ETD

Archivio digitale delle tesi discusse presso l'Università di Pisa

Tesi etd-08262018-113702


Tipo di tesi
Tesi di laurea magistrale
Autore
LOLAICO, MARCO
URN
etd-08262018-113702
Titolo
Optimization of wireframe DNA nanostructures for biomedical applications
Dipartimento
BIOLOGIA
Corso di studi
BIOTECNOLOGIE MOLECOLARI
Relatori
relatore Prof.ssa Ori, Michela
Parole chiave
  • DNA origami
  • DNA nanostructures
  • nanotechnologies
  • molecular dynamics simulations
Data inizio appello
24/09/2018
Consultabilità
Non consultabile
Data di rilascio
24/09/2088
Riassunto
DNA nanotechnology is the field in which DNA molecules are used as building blocks for self-assembly into artificial nanostructures. After the invention of the field in the late '80s by Ned Seeman, the discipline has shown promising results to produce complex structures at the nanoscale. In 2006 the landscape of the field was revolutionized by the introduction of DNA origami, where a long (~7000 bases), viral single-strand DNA (the "scaffold" ) is folded into the desired pattern thanks to the addition of shorter (20-60 bases) DNA single strands (the "staple strands"). This technique was first used to create 2D flat sheets and was later modified to allow the creation of 3D structures by bundle DNA helices in a lattice.

The design space was expanded in 2015 to wireframe structures where a single helix represents a single edge of the structure. This approach uses polyhedral meshes as bases and, through the use of a software package, can produce 3D DNA nanostructures. The method has been later expanded to produce also 2D flat sheets.
The helices in these polyhedral structures are not packed on a tight lattice, so they can assemble and remain stable at physiological salt conditions, unlike previously reported 3D DNA origami. At the same time, it appears from electron microscopy that these structures are not as rigid as the ones based on the tight packing of helices. This can be a problem for some applications that require more rigid structures, for example, the use for study cellular membrane proteins or the use in drug delivery.

The aim of my work is to study the effect of new design choices on the rigidity of DNA wireframe structures. In particular, in this work, internal edges have been used to improve the mechanical rigidity of the nanostructures. These additional edges are formed only by staples strands, not connected to the scaffold strand.

I modified the original software package to permit the introduction of the internal edges into the structure. Using this software, I designed different nanostructures, specifically different rods structures. In these structures, the rigidity can be evaluated by looking at the length of the structure. Different styles have been created for the internal edges, trying to find the best one. In order to test our design choices, I carried out both experimental and \textit{in silico} analysis. The rigidity of the structure has been studied using a coarse-grained molecular dynamics simulation package called oxDNA. In parallel, I studied the correct folding of these nanostructures and the structures have been characterized by agarose gel electrophoresis, transmission electronic microscopy and atomic force microscopy. The kinetics of folding has also been studied in order to overcome experimental issues, like aggregation and low yield.

Combining both in silico and experimental studies I found that the internal edges can be used to improve the rigidity of DNA origami wireframe structures, although additional analysis needs to be done to improve yield and stability, for example in physiological salt conditions. Different design choices can affect in different ways the folding and the aggregation of the structures, leaving a wide space of study for what concern the behaviour and the folding kinetics of nanostructures.
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