DESIGN

What is to accomplish?

The DNA origami design is a functionalized cylinder and was realized by arranging DNA helices on a square lattice. The structure was designed using caDNAno version 2.2.0.

CaDNAno is an open source software with a graphical user interface which aids in designing any 2D or 3D shape or pattern.1

 

Since it is the goal of this project to investigate glycan-receptor interactions, the DNA structure has to meet certain demands:

 

No open ends

As the structure should exhibit great stability, especially with respect to cell applications, it was designed as a closed ring in order to avoid accessible ends of helices. This prevents nucleases, enzymes that are naturally present in and around the cell and which are responsible for the degradation of DNA, from working.2

 

High stability under cellular conditions

As the goal is to determine interactions under cellular conditions, the structure's stability should be able to cope with the typical cells’ environment. Therefore, the structure should exhibit a high stability in general but also concerning low magnesium concentrations.

These aspects justify the choice of designing the DNA structure with two inner rings.2

 

Sufficient surface area for attaching points

Due to the spacial requirements for sugar molecules and fluorophores, the design is not only a single layer ring but a cylinder and hence provides sufficient space for numerous attaching points.2

 

How does it look like?

Figure 1 displays the cross section of the cylinder wall. Based on the p7560 scaffold, the structure consists of three layers and leads to a cylinder with two inner rings. The structure's diameter measures 47.62 nm for the outer ring, 42.22 nm for the middle one and 39.52 nm for the inner ring.

The third layer was designed to prevent a decrease in the structure's stability. It permits an even distribution of targeted insertions and deletions, which are responsible for the desired curvature. Two layers would only allow distributing insertions and deletions in such a way that an unilateral strain would emerge. This is only possible to compensate by a deletion pattern on the third layer.2

As more deletions/insertions lead to higher curvature 3 the inner ring must have the smallest diameter and consequently features a larger amount of deletions than the middle ring. This was achieved by introducing deletions every 8 bp in helix 0,7,12,17 whereas helix 1,6,11,16 displays deletions every 16 bp. Cutting the inner rings is not sufficient. In order to accomplish a proper curvature, the outer ring simultaneously has to show insertions every 16 bp. The ends of the rings are connected with staples ("Connectors").

The connector staples are differently extended at the two ends of the origami design. The energy which is necessary to attach a staple strand to the scaffold depends on its length. Assembling the scaffold strand with staples of long base sequence demands more energy than attaching shorter staple strands. Hence, high folding temperatures foster the binding of long staples whereas short staples rather bind at lower temperatures.

To increase the probability that the connector staple strands do not crosslink multiple scaffolds, the connector staples has a short end and a long end. Thus, these oligonucleotides bind at different temperatures and times during the folding process. That reduces the probability of the structures to cluster.2

 

How is it functionalized?

The structure is functionalized by a systematic incorporation of click oligonucleotides ("click oligos") - staple strands that are chemically modified by carrying an alkyne group at the 3 ‘ - end in order to attach to the sugar molecules.

The origami contains eight click oligos on each helix 4, 9 and 14 in a regular distance of 53/54 bp and thus has a capacity of 24 sugar attaching points.

The selected staple strands are supposed to be orientated outwardly by their 3’ - end.

 

The regular spacing between the sugar attaching points allows for the creation of sugar patterns on the structure. As it is not necessary to exploit the whole capacity of 24 attaching points, it is possible by regularly skipping of some attaching point to generate an origami with particular sugar pattern on its surface.

