Relaxase engineering for multiprotein assembly on DNA nanostructures
ABSTRACT: In the last decade, nanostructures made from DNA have been created with any imaginable shape. For the application of these DNA-based nanostructures (DNA origamis) in biomedicine, new approaches are required for covalent coupling of proteins to DNA. In this thesis, we focused in the applica...
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| Format: | doctoral thesis |
| Publication Date: | 2016 |
| Country: | España |
| Institution: | Universidad de Cantabria (UC) |
| Repository: | UCrea Repositorio Abierto de la Universidad de Cantabria |
| Language: | English |
| OAI Identifier: | oai:repositorio.unican.es:10902/8582 |
| Online Access: | http://hdl.handle.net/10902/8582 |
| Access Level: | Open access |
| Keyword: | Relaxasas Conjugación bacteriana Origami de ADN Nanotecnología Bioconjugación Relaxases Bacterial conjugation DNA origami Nanotechnology Bioconjugation |
| Summary: | ABSTRACT: In the last decade, nanostructures made from DNA have been created with any imaginable shape. For the application of these DNA-based nanostructures (DNA origamis) in biomedicine, new approaches are required for covalent coupling of proteins to DNA. In this thesis, we focused in the application of relaxases for site-specific covalent conjugation of proteins to single stranded DNA extensions on DNA origamis. Relaxases are involved in DNA processing for bacterial conjugation, which is a process of DNA transfer from a donor to a recipient cell. Relaxases are capable of forming a covalent phosphotyrosine bond with specific DNA sequences. Three of the four relaxases investigated, TrwCR388 TraIR100 and MobAR1162, showed good binding performance to DNA origamis with high specificity and orthogonality. We have also studied the relaxases as a new tool for polymerization of single and double stranded DNA (dsDNA), or even polymerization of DNA origamis. Our goal was also the improvement of the reaction performed by relaxases. For this purpose, we have modified the recognition sequence of relaxases with secondary structures mimicking the stem-loop recognized and cleaved by replicases. We have also used substrates with a permutation between the nic site and the inverted repeat (reverse conformation), and also substrates with dsDNA around the nic site. We found that the percentage of covalent complexes with TrwCR388 improved significantly with these substrates but not all the evaluated relaxases increased the yield. These studies have helped to gain more insight into the application of relaxases in the field of biotechnology. |
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