Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system.
We propose a novel method to prepare a DNA-protein conjugate using histidine-tag (His-tag) chemistry. Oligo-DNA was modified with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni(2+). Investigations using a microplate which displayed a complementary DNA-strand revealed that a NTA-modified DNA-protein conjugate was formed and immobilized in the presence of Ni(2+) on the microplate. We then adopted alkaline phosphatase (AP) as a model protein, and application of the DNA-AP conjugate was demonstrated in a thrombin aptamer-based detection system with a detection limit of approximately 10 nM.
Shimada
Josui
J
Department of Applied Chemistry, Graduate School of Engineering, Center for Future Chemistry, Kyushu University, 744 Moto-oka, Fukuoka, 819-0395, Japan.
Maruyama
Tatsuo
T
Hosogi
Takuya
T
Tominaga
Jo
J
Kamiya
Noriho
N
Goto
Masahiro
M
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results
D052157:Aptamers, Nucleotide; D011506:Proteins; D011994:Recombinant Proteins; D006639:Histidine; D004247:DNA; D000469:Alkaline Phosphatase; D009571:Nitrilotriacetic Acid