Staining of DNA nanostructures
with ruthenium surfactant
Bachelor thesis
Patrick Borgeld
2009
DNA double helix structure
9 february 2009 – 29 may 2009
Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen
Prof. A. Herrmann, A. Bastian
INDEX
1 STAINING OF DNA-NANOSTRUCTURES...- 1 -
1.1 DNA... -2-
1.2 DNA- SURFACTANT COMPLEXES... -4-
1.3 DNA NANOSTRUCTURES... -6-
1.4 TRANSMISSION ELECTRON MICROSCOPY... -9-
2 PROJECT OBJECTIVES ...- 10 -
3 RESULTS...- 12 -
4 CONCLUSIONS AND OUTLOOK...- 14 -
5 MATERIALS AND METHODS ...- 16 -
5.1 MATERIALS... -16-
5.2 EXPERIMENTAL... -16-
6 SPECTRA ...- 19 -
7 REFERENCES ...- 22 -
8 GLOSSARY ...- 23 -
Fig. 1: Displaying of the DNA nanostructures by AFM (a) and cryo-TEM (b).
These techniques provide relative low contrast pictures. For AFM the resolution is limited by the tip size. DNA has a low electron density and results in a low contrast TEM images. High contrast images can be obtained by TEM, if many scans are overlayed. However, it takes around six weeks to accomplish the desired result.
In this work, the contrast of the TEM images of DNA nanostructures will be improved by introducing a high electron dense atom to DNA. To accomplish this, a DNA-surfactant complex will be synthesized containing a ruthenium atom.
The work of F. Wurm and F. M. Kilbinger has shown that this method works for making micelles visible on a high contrast TEM picture [2]. They synthesized micelles of poly(ethylene oxide)-block-poly(propylene oxide) with ruthenium atoms attached to the vinyl ether end groups of the poly(ethylene oxide) on the outside of the micelles. These micelles were made visible on TEM pictures with high contrast.
In context DNA, DNA nanostructures and surfactant complexes will be described in the next chapters.
1.1 DNA
DNA is most commonly known as the carrier of the genetic information of all living organisms. The structure of DNA was unknown until in 1953 the double helix structure of DNA was confirmed by James D. Watson, Francis Crick, Maurice Wilkins and Rosalind Franklin (fig. 2) [3]. The formation of the double stranded DNA is caused by hybridization of two single-stranded DNA, which is caused by hydrogen bonding between the bases. These two strand run in anti-parallel direction. One runs in 3´-5´ direction, and the other 5´-3´- direction.
Fig. 2: Double stranded DNA
The DNA chain is approximately 20 Angströms wide and one rotation corresponding to ten nucleotides is approximately 34 Angströms long. The strands of DNA are called polynucleotides, which are coiled around a common axis. A nucleotide contains a diesterphosphate group, a sugar and a base. The phosphate-sugar backbone lies on the outside of the DNA helix and is negatively charged. The bases are on the inside of the helix.
There a two types of bases, purines and pyrimidines (fig. 3). The base pairing takes place between purines (adenine and guanine) and pyrimidines (thymine and cytosine). Adenine (A) forms two hydrogen bonds with thymine (T) and guanine (G) forms three hydrogen bonds with cytosine (C).
Fig. 3: DNA backbone
Hydrogen bonds can be formed and broken easily, because the bonds are not covalent. In DNA, these hydrogen bonds can be broken thermally or mechanically.
Furthermore double stranded DNA occurs depending on the environment in different conformations. In fig. 4 the three well known A-, B- and Z-DNA are shown.
Fig. 4: Conformations of DNA, A-DNA, B-DNA, Z-DNA
1.2 DNA- surfactant complexes
As described in chapter 1.1 the negative charged diesterphosphate groups of the DNA exhibit the phosphate-backbone of the DNA. Therefore the negative charges of the phosphate groups are accessible for positively charged ions, like sodium or ammonium ions. This property will be used to create DNA-surfactant complexes.
Positively charged surfactants can be coordinated to the negatively charged backbone of the DNA-helix. In recent studies different kinds of surfactants have been synthesized and coordinated to DNA for different kinds of purposes. For example, Tanaka and Okahata have created a DNA-surfactant complex for analysis of conformational changes depending on the water content [4]. They have synthesized a lipid, containing an ammonium end group and four ester groups. This lipid is added to DNA forming the desired complex (fig. 5).
