DNA DIRECTED ASSEMBL · DNA-heme conjugates for the reconstitution and DNA directed assembly of redox enzymes
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2005-05-01 → 2007-04-30
- EU contribution
- €146,320
- Participants
- 1
- Scheme
- IIF
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Results in brief
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.
Data: CORDIS, © European Union
Project objective
Currently there is a great interest in using DNA as scaffold for the assembly of nanoscale arrays. The DNA directed assembly of proteins at solid substrates (glass or metal) or at the surface of nanoparticles is an exciting area of DNA nanotechnology, which will open novel ways of utilising the large variety of highly functional proteins. Nowadays the scientific and technological interest is rapidly moving towards the production of enzyme arrays. The proposed project is aimed to explore a new concept for generating micro and nanostructured redox enzyme -DNA conjugates by means of DNA directed assembly. This will be achieved by covalently linking heme with a single stranded DNA and use of the resulting DNA-heme conjugate for reconstitution of heme dependent enzymes. Subsequently, the DNA tagged enzymes will be immobilised on gold nanoparticles, which contain complementary capture DNA.The redox enzymes adsorbed on the gold surface will be characterised by electrochemical and spectroscopie means, including novel sensitive technique; surface enhanced resonance Raman scattering (SERRS). Additionally, the DNA-enzyme-gold nanoparticle conjugates will be immobilised at macroscopic gold electrode byDNA hybridisation, taking the advantage of the second coding DNA sequence attached to the gold nanoparticles. These experiments should provide the basis for continuing research devoted to the DNA directed assembly of micro and nano-structured enzyme arrays, which have many possible applications in the area of biosensing and material's research. Moreover, the results of this work will also provide information useful for the production of micro-arrays that can be utilised for screening of enzymes in the unknown sample as well as for the investigation of the activity of enzyme libraries.
Original text from CORDIS.
Participants
- UNIVERSITÄT DORTMUND · DORTMUNDCoordinatorCity levelGermany
Links
Data: CORDIS, © European Union