RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed “aptamers,” were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs.

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 °C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium ΔG°, ΔH°, ΔS°, ΔC°P, and activation ΔG[Dagger] of S45A at 37 °C are −13.7 ± 0.1 kcal/mol, −21.1 ± 0.5 kcal/mol, −23.7 ± 1.8 cal/mol K, −223 ± 12 cal/mol K, and 20.0 ± 2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the ΔH[Dagger] and ΔS[Dagger] of dissociation, 25.8 ± 1.2 kcal/mol and 18.7 ± 4.3 cal/mol K. The large increases of ΔH[Dagger] and ΔS[Dagger] in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.