Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qbeta replicase efficiently binds small...

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Coliphage Qbeta replicase is a key enzyme in viral RNA replication.
• Understanding the precise mechanisms of RNA binding by Qbeta replicase is crucial for studying viral processes.

Purpose of the Study:

• To develop and utilize a sensitive fluorometric assay for studying small RNA binding to Qbeta replicase.
• To determine the binding affinities of various RNAs and DNA oligonucleotides to Qbeta replicase.
• To elucidate the molecular basis of nucleic acid recognition by Qbeta replicase.

Main Methods:

• A pyrene-5'-end-labeled DNA oligonucleotide probe was synthesized and used in a fluorometric assay.
• The assay monitored changes in pyrene fluorescence upon binding of the probe to Qbeta replicase.
• RNA displacement and reverse titration assays were employed to determine binding affinities (equilibrium dissociation constants).

Main Results:

• Stoichiometric binding of the probe to Qbeta replicase caused a significant increase in pyrene fluorescence.
• Replicable RNA addition led to probe displacement, decreasing fluorescence and indicating binding affinity.
• Equilibrium dissociation constants for various RNA strands and tRNAs were in the nanomolar range.
• Qbeta replicase binding site recognizes pyrimidine residues and does not require a 2' hydroxyl group.
• Binding affinity is influenced by base pairing, with thymine interaction lost in A.T pairs but cytosine retained in Watson-Crick pairs.

Conclusions:

• The developed fluorometric assay is sensitive enough for real-time detection of RNA amplification by Qbeta replicase.
• Qbeta replicase exhibits discriminative binding affinities for different RNA strands and non-replicable tRNAs.
• The findings provide insights into the sequence and structural requirements for nucleic acid recognition by Qbeta replicase.