Homogeneous DNA sensing using enzyme-inhibiting DNA aptamers.
A novel aptameric enzyme subunit (AES) which can detect target DNAs has been developed. AES is an enzyme-inhibiting aptamer bearing a target-molecule binding site which can allosterically control enzymatic activity. The thrombin-inhibiting aptamer bearing a G-quartet structure was chosen as the enzyme-inhibiting aptamer. The stem-and-loop structure, which contains a strand complementary to the target DNA, was inserted into the G-quartet structure of the thrombin-inhibiting aptamer to disrupt the G-quartet structure through the hybridization of the target DNA with the complementary strand in the AES. The disruption of the G-quartet structure led to a decrease of the inhibitory activity of the whole aptameric complex. Using this designed aptamer, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution without bound/free separation, and the lower detection limit was 20 nM.
Yoshida
Wataru
W
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-machi, Koganei, Tokyo 184-8588, Japan.
Sode
Koji
K
Ikebukuro
Kazunori
K
D052158:Aptamers, Peptide; D001483:Base Sequence; D015374:Biosensing Techniques; D004247:DNA / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D021122:Protein Subunits / Q000032:analysis; D017422:Sequence Analysis, DNA / Q000379:methods; D013917:Thrombin / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics
D052158:Aptamers, Peptide; D021122:Protein Subunits; D004247:DNA; D013917:Thrombin