DNA nanocarrier for investigation of glycan-cell interactions
The unique glycosylation pattern of biopolymers and its recognition by the cells plays a crucial role in cellular pathways and the induction of signal transduction.
Recent and ongoing research have identified the importance of glycosylation in a wide range of diseases and illuminated many particular aspects of glycan-receptor interactions, however, knowledge of specific glycosylation patterns is missing in many instances. Therefore, the goal of this project was the development of a basic tool to specifically investigate binding behavior of a variety of cell types in response to nanometer-precise arrangements of a class of sugar molecules.
DNA origami provides an ideal platform to design a nanocarrier for sugar molecules. The designed structure allows quantitative and high-throughput analysis of these interactions by being highly modifiable in three different ways: a) geometrically by realizing sugar patterns on the structure’s surface, b) chemically by altering the type of attached carbohydrates and c) spectroscopically by implementing differently excitable fluorophores.
Motivation and Background
The communication of cells plays a crucial role in understanding live, especially in a multicellular environment. One way cells communicate and apprehend their environment is through production and recognition of distinct glycosylation patterns on proteins, lipids and other organic molecules. Glycosylation is the enzymatic process of attaching a polysaccharide to one of these cellular structures. Polysaccharides are one of the key components of cells along with DNA, RNA and proteins. Their attachment to proteins are a major determinant of their function and location in the cell. Most membrane and secretory proteins in eukaryotic cells are modified in this way.1 Glycosylation is one of the most abundant modifications of proteins, co- as well as post-translationally. It has a wide range of indications for the proteins fate. It has been shown that glycosylation plays an important role in protein stability and folding1, but also that it is possible to control a stem cell’s fate by presenting the right glycosylation pattern, imitating the extracellular matrix.2 It also plays a pivotal role in severe diseases like infections and cancer. Through glycan-receptor mediated cell-cell adhesion, our immune system is capable of detecting and neutralizing foreign cells.3-5 Due to the sheer endless number of possible combination of monosaccharides, their complex modification, difficulties in their characterization and manipulation, glycomics is a still developing field in cell biology. The question of how exactly cellular receptors recognize and process this multitude of information still remains elusive.5
In order to investigate the distinct binding mechanism between cells and glycans or patterns of these molecules, it is necessary to produce well defined structures with the possibility of attaching molecules on a desired location. DNA nanotechnology, an area of research which uses the unique properties of DNA, such as its capability to self-assemble, to be programmable and biocompatible and its technique like DNA origami provides an ideal basis for this.
• Design of a nanocarrier
The project aims to develop a tool for highly specific identification of carbohydrate - protein interactions. Therefore, the goal is the construction of a DNA origami, which is stable in physiological conditions, and its functionalization through different sugar patterns generated on its surface. To satisfy those requirements a cylindrical origami without open DNA ends and large surface area for possible functionalization was designed.
• Functionalization with carbohydrates
The sugar coated origami structure should enable addressing cell types equipped with certain glycan receptors that should bind these functionalized origami structures via their carbohydrates. The individual sugar pattern generated on the origamis surface should identify the presence and localization of those receptors on the surface of selected cell types.
• High-throughput and quantitative analysis of glycan-cell interactions
By the principle of exclusion, researchers can apply the origami to the cell culture of interest and test if the cells shows binding behavior to a carbohydrate which the origami presents on its surface. Visualizing and statistical analyzing of the interactions is achieved by fluorescence microscopy and FACS measurements.
Experimental issues in realizing these goals constitute the design as well as the optimization process of the structure, so that the origami can act as a carrier for the carbohydrates. The intended cell applications prescribe certain demands which the design must meet.
"Life performs computation"
As said by Paul C. Rothmund, developer of the DNA-Origami technique, in 2006, this statement precisely expresses the basic concept at the heart of DNA origami. Beyond its significance in nature, DNA gained a new scope of application during the last decade which exploits the DNA's property to be based on a programmable code and thus uses it as building material.
Until 2006 methods using DNA to construct nanoscale structures still left room for improvement as it was time consuming and was subject to stoichiometric problems such as low yields. Those disadvantages originated from the interaction of numerous short oligonucleotides.
