Gc Gc Cg Gc Au Cg Gc Ua Cg Au Cg Cg Ug Uc

E. coli 16S A-site

16S A-site Control

FIGURE 3.3 Sequence and secondary structures of the 27-mer 16S A-site RNA construct (16S) and the 28-mer control RNA construct (16Sc) in which the internal bulge of 16S has been replaced with a duplex region. Base numbering is in reference to full length Escherichia coli 16S rRNA.

16S (MWmin = 8635.1790) contains the essential components of the 16S rRNA A-site, especially the internal A-bulge and the noncanonical U-U base pair. The 16Sc (MWmin = 9300.3388) is a duplexed RNA with the same tetra loop as the 16S. In addition, 16Sc contains an 18-atom hexaethylene glycol chain attached to the 5' terminus of the oligonucleotide that shifts the mass of the construct and minimizes overlaps between ligand complexes with 16S and 16Sc [16]. By screening against these two targets simultaneously, ligands that bind specifically to the internal bulge of 16S can be identified. If a ligand binds equally well to both constructs, then the ligand probably binds to a duplex region or loop region that the two constructs have in common. Thus, the ratio of the abundance of 16Sc to 16S can be used to monitor the specificity of ligand binding. If the ligand binds the 16S specifically, then the 16Sc/16S ratio will increase, since the abundance of 16S will decrease relative to that of 16Sc. Conversely, if a ligand binds the two targets equally well, then the 16Sc/16S ratio will not change, since the abundance of both 16S and 16Sc will decrease. Similarly, in the absence of ligand binding to either target, the 16Sc/16S ratio will not change.

A bacterial fermentation broth from S. rimus sp. paromomycinus was fractionated with high-performance liquid chromatography (HPLC), and the individual fractions were screened against 16S and 16Sc. Two ¡L aliquots of eachLC fraction was combined with a 15-xL solution containing 2.5 ¡M each 16S and 16Sc in 100 mM ammonium acetate and 33% isopropyl alcohol. This mixture was vortexed and incubated for 60 minutes at room temperature prior

16Sc

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