Multiple combing with spermidine

The grafting densities obtained by simple deposition are limited by the entropic repulsion between DNA coils. Spermidine assisted molecular combing, as dis-cussed already in chapter 5.2.2, is a method that allows to deposit long-chain

(a)

(b) (c)

(d) (e)

Figure 5.8: a) A bare ITO surface silanized with APTES and coated with glu-taraldehyde and fluorescent streptavidin. b) Biotin-modified λ - DNA is first incubated on ITO surface with electric field on so that DNA is pushed against the streptavidin coated surface. In the figure the field is off and the sample is shown directly after the incubation. c) The field is reversed so that unbound DNA is washed away and the end-grafted DNA is stretching with the field in-duced flow. The end-graft density of ∼ 0.3 molecules/µm² inside the ”island” is estimated by counting the fluorescent spots. This is the same spot as in (b). d) The same experiment as in (b) but the ITO surface has additionally 5 nm layer of SiO₂ on top of ITO. The result show similar end-graft density but here the surface coverage is more homogeneous. e) The same experiment as in (c) but the ITO surface has additionally 5 nm layer of SiO₂ on top of it. All the snapshots are 30 µm × 40µm.

DNA densely to a solid substrate. The reduction of the excluded polymer vol-ume accompanied by stretching and deposition of DNA chains to the surface upon molecular combing thus seems ideal to increase tethering density as subse-quent generations of DNA chains are deposited into the gaps between previously combed chains by multiple combing.

As already shown in Fig. 5.1 (b) the combing can be performed several times meaning that the remaining problem is to find out a method which allows to re-lease the combed molecules in a controlled manner. By following the development of the samples on which DNA was combed, we found that from the epoxy and carboxyl group coated surfaces the release of the molecules seemed to be very slow taking days whereas from the streptavidin coated surface the release was much faster varying from minutes to hours. However, these observations are only preliminary results as, for example, on the streptavidin surfaces the concentra-tion of spermidine and the fact whether DNA was purified by PEG precipitaconcentra-tion or not seemed to have an effect on release rate. Furthermore the mechanism of release of the combed molecules is also unclear, we speculate that it is the degra-dation of spermidine which drives the release. This is supported by the fact that the success of combing was very sensitive to the age of the spermidine.

Instead of waiting that the molecules release from the surface, we found that by using polyacrylic acid (PAA) spermidine-combed DNA molecules could be released from the surface. The use of PAA was first reported by Mel’nikow et al. [66] who reported that the condensation of DNA with CTAB (cetyltrimethy-lammonium bromide) is reversible by using PAA (polyacrylic acid). Since CTAB is toxic we tested first whether the molecules combed with spermidine would de-tach from the surface by using PAA. As a first result we show Fig. 5.9 where on carboxylated surface combed amino-modified λ - DNA is released with PAA¹⁶. PAA was added directly on combed surfaces so that the end concentration was 2 mM in 0.5×TBE. In Fig. 5.9 (a) is the surface before addition of PAA, situation directly after addition of PAA is presented in Fig. 5.9 (b) and finally two hours after the addition of PAA is in Fig. 5.9 (c).

It is easy to notice how the PAA reduces the amount of combed molecules at the surface. The both mechanisms: how the combing works and how the release from the surface works are still unclear. The combing mechanism is apparently so that, when the spermidine¹⁷is brought to the solution, it binds to the negatively loaded DNA backbone and changes the charge of the DNA. As the flow pulls the DNA molecule long, now positively charged, DNA is attached to the surface which is negatively charged. The release mechanism then again could be so that the PAA molecule, which has carboxyl group in each monomer being then negatively charged¹⁸ long polymer, has a bigger affinity to the spermidine as the

16Average molecular weight ∼2000.

17+3 valued ion when the pH of the buffer is roughly 7 [67].

18PAA has pKa = 4.25 [68] and we use buffer with pH 9 meaning that the carboxyl groups are deprotonated.

(a) (b)

(c)

Figure 5.9: a)Carboxylated surface which was incubated with amino-modifiedλ - DNA and later combed once with spermidine. The sample was stored two days in TBE at 4^◦C. During this time there was no detachment of molecules from the surface. The kinks of the combed DNA are due to pipetting: the spermidine added buffer was pipetted directly by hand to the samples and therefore the end result was not always parallel. b) Directly after addition of PAA to the sample (in end concentration of 2mM): The combed λ - DNA molecules start to wiggle and detach their backbones from the surface. c)After two hours there is only few molecules attached at the surface. All the snapshots are 30 µm × 40 µm. The snapshots are not taken from the same location but are from the same sample spot.

phosphordiester in the DNA backbone has to the spermidine. Therefore PAA acts as a chelator¹⁹in the solution by complexing the spermidine ions.

The question arises whether the DNA molecules released by PAA are end-attached. We have thus combed a carpet with spermidine (Fig. 5.10 (a)) which was then released with PAA (Fig. 5.10 (b)). This released carpet was then combed a second time with spermidine (Fig. 5.10 (c)), in a direction roughly per-pendicular to the first combing direction. Incompletely detached DNA molecules would then appear as right-angle kinks or loops. However, the resulting structure contains only straight and nearly straight molecules, indicating that PAA-induced release restores end-tethering.