The DNA origami technique eschew this problems by using one long single DNA strands of a known base sequence ("scaffold") and numerous short oligonucleotides ("staples"). Staples are approximately 20 - 60 basepairs (bp) long and serve as small "clips" in order to mould the scaffold strand in a desired shape.6
Because of its length it is technically difficult to synthesize the scaffold strand. Therefore one uses a virus, typically the M13mp18, which naturally carries a 7249 bp long single-stranded genome6. Along with the scaffold the staples complete the double helical structure.
It is the exclusive character of the base pairing which permits to exactly address certain regions of the scaffold strands with the staples. Hence, "programming" the shape requires choosing a staple's base sequence complementary to two distinct regions of the scaffold which generates two locally distant binding sites on the scaffold strand. Consequently the short oligonucleotides can compress the scaffold in an array of parallel helices or any other two-or three-dimensional shape.
3D-designing became possible in 2009 by constraining DNA helices either to a honeycomb or to a square lattice.7
Studies completed by Dietz et al. evince how to achieve twist and curvature by controlled incorporation of basepair insertion and deletion in the DNA origami design.8 That plays a central role in the designing procedure in this project.
The copper catalyzed azide alkyne cycloaddition (CuAAC) process has emerged as the premier example of click chemistry, a term coined in 2001 by Sharpless to describe a set of ‘near-perfect’ bond-forming reactions useful for rapid assembly of molecules with desired function.9
It has since then been used in a wide range of application due to its reliability and simplicity to make covalent connections between building blocks containing various functional groups.10
One of the big advantages of this method are the mild reaction conditions in an aqueous and alcohol-based solutions, that makes it perfect for bioconjugation applications. It has also been wieldy adopted in the fields of organic synthesis, medicinal chemistry, surface and polymerchemistry.10
Although the reaction in its application is simple and very reliable, the exact mechanism still remains elusive.11
For our project alkine modified DNA staples where fused with azide modivied sugars (s. figure 1 and 2).
Figure 1: Copper catalyzed coupling the azide modified sugar (Galβ1-4GlcNAcβ1-3)3β-CH2CH2N3 (Tri-LN) with an alkine DNA.
Figure 2: Copper catalyzed coupling the azide modified sugar Neu5Acα2-3Galβ1-4[Fucα1-3]GlcNAcβ- CH2CH2N3 (SLex) with an alkine DNA.
1 Varki, A. et al. Essentials of glycobiology, 1999. Cold Spring Harber Laboratory Press, New York (1998).
2 Pulsipher, A., Griffin, M. E., Stone, S. E. & Hsieh-Wilson, L. C. Long-lived engineering of glycans to direct stem cell fate. Angew Chem Int Ed Engl 54, 1466-1470, doi:10.1002/anie.201409258 (2015).
3 Gupta, G. & Surolia, A. in Biochemical Roles of Eukaryotic Cell Surface Macromolecules 1-13 (Springer, 2012).
4 Dube, D. H. & Bertozzi, C. R. Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov 4, 477-488, doi:10.1038/nrd1751 (2005).
5 Taniguchi, N., Endo, T., Hart, G. W., Seeberger, P. H. & Wong, C.-H. Glycoscience: Biology and Medicine. (Springer Japan, 2015).
6 Rothemund, P. W. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297-302 (2006).
7 Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414-418, doi:10.1038/nature08016 (2009).
8 Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725-730 (2009).
9 Kolb, H. C., Finn, M. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angewandte Chemie International Edition 40, 2004-2021 (2001).
10 Hein, J. E. & Fokin, V. V. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and beyond: new reactivity of copper(i) acetylides. Chemical Society Reviews 39, 1302-1315, doi:10.1039/B904091A (2010).
11 Spiteri, C. & Moses, J. E. Copper-catalyzed azide-alkyne cycloaddition: regioselective synthesis of 1,4,5-trisubstituted 1,2,3-triazoles. Angew Chem Int Ed Engl 49, 31-33, doi:10.1002/anie.200905322 (2010).