Fig. 5: DNA-surfactant complex synthesized by Tanaka and Okahata
An other example is to investigate the transfection-related mechanisms at the molecular and the self-assembled level. Safinya et. al. mixed DNA with the lipid DOTAP (dioleoyl phosphatidylethanol amine) and a helper-lipids DOPE (dioleoyl trimethylammonium propane) and DOPC (dioleoyl phosphatidylcholine) [5]. These complexes mimic natural viruses in their ability to carry extracellular DNA across outer cell membranes for gene delivery. When the concentrations of DOPE and DOPC are varied, the structure can transform reversibly into a lamellar structure or a inverted hexagonal structure (fig 6).
Fig. 6: A lamellar structure (left) and an inverted hexagonal structure (right)
The advantage of these complexes is the hydrophobicity caused by the surfactant, which can be used to make the DNA soluble in organic layers. This opens a new field of application of DNA, for example to incorporate DNA in organic materials. In this work the surfactants are used to attach high density atoms to the DNA (see chapter 2).
1.3 DNA nanostructures
DNA molecules have interesting features, which can be used for the design of nanostructures.
There are two types of design possibilities. The first are top down systems where manipulations on microscopic scale are performed to create patterns. The second are bottom up constructions, were molecules usually self assemble due to their molecular recognition properties. Since the 1970s the possibilities were explored. For the first time molecules were joined together with sticky ends.
For example in figure 7 the self-assembly of branched DNA molecules into a cubical shaped structure is depicted. First, four complementary DNA strands self-assemble into a branched junction (step a). Due to the sticky ends several junctions then self-assemble into a cubical- shaped structure (step b and c) [6].
Fig. 7. Assembly of DNA molecules into a stick-cube
The next step would be to form two dimensional arrays. This required a stiffer motive than the stick-cube. This motif can be created by a DNA double-crossover (DX) molecule (fig. 8).
This molecule contains two double helices connected to each other twice through crossover points, the so called Holliday Junctions (HJ). Meiotic DX (MDX) align and cross over to swap genetic information in living systems.
An analogue DX (ADX) to the MDX, contains two strands (red), two crossover strands (blue) and one central crossover strand (green). This structure is used as a tiling unit in the stiff motive. The ADX, labelled A, and a variant, labelled B* are the two types of units that has been used. B* contains an extra DNA domain extending from the central crossover strand.
This extra domain is positioned perpendicular to the ADX molecule. Both units, A and B*, have sticky ends and will form the desired structure[6].
Fig. 8: Two dimensional DNA structure, formed from DNA tiles.
Another bottom-up approach to create a desired two dimensional DNA nanostructure is to fold DNA [7]. The method for folding DNA in to a desired structure is to raster fill the design with a 7-kilobase single stranded scaffold and to hold this scaffold in to place with staple strands. A staple strand is a short polynucleotide chain that is complementary to different parts of the scaffold. These pieces can self-assemble in one step to the desired structure. To design different kinds of structures, first of all a hand made model was created. When the desired shape was established, a computer program aided to make the required DNA- sequence. In each structure multiple angles occur that the DNA has to overcome. After the self assembly of the DNA pieces, the structure can be analysed, commonly by AFM imaging techniques. One example of a desired structure is shown in figure 9.
Fig. 9: a) A DNA handmade origami shape. b) A computer model. c and d) Analysis by AFM.
Not only two dimensional shapes can be produced. From the principle of sticky-ends, three dimensional arrays can be synthesized [1]. For example a dodehedron structure can be formed (fig 10). First three types of DNA single strands will form into a symmetric three-point-star motif (step a). These motives can form into a three dimensional structure in a one pot self- assembly (step b).
Fig. 10: Formation of a dodahedron DNA structure
The analysis of these structures have proven to be time consuming and difficult by TEM imaging techniques. To increase the resolution of these nanostructures by TEM, a high electron dense atom has to be introduced to these structures.
scattered. In areas with low electron density, the electrons can pass through the sample and are detected by an electron microscope (fig. 11). These different areas form a contrast to each other [8]. When the electron density of an area is increased, the contrast will be increased on the formed image.
