SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5 Protein

• Published in: Biochemistry (Easton) 2001-08, Vol.40 (31), p.9300-9310
• Main Authors: Wen, Jin-Der, Gray, Carla W, Gray, Donald M
• Format: Article
• Language: English
• Subjects: Bacteriophage M13 - genetics; Base Sequence; Circular Dichroism; Cloning, Molecular - methods; DNA Primers - genetics; DNA Primers - metabolism; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNA, Viral - genetics; DNA, Viral - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; gene 5 protein; Inovirus - genetics; Ligands; Molecular Sequence Data; Oligonucleotides - genetics; Oligonucleotides - metabolism; Osmolar Concentration; Peptide Chain Initiation, Translational - genetics; Phage Ff; Protein Binding - genetics; Salts; Sequence Analysis, DNA - methods; Sequence Deletion; Sodium Chloride; Viral Proteins - genetics; Viral Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010109z

The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 °C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.