Direct In Vitro Selection of Hemin-Binding DNA Aptamer with Peroxidase Activity

A novel hemin-binding DNA was synthesized using in vitro selection (SELEX) method. The selected DNA not only bound to the hemin. but also had peroxidase activity when complexed with hemin. A pool of 104-nt single-stranded DNA (ssDNA) molecules containing a randomized sequence of 60nt was synthesized. The DNA pool having random sequences was incubated with a hemin-immobilized affinity column. Bound DNAs were eluted with hemin solution and amplified by PCR with biotin-labeled primers. ssDNAs were isolated from the biotin-labeled double-stranded DNA (dsDNA) molecules after each selection process. After four rounds of selection process, the selected DNAs were cloned and sequenced. Four DNA aptamers were chemically synthesized from the sequenced clones. Two aptamers exhibited binding, affinity to hermin and peroxidase activity. A 21-nt oligonucleotide was designed from the aptamer sequence that formed a complex with hermin and exhibited high peroxidase activity.