Fig 11: Electron microscope
2 Project objectives
To achieve high contrast TEM- images of DNA-surfactant complexes, the targets of this thesis are the synthesis of a hydrophobic ammonium bromide salt containing a vinyl ether group, synthesis of DNA-surfactant complex and to attach a high electron dense atom to this construct. A very useful agent to introduce ruthenium, which is a high electron dense atom, is the Grubbs catalyst 1st generation (1) [2]. This catalyst reacts irreversible with vinyl ether group by creating a covalent binding between the ruthenium and the surfactant (fig. 12).
Fig. 12: Reaction of Grubbs catalyst and vinyl group
The pathway of the synthesis of ammonium salt 4 is shown in figure 13. The synthesis consists of two steps, including vinylation of the bromoalcohol 2 to the vinyl ether 3 with vinyl ethyl ether and followed by substitution reaction of the alkylbromide 3 to the ammonium salt 4. The surfactant (4) that will be created contains two long hydrophobic chains. This will increase the solubility of the DNA complex in organic solvents. This is necessary to perform the reaction to create complex 6. The catalyst will react with water. The synthesis route of the DNA-surfactant complex containing the staining agent is depicted in figure 13.
N+
CH3
O CH2 C
H3
C H3
9 N+ 13
CH3
O Ru
C H3
C H3
PCy3 PCy3 Cl
9 Cl 13
(6) (5)
Ru PCy3 PCy3 Cl
Cl Ph
Fig. 13: Reaction scheme
The second objective is to form DNA-surfactant complex 5 and prove its solubility in organic layer. The final objective of this thesis is to display the DNA-molecule on a TEM image, using Grubbs catalyst 1st generation as a staining agent. The Grubbs catalyst will be added to DNA-surfactant complex 5 to form complex 6.
3 Results
In case of the synthesis of the ammonium bromide salt the shown pathway in fig. 14 is successful with a yield of 8% over two steps. The reaction between 11-bromoundecanol (2) and ethyl vinyl ether has to be performed [9]. This reaction results using mercury acetate as Lewis acid in a yield of 11%. The substitution reaction between the bromoalkane 3 and N,N- dimethylhexadecylamine to form the desired ammonium salt 4 results in a yield of 72% [9]. Also the creation of the complex is successful. Two different lengths of DNA (80bp and 3000bp) are used for the formation of complex 5. To synthesize complex 5, both DNA and ammonium salt 4 need to be dissolved in water [10]. DNA forms a highly viscous solution.
Both solutions are put together to form a suspension. The complex with eighty base pairs results in a yield of 59%. The complex with a three thousand base pairs formed a lower viscous solution in water and resulted in a higher yield of 84%.
Br OH
9
C
H2 O CH3
Br O CH2
9
C H3
N
CH3
CH3 13
N+
CH3
O CH2 C
H3
C H3
9 13 N+
CH3
O CH2 C
H3
C H3
9 13
N+
CH3
O Ru
C H3
C H3
PCy3 PCy3 Cl
9 Cl 13
(2) (3) (4)
(6) (5)
Ru PCy3 PCy3 Cl
Cl Ph
11%
Hg(CF3COO)2
6 h, N2, RT acetonitrile, benzene
10 days, 82oC
72%
DNA, H2O 24h, RT
80 bp, 59%
3000 bp, 84%
2h, RT
Fig. 14: Results of synthesis
For the reaction between the complexes and the ruthenium catalyst, the DNA complex has to be dissolved in either dry dichloromethane or dry THF [2]. The 80 bp DNA complex 5 is in a low amount soluble in these solvents. The 3000 bp DNA-complex 5 is soluble in THF, DMSO and dichloromethane. In these solvents a gel is created. For the reaction with the ruthenium catalyst, only the solution is chosen of 3000 bp DNA-complex in dichloromethane because of its solubility in organic layer.
DNA-surfactant complexes are shown, but small packages. This can be the desired structure (6). DNA it self has a width of around two nanometers. One ammonium salt molecule has a length of around 1,5 nanometers. The structure on the image has a average width of around five nanometers. This is the synthesized DNA-surfactant complex 6 (1,5 nm + 1,5 nm + 2 nm
= 5 nm). This surfactant was not suitable for the staining of DNA, single molecules can not be seen.
