Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qβ replicase efficiently binds small RNAs by recognizing pyrimidine residues

Abstract

Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qβ was studied utilizing a fluorometric assay. A DNA oligonucleotide probe of sequence 5′-d(TTTTTCC) was 5′-end-labeled with pyrene. In this construct, the proximal thymine residues efficiently quench the fluorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state fluorescence increases by two orders of magnitude, the fluorescence anisotropy increases, and a long fluorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state fluorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely reflects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qβ replicase. The effect was utilized to access binding affinities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16°C, equilibrium dissociation constants for different template midi- and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qβ replicase during replication, exhibited discriminative affinities, while their hybrid bound less efficiently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qβ replicase does not require a 2′ hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A·T base-pair, while that with cytosine is retained after Watson-Crick base-pairing. These findings can explain the affinities of RNA-Qβ replicase interactions reported here and in earlier investigations. The sensitivity of the described fluorometric assay allows detection of RNA amplification by Qβ replicase in real-time.