SELEX selection of high-affinity oligonucleotides for bacteriophage Ff gene 5 protein.
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 degrees 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.
Wen
J D
JD
Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688, USA.
Gray
C W
CW
Gray
D M
DM
D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017912:Inovirus / Q000235:genetics*; D008024:Ligands; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D009994:Osmolar Concentration; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012492:Salts; D017422:Sequence Analysis, DNA / Q000379:methods; D017384:Sequence Deletion; D012965:Sodium Chloride; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008024:Ligands; D009841:Oligonucleotides; D012492:Salts; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; D012965:Sodium Chloride