In conclusion we have developed a method, which can lead to very high density carpets by using multiple combing and incubation followed by detachment of all the molecules. Up to now we have concentrated to reproduce the single combing and detach procedure. This alone turned out to be rather difficult since the chemicals are very sensitive and degrade easily. However, we believe that the protocol presented here is finally a reliable one for further experiments.

5.4 Conclusions

In this chapter we have used fluorescence microscopy with combing and microar-ray reader to characterize densities of the end-grafted DNA carpets. For the carpets first combed and then analyzed with fluorescence microscopy we got that they were 0.2−0.3 molecules/µm² dense. For comparable carpets now analyzed with microarray reader, we got that they were 0.08 molecules/µm² dense. The discrepancy between the results shows mainly how difficult it is to gain exact values for the end-grafting density. Nevertheless, the results show us roughly the range of surface densities that can be reached through the packing of random coils. It is worthwhile to point out here that this is still far from the theoretical value which could be reached if we take the packing to be limited by the entropic repulsion between DNA coils. The theoretical end-grafting density for YOYO-1 loadedλ - DNA, R_g = 0.8µm, is roughly 1/R²_g = 1.6 molecules/µm². Up to now we have not found an explanation for this discrepancy. Therefore the important result here was that we have developed two additional techniques (microarray reader and combing) to analyze the end-graftedλ- DNA carpets since up to now we have only used fluorescence microscopy directly to analyze the carpet densi-ties. Furthermore the microarray reader was used also to study the kinetics ofλ - DNA carpets formation, revealing the characteristic time scales of the process.

Additionally we have also studied the possibilities to enhance the surface end-grafting densities by depletion forces, by electrophoresis and by multiple combing on hydrophilic surfaces. The use of depletion forces did not produce high density carpets but with the use of electric field we were able to achieve carpets of

19Metal/Positive ion scavenger.

(a) (b)

(c)

Figure 5.10: a) Biotin-modified DNA was combed on a streptavidin function-alized surface. b) The carpet was released with PAA. c) In the following step the carpet was combed again. As the resulting structure contains only straight and nearly straight molecules, indicating that PAA-induced release restores end-tethering. All the snapshots are 30 µm × 40 µm. The snapshots are not taken from the same location but are from the same sample spot.

roughly 0.3 molecules/µm² on functionalized conducting transparent substrates.

However, as we did not use BSA blocking here the amount of unspecific grafting is unclear. Additionally, during these experiments we were not aware of the possible problem with free oligos and therefore redoing some of the experiments could provide better results. Finally, we found a method how specifically end-grafted DNA can be several times combed with spermidine on a hydrophilic surface. The subsequent generations of combed DNA chains can be released with polyacrylic acid in a controlled manner. This provides a completely new method to produce high density carpets. However, the work on hydrophilic combing is not complete and is a subject for further investigations.

Evanescent wave dynamic light scattering on DNA carpets

Abstract

In this chapter we will present characterization of an evanescent wave dynamic light scattering (EWDLS) setup with a simplified data analysis method which makes additional mixing of local oscil-lator to the dynamic part of the detected scattering signal unnec-essary. The functionality of the setup was tested by reproducing measurements of Lan et al. [69] on colloidal particles in the vicinity of a wall. Furthermore EWDLS was used to study the dynamics of 2 kbp DNA end-grafted on a surface. Our results show that we have measured a scattering signal from a monolayer of end-grafted DNA molecules but further analysis of the data was not possible.

This is due to the lack of mechanical stability of the setup which has caused corruption of the data.

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6.1 Introduction

An evanescent optical wave is created when a ray of light is reflected from an in-terface between a high-index and a low-index medium at an angle exceeding the critical angle. The evanescent wave penetrates roughly a wavelength into the low-index material beyond the reflecting surface acting as an optimal light source for the study of surface specific dynamic phenomena. Typical uses are, for example, excitation of fluorescent dye molecules close to a surface with evanescent wave, giving information about cytoskeletal contacts of a cell to a substrate [70]. Fur-thermore evanescent waves have been used in various light scattering experiments to study the interfacial structure and dynamics of a polymer monolayer [71], Brownian dynamics of colloidal suspension close to a wall [69] or the interaction of a single particle with a wall [72]. In all these applications the common feature is a drastic decrease of the scattering volume in the direction perpendicular to the interface through the evanescent wave illumination profile which suppresses the background signal from the bulk. This fact can be used to gather information of the effect of the interface to the sample or to study phenomena that occur at the interface. As we discussed in the general introduction 1, one of the main conditions in our DNA stretching and structure determination experiment is a measurable signal from an end-grafted DNA layer. The use of an evanescent wave as incident light source has the advantage that the surface attached DNA is in the scattering volume whereas DNA in the bulk can be washed away. Therefore our objective in this work was to show that such a high density end-grafted DNA carpet can be produced that a light scattering signal in an evanescent wave il-lumination can be detected. In this chapter we will first discuss the theory of the EWDLS and then present the setup, its characterization, sample preparation and finally the results of the scattering experiments on DNA carpets.