 

a) Carbohydrates

The carbohydrates used in this project as ligands for special receptors were purchased from CFG functional glycomics gateway and by Sussex research, Ottawa, Canada. The saccharides are equipped with a synthetic spacer 2-azidoethyl ("Sp") which is compatible to the alkyne group of the click oligonucleotides. The following saccharides are involved in the experiments:

 

These saccharides have been selected based on their biological function. The biological function of Lec, 3’ SLex and 2 - Azidoethyl α- Mannopyranoside is partially revealed. Sialyl Lewis X (SLex) is supposed to be one of the most important carbohydrates in the human body as it is a blood group antigen. It is the ligand for different types of selectins, a special type of lectin. SLex interacts with L-selectin to mediate the adhesion of leukocytes to endothelial cell. The binding of SLex to E-selectin is necessary for inflamed tissue to be able to recognize leukocytes whereas binding to P-selectin supports interaction of activated platelets with leukocytes.4 SLex is also an important participant in human fertilization as it is predominantly appearing on the oocyte's coating.5

2 - Azidoethyl α - Mannopyranoside is a mannose-like carbohydrate and interacts with the mannan- binding lectin ("MBL"). MBL is a C-type lectin and plays an essential role in the innate immunity as it identifies patterns of carbohydrates presented on pathogenic cell surfaces.6

The interaction between these carbohydrates and their receptors will be tested with the functionalized origami structure.

First, it is aimed to reproduce the results of these previous studies that had revealed the binding behavior of these carbohydrates. If this is successful, one can begin applying the structure coated with carbohydrates such as P1 tri and Tri-LN - carbohydrates of unknown binding behavior - to specific cell lines that are promising. In doing so, the origami structure will be coated with sugar patterns.

 

The sugar patterns will differ in sugar density and sugar type. The amount of different sugar types presented on a pattern is also varied. Assuming that both, the cell and the origami structure exhibit circular shapes, only a quarter of the structure can be in contact with the cell at the same time. Considering this aspect, the sugar pattern consists of small units that repeat four times.2

 

The sugar patterns will differ in sugar density and sugar type. The amount of different sugar types presented on a pattern is also varied. Assuming that both, the cell and the origami structure exhibit circular shapes, only a quarter of the structure can be in contact with the cell at the same time. Considering this aspect, the sugar pattern consists of small units that repeat four times.2

 

 

b) Fluorophores

Additional modification of the structure is made by integrating fluorescent dyes. Similar to the sugar oligonucleotides the structure contains a maximum of 24 possible attaching points for fluorophores. On each helix 0, 7, 12, 17 six staples are designed in a way that fluorophores can be easily attached by an enzymatic method.

To label DNA origami with fluorophores, the respective oligos are labelled with the dyes before the folding process of the whole structure is initiated.2

 

[1]   DOUGLAS, Shawn M.; MARBLESTONE, Adam H.; TEERAPITTAYANON, Surat; VAZQUEZ, Alejandro; CHURCH, George M.; SHIH; William M.: Rapid prototyping of 3D DNA-origami shapes with caDNAno. In: Nucleic Acids Research (2009), June

[2] Kilwing, Luzia: DNA origami probe for studying carbohydrate-lectin interactions. Bachelorthesis, 2015

[3] DIETZ, Hendrik; DOUGLAS, Shawn M.; SHIH, William M.:  Folding DNA into twisted and Curved Nanoscale Shapes. In: Science 325 (2009), August, no. 5941, 725-730. - DOI 10.1126/science.1174251

[4] VARKI, Ajit:  Biological roles of oligosaccharides: all of the theories are correct. In: Glycobiology 3 (1993), January, No. 2, 97-130. -DOI 10.1093/glycob/3.2.97

[5]  PANG, Poh-Choo; CHIU, Philip C.N.; LEE, Cheuk-Lun; CHANG, Lan-Yi; PANICO, Maria ; MORRIS, Howard R.; HASLAM, Stuart M.; KHOO, Kay-Hooi; CLARK, Gary F.; YEUNG, William S. B.; DELL, Anne:

 Human Sperm Binding Is Mediated by the Sialyl-Lewisx Oligosaccharide on the Zone Pellucida. In: Science 333 (2011), September, no. 6050, 1761-1764. -DOI 10.1126/science.1207438

[6] FRASER, Iain P.; KOZIEL, Henry; EZEKOWITZ, R. Alan B.: The serum mannose-binding protein and the macrophage mannose receptor are pattern recognition molecules that link innate and adaptive immunity. In:  Elsevier 10 (1998), October, no. 5, 363-372.-DOI 10.1006/smim.1998.0141