Design of the structure
To realize the requested goals - observation of changes in twist and length - the following aspects have to be considered.
Proper folding Solid and flexible regions Accessibility for FRET measurements and TEM Modifiability
To obtain good yields the structure is designed based on one single scaffold strand. To minimize aggregation or misfolding of the structure it was folded with and without poly-T staples at the ends. Poly-T staples are usually used to prevent unspecific base stacking.
Solid and flexible regions
To report conformational changes it is necessary to have flexible regions within the origami. The solid parts on the other side are supposed to act as a rigid reference to report the direction of the change and allow an easy orientation of the structure.
Accessibility for FRET measurements and TEM
All DNA origamis exhibit a stiffness sufficient for observation via TEM. For FRET measurements it is crucial to supply positions for fluorophores within the Förster distance (in this case ca. 6.5 nm). The changes in twist and length should affect different FRET pairs in a defined way. Additionally the labels need to remain in the dynamic range for FRET. To measure twisting seperatyl form chages in length the fluorophores need to be on the same horizontal level in comparison to the solid core.
Fluorophores as well as biotin for immobilization must be attached to specific sites.
A set of staples was considered to be at reasonable positions for the fluorophores. These staples can be modified using a terminal transferase which adds labled nucleotides at the 3' end
Biotinylated staples, available in the lab, were attached to adapter stapels
Regarding these considerations we designed a U-shaped origami. This shape enables the positioning of the fluorophores at the same height. The arms are consist of a six helix bundle, thus providing the flexible region required. The bottom part (the base) connecting the two arms is a 18 helix bundle and therefore the stiffer region of our origami.
The structure needs both, solid core and flexible regions which can be used to report the conformational changes. Also the observation of the chages via TEM has to be possible with the structure. arme als reporter, schmale arme in vgl zu sockel twist, twist unabhängig von längenänderung messbar. Spreizen der Struktur??
There are several experiments necessaray to investigate and validate the functionality of the structure using before all the TEM and spectroscopic techniques.
Flexibilität der DNA, Spermine, Ethidium bromide, DAPI
Warum und was wurde untersucht. was ist möglich. Erwartungen.