Study Of DNA Bisintercalators Using Atomic Force Microscopy Biology Essay
The prevalence of Atomic Force Microscopy (AFM) is increasing in studying protein and DNA interactions. We have used AFM to study the interactions between DNA and three different bisintercalating dimers (LH4.40, LH4.42, and LH4.44). AFM images provided contour lengths and volume of DNA to be used as a measurement of bisintercalation. Binding was observed using a pBR-322 DNA fragment of 749-base pairs in length. The contour length of the DNA fragment was found to increase in the DNA-LH4.40 complex. In contrast to the increase in length, the morphology of the DNA produced equal proportions for simple and complex molecules.
Keywords: Atomic force microscopy; Bisintercalation;
Intercalation was first proposed by in 1961 and is characterized by noncovalent stacking between adjacent base pairs (Lerman, 1961). This interaction occurs with π-orbitals of the base pairs in DNA and sometimes is combined with hydrogen bonding pairs (Graves and Velea, 2000). Typically intercalating drugs possess flat, heteroaromatic ring systems that can insert between two adjacent base pairs in a helix. Additionally the basic chains linked to the chromophores are significant in the selectivity and affinity the drug may have. The DNA-drug complex becomes stabilized by π- π stacking interactions between the drug molecule and the DNA bases that surround it (Brana et al 2001). The planes of DNA base pairs are 3.4Ǻ apart with 10 base pairs present in each turn of the helix. Thus each base pair is rotated 36o compared to its neighbour. An interaction between DNA and intercalators results in structural modification, this modification results in the unwinding of the helix and lengthening of the DNA by ~ 1 bp spacing i.e. ~ 3.4Ǻ (Gale et al., 1981).
More complex effects on the properties of DNA are being investigated. From initially studying intercalating drugs, the field has advanced into studying bisintercalating drugs and the effect they have on DNA. Ditercalinium, an anti-tumour drug, has brought about a specific interest in studying its mechanism of cytotoxicity and its binding mode to DNA. In contrast to other anti tumour drugs that bind DNA, ditercalinium causes a malfunction of the DNA repair systems, rather then inhibiting DNA replication or transcription (Berge et al, 2002).
Previous research has also shown extensive study into echinomycin, the first bisintercalator identified, which binds to DNA by inserting two planar chromophores between base pairs (Waring and Wakeling, 1974). Echinomycin, the ‘father’ of an extended family of natural products, interacts with DNA by a similar mechanism. This extended family including luzopeptins, quinoxapeptins and thiocoraline, have brought about an interest, where thiocoraline has progressed into clinical trials (Dale and Ichiwaka, 2000). In a further attempt at investigating bisintercalators, the interaction between luzopeptin B and DNA was observed, in anticipation that the study would provide an insight into the binding mode of the antibiotic (Berge et al, 2002). Results showed a general preference for binding to AT-rich DNA (Bailly et al., 2000). More notably, there was an increasing prevalence of complex morphology among the DNA molecules.
AFM is now regularly being used to examine ligand-protein and ligand-DNA interactions. Originating in 1986, a typical AFM system (Figure. 2.) is made up of a cantilever probe, a sharp tip mounted to a Piezoelectric (PZT) actuator, a position sensitive photo detector and a laser beam which is reflected off to provide feedback (Binnig et al. 1986). With the tip above the sample, the surface is scanned as the tip moves up and down the length of the surface. The reflective cantilever deflects the laser beam and the magnitude is detected by a photomultiplier array. The image produced as a result is a topological map of the sample surface. (Edwardson and Henderson, 2004).
Figure. 1. Typical AFM system (Edwardson and Henderson