In contrast to the experiments of Smith et al. [1] and Cluzel et al. [2] we have to stretch, not a single molecule, but several thousands of molecules in order to get measurable signal for the structure determining measurement. In a suc-cessful experiment DNA molecules are stretched between two separate surfaces and simultaneously the structure of the DNA is determined for example by
X-ray diffraction as presented in Fig. 1.2. What are then the requirements for a successful experiment?
Let us look carefully at the DNA sample preparation:
• First of all we have to introduce end-modifications on both ends of the DNA molecule so that there is a handle for end-grafting.
• The end-modifications have to be covalently linked to the rest of the molecule so that force can be applied over them.
• The end-modification process should also be efficient so that both ends of all the molecules really carry their modifications.
• The sample preparation should provide DNA molecules which are monodis-perse in length so that the S state is reached simultaneously by all the molecules as the two surfaces are moved apart.
• The substances used to prepare the DNA samples can distort the exper-iment in later phase so an effective purification method has to be also developed.
There are also very hard criteria for successful DNA end-grafting surface chemistry:
• The surfaces have to carry functional groups which are first of all strongly enough, i.e. covalently linked to the surface.
• The functional groups have to be able to react with the DNA end-modifications creating bonds that are also stable enough so that the S state can be reached.
• The functional groups on the surface are allowed to react only with one kind of DNA end-modification at a time. When both of the end-modifications would react already with one surface, we would have loops of DNA, instead of DNA molecules spanning from one surface to the other.
• The functionality should stay reactive so long that dense DNA carpets can be formed. It is clear that even the best surface chemistry will not help when the end-grafting densities of the DNA carpets are not high enough to produce measurable signal for the structure determination.
In this work we will concentrate to fulfill the criteria explained above since we believe that the existing apparatus build by M. Clausen [9] meets from the mechanical point of view the needs of the experiment. In chapter 2 we show that the previously developed surface end-grafting methods [8] are not stable enough for the multi-molecule stretching experiment as described above. Furthermore in
generator : KaCu = 0.154 nm
Sample to detector distance Rotating anode
-generator : KaCu = 0.154 nm
30 cm < D < 1 m Pinhole :
5 mm diameter
Confocal optics Surface Force Apparatus Detector (image plate) Flux :
1.38 x 106 photons/seconds X-rays
Figure 1.2: In a successful experiment dense carpet of both ends grafted DNA molecules are being stretched and simultaneously X-ray diffraction is performed revealing the structure of the overstretched DNA or DNA-protein complex (with the courtesy of A. Zinck).
chapter 3 we show that previously used sample preparation protocol [8] is not optimal: we have instead of homogeneous both ends modified λ- DNA molecules very inhomogeneous samples. The problems of the sample inhomogeneities and the stable surface end-grafts are treated and solved in chapters 3 and 4. Fur-thermore in chapter 4 we give also additional possibilities to covalently end-graft DNA on various types of surfaces. In chapter 5 we present methods to character-ize the surface end-grafted DNA carpets, and additionally we show also methods how to enhance the end-grafting density. Finally in chapter 6 we show that a scattering signal, which is inevitable for structure determination, can be recorded from an end-grafted DNA carpet.
Confocal Microscopy and
Mechanical Stretching of DNA
Abstract
In this chapter we will present basic techniques to modify DNA molecules with oligonuleotides, silanize surfaces and finally end-graft modified DNA molecules specifically on functionalized sur-faces. Furthermore we will present an experimental setup with which we studied the mechanical stability of the DNA-surface an-choring by stretching the molecules and observing the rupture lengths. We present rupture length distributions for λ - DNA molecules which were end-grafted from one end with gold-thiol linkage and from other end with biotin-streptavidin linkage to the surface. Additionally we study the effect of multi-biotin end-modification in the end of theλ - DNA molecules, instead of single end-modification, on the rupture length distribution. Our results show that the present end-grafting techniques are not fulfilling the conditions that are needed for the multi-molecule stretching exper-iment as presented in the general introduction 1.
11
2.1 Introduction
There are only a few experimental techniques available to study the quality of the specific end-graft of a single DNA with a surface. The possibilities presented in the literature rely almost always to the fact that DNA is attached on one end to a colloidal bead or to a AFM tip and on the other end to a solid support or to a another colloidal bead. The bead/tip can then be manipulated with a optical/magnetical tweezer [10] [11], with a atomic force microscope (AFM) [12]
or with a micropipette [2] while the other end of the DNA molecule is held fixed.
The common feature for all of these experiments is the fact that they are all single molecule experiments. Single molecule experiments are done in order to see the actual behavior of molecular individuals while the classical macroscopic experiments tend to average over the molecular conformations. For example the B-S transition of stretched DNA reported first by Cluzel et al. [2] and Smith et al. [1] are beautiful examples of single molecule experiments where the force-extension behavior of single DNA is measured but no structural information of the stretched state is gained. In order to gain the structural information classical macroscopic experiments have to be done. Our goal is basically to combine these two point of views: (i) to stretch a single molecule with both ends grafted and (ii) to do it on large number of molecules in parallel. So the requirements for the DNA functionalization and the surface preparations are very demanding.
In this chapter we review the basic experimental techniques used already by Lehner [8] to modify DNA and end-graft it onto a surface. Furthermore we present a simple but effective setup with which we study the quality and the possible problems in the DNA end-grafting to the surface.