Selection of genomic sequences that bind tightly to Ff gene 5 protein: primer-free genomic SELEX.

Single-stranded DNA or RNA libraries used in SELEX experiments usually include primer-annealing sequences for PCR amplification. In genomic SELEX, these fixed sequences may form base pairs with the central genomic fragments and interfere with the binding of target molecules to the genomic sequences. In this study, a method has been developed to circumvent these artificial effects. Primer-annealing sequences are removed from the genomic library before selection with the target protein and are then regenerated to allow amplification of the selected genomic fragments. A key step in the regeneration of primer-annealing sequences is to employ thermal cycles of hybridization-extension, using the sequences from unselected pools as templates. The genomic library was derived from the bacteriophage fd, and the gene 5 protein (g5p) from the phage was used as a target protein. After four rounds of primer-free genomic SELEX, most cloned sequences overlapped at a segment within gene 6 of the viral genome. This sequence segment was pyrimidine-rich and contained no stable secondary structures. Compared with a neighboring genomic fragment, a representative sequence from the family of selected sequences had about 23-fold higher g5p-binding affinity. Results from primer-free genomic SELEX were compared with the results from two other genomic SELEX protocols.