Fig. 15: TEM image of DNA with ruthenium complex. 25 mg of Grubbs 1st in 1 mL of the DNA-surfactant complex solution, 100x diluted
4 Conclusions and Outlook
The synthesis of the ammonium salt was proven successful by analysis by 1H-NMR, 13C- NMR and by mass-measurements. After the successful formation of the DNA-surfactant complex, the ruthenium catalyst was added and a TEM image was obtained (fig. 15). On this picture small packages could be seen, which had a average width of six nanometers. This is the desired DNA complex 6. With the used surfactant it was not possible to visualize single molecules in TEM. The target was to create a TEM image were single molecules could be seen like in figure 16.
N+
CH3
O Ru
C H3
C H3
PCy3 PCy3 Cl
9 Cl 13
N+
CH3
O Ru
C H3
C H3
PCy3 PCy3 Cl
9 Cl 13
N+
CH3
O Ru
C H3
C H3
PCy3 PCy3 Cl
9 Cl 13
N+
CH3
O Ru
C H3
C H3
PCy3 PCy3 Cl
9 Cl 13 N+
CH3
Ru O PCy3
PCy3 Cl
C H3
Cl CH3
13
9
N+ CH3
Ru O PCy3
PCy3 Cl
C H3
Cl CH3
13
9
N+ CH3
Ru O PCy3
PCy3 Cl
C H3
Cl CH3
13
9
N+ CH3
Ru O PCy3
PCy3 Cl
C H3
Cl CH3
13
9
Fig. 16: DNA-surfactant complex.
One reason why no single molecules can be seen, can be the long distance between the ruthenium and DNA. The chain containing the vinyl ether group has to be shortened and at the same time one chain has to be long enough to maintain the solubility of DNA in organic solvents. An example of such structure is shown in figure 17.
Fig. 17: An example of a new surfactant
5 Materials and Methods
5.1 Materials
Chemicals
All solvents were purchased of pro analysis quality. All reactants and solvents were purchased at Sigma Aldrich and Acros Organics.
TEM images were obtained by Philips CM-10.
GC/MS data were recovered by Hewlett Packard HP 6890 Series GC Systems.
NMR spectra were measured with Oxford NMR devices. 1H-NMR was measured with either the Oxford AS400 or the Oxford AS300. 13-NMR spectra were measured by the Oxford AS200.
5.2 Experimental
11-Bromoundecanylvinylether (3) [9]
To a solution of 11-bromoundecanol (5 g, 184 mmol) in fresh distilled ethyl vinylether (500 mL) was added mercury trifluoroacetate (0.6 g). The reaction mixture was boiled under reflux with stirring under nitrogen flow for 6h.
The mixture was cooled to RT and anhydrous potassium carbonate (5 g) was added. Ethyl vinylether was removed in vacuo. The residue was taken up in dichloromethane (45 mL) and water (45 mL). The organic layer was washed with brine (45 mL) and water (2x 45 mL). The solvent was removed in vacuo. After purification by column chromatography (Hex/DCM3:1) a colourless oil was obtained.
C13H25BrO (277.20 g/mol)
Yield: 7.3g (26 mmol, 11%) 11-bromoundecanylvinylether 3, Rf: 0.58 (Hex/CH2Cl2 3:1).
1H-NMR (CDCl3, 300MHz): δ[ppm] = 6.47 (m, 1H, 2-H); 4.17 (d, 2J= 14 Hz, 1H, 1-H);
3.97(d, 2J= 6.9 Hz, 1H, 1-H); 3.68 (t, 3J= 1.8 Hz, 2H, 3-H); 3.41 (t, 3J= 7.5 Hz, 2H, 14-H), 1.86-1.30 (m, 18H, 4-H, 5-H, 6-H, 7-H, 8-H, 9-H, 10-H, 11-H, 12-H).
Br O CH2
9 1*
2*
3*
4* - 13*
14*
A mixture of 11-bromoundecanylvinylether (0.85 g; 3,1 mmol), N,N-dimethylhexadecylamine (1 g, 3.6 mmol, 1,2 mL), acetonitril (24 mL) and benzene (5 mL) were refluxed for ten days. The mixture was cooled overnight at 4oC. The crude product was filtered and washed with cold ether. A white solid was obtained.
