RNA aptamers that specifically bind to a 16S ribosomal RNA decoding regionconstruct

RNA-RNA recognition is a critical process in controlling many key biologica l events, such as translation and ribozyme functions. The recognition proce ss governing RNA-RNA interactions can involve complementary Watson-Crick (W C) base pair binding, or can involve binding through tertiary structural in teraction. Hence, it is of interest to determine which of the RNA-RNA bindi ng events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it pos sesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA, It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest b inding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem-loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem-loop region of the A-site 16S rRNA, Point mutations on each of the three nucleotides on th e stem-loop of the aptamer abolish its binding capacity. These studies sugg est that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site dec oding region of 16S rRNA through their respective stem-loops. The most prob able mode of interaction is through complementary WC base pairing, commonly referred to as a loop-loop 'kissing' motif, High affinity binding to the o ther structural motifs in the decoding region were not observed.