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

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• 作者：Jin-Der Wen ; Carla W. Gray ; and Donald M. Gray
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：August 7, 2001
• 年：2001
• 卷：40
• 期：31
• 页码：9300 - 9310
• 全文大小：258K
• 年卷期：v.40,no.31(August 7, 2001)
• ISSN：1520-4995

文摘

The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used toidentify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and37 mages/entities/deg.gif">C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiplecopies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p hashigher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-richsequence, named I-3, was found to have g5p-binding properties that were correlated with a structuraltransition. 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 ofG-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than forthe single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first ofwhich was the formation of an apparent initiation complex consisting of one I-3 strand and about threeg5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiationcomplex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation ofg5p binding and also provide the actual g5p-binding sites.