C30H64BrNO (534.7 g/mol)
Yield: 1.2g (2 mmol, 72%) N,N-dimethyl-N-(11-(vinyloxy)undecyl)hexadecan-1-aminium bromide 4
1H-NMR (CDCl3, 300MHz): δ[ppm] = 6.47 (m, 1H, 2-H); 4.17 (d, 2J= 14.1 Hz,1H, 1-H);
3.97 (d, 2J= 6.6 Hz, 1H, 1-H); 3.67 (t, 3J= 6.3 Hz, 2H, 3-H); 3.51 (m, 4H, 13-H, 16-H); 3.43 (s, 6H, 14-H, 15-H); 1.66- 1.30 (m, 46H, 4-H, 5-H, 6-H, 7-H, 8-H, 9-H, 10-H, 11-H, 12-H, 16-H, 17-H, 18-H, 19-H, 20-H, 21-H, 22-H, 23-H, 24-H, 25-H, 26-H, 27-H, 28-H, 29-H);
0.89 (t, 3J= 5.4 Hz, 3H, 30-H).
13C-NMR (CDCl3, 50 MHz): δ[ppm] = 152.1 (C2); 86.3 (C1); 68.2 (C13, C16); 64.1 (C3);
51.5 (C14, C15); 32.2-23.1 (C4, C5, C6, C7, C8, C9, C10, C11, C12, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29); 14.9 (C30)
Formation of DNA complex (5) [10]
N,N-dimethyl-N-(11-(vinyloxy)undecyl)hexadecan-1-amonium bromide (431 mg, 0,79 mmol) was dissolved in water (25 mL). DNA (259mg, 80 bp (2 µm), 3000 bp (0,17 mmol)) was dissolved in water (25 mL). To dissolve the salt and the DNA in water the mixtures were shaken overnight. The solutions were put together and shaken overnight. The precipitated complex was washed with water (3x 15 mL). The white solid was obtained after drying at atmospheric pressure at RT.
Yield: 80 bp: 0.38 g (59%), 3000 bp: 0.5 g (84%)
N+
CH3
O CH2
C H3
C H3
9 13
1*
3* 2*
4* - 12 * 14* 13*
15* 16*
17* - 29*
30*
Staining procedure (6)[2]
DNA-surfactant complex (5 mg) was dissolved in THF and or dichloromethane (0.2 mL). To the solution was added 1st generation Grubbs catalyst (1.1 eq, 6 mg). The solution was shaken for 2 hours. A TEM-image was taken.
13C-NMR
GC
MS
13C-NMR
7 References
[1] W. Jiang, C. Mao, Nature, 2008, 452, 198-201
[2] F. Wurm, F. M. Kilbinger, J. Am. Chem. Soc., 2008, 130, 5876-5877 [3] R. Dahm, Developmental Biology , 2005, 278, 274-288
[4] P. A. Levene, Journal of Biological Chemistry, 1919, 40, 415
[5] J. M. Berg, J. L. Tymoczko, L. Stryer, Biochemistry, WH Freeman and Company, 5th edition, 1975, 117-139
[6] K. Tanaka, Y. Okahata, J. Am. Chem. Soc., 1996, 44, 10679-10683 [7] I. Koltover, C. R. Safinya, Science, 1998, 281, 78-81
[8] N. C. Seeman, Nature, 2003, 421, 427-431 [9] P. W. K. Rothemund, Nature, 2006, 297-302
[10] L.C. Sawyer, D.T. Grubb, Polymer Microscopy, Chapman and Hall, 1987
[11] A.W. Agar, D. Chescoe, R.H. Alderson, Principles and Practice of Electron Microscope Operation, 1974
[12] C. G. Swain, M. M. Kreevoy, J. Am. Chem. Soc., 1954, 77, 1122-1128 [13] K. Tanaka, Y. Okahata, J. Am. Chem. Soc., 1996, 118, 10679-10683
AFM atomic force microscopy
bp base pairs
C cytosine
CDCl3 deuterated chloroform
d doublet
DCM dichloromethane
DNA deoxyribonucleic acid
DOPC dioleoyl phosphatidylcholine
DOPE dioleoyl trimethylammonium propane
DOTAP dioleoyl phosphatidylethanol amine
DX double crossover
et. al. et alumni
EtOAc ethyl acetate
eq. equivalent
fig. figure
G guanine
GC gas chromatography
Hex n-hexane
HJ holiday junction
J J-coupling constant
m multiplet
MDX meiotic double crossover
MS mass spectroscopy
NMR nuclear magnetic resonance
ppm parts per million
prof. professor
Rf ratio of fronts
RT room temperature
T thymine
t triplet
TEM transmission electron microscopy
THF tetrahydrofuran
