Final Activity Report Summary - DNA DIRECTED ASSEMBL (DNA-heme conjugates for the reconstitution and DNA directed assembly of redox enzymes)
The main objective of the project was to use DNA modified heme cofactor to produce semisynthetic DNA-enzyme conjugates, enzymes containing single stranded DNA which can then be used as a handle in programmable enzyme immobilisation (DNA directed immobilization or DDI). This was achieved by developing chemical methodology to synthesise covalent heme-DNA adducts. Heme is a known enzyme cofactor, a molecule that plays a significant role in the function of many important enzymes like peroxidases and cytochromes.
Purified and characterised heme-DNA adducts were used to replace natural heme cofactors in myoglobin and horseradish peroxidase and the enzymatic activity of novel, semisynthetic constructs was studied using kinetic models. Remarkable increase of peroxidase activity was observed in the case of myoglobin - DNA conjugate. DNA strand introduced by chemical means to the enzyme was utilised to attach enzymes to different surfaces in highly selective and efficient way using DNA-complementary DNA hybridisation. Using this methodology, horseradish peroxidase was immobilised on the surface of the gold microelectrode and the electrode containing chip and the activity of the enzyme monitored electrochemically.
This was a successful proof of principle which showed that DNA directed immobilisation is well suited to produce much larger arrays of proteins and lead towards the development of electrochemical enzyme arrays for screening of enzymatic activity.
Purified and characterised heme-DNA adducts were used to replace natural heme cofactors in myoglobin and horseradish peroxidase and the enzymatic activity of novel, semisynthetic constructs was studied using kinetic models. Remarkable increase of peroxidase activity was observed in the case of myoglobin - DNA conjugate. DNA strand introduced by chemical means to the enzyme was utilised to attach enzymes to different surfaces in highly selective and efficient way using DNA-complementary DNA hybridisation. Using this methodology, horseradish peroxidase was immobilised on the surface of the gold microelectrode and the electrode containing chip and the activity of the enzyme monitored electrochemically.
This was a successful proof of principle which showed that DNA directed immobilisation is well suited to produce much larger arrays of proteins and lead towards the development of electrochemical enzyme arrays for screening of enzymatic activity.