslated region of an expression plasmid with a (f-galactosidase reporter gene. Chinese hamster ovary (CHO) cells were transfected with the expression plasmid and grown in the presence of H33258. Quantification of P-galactosidase activities 24 hours after transfection revealed that translation was prevented at concentrations of 5-10 mM of H33258 [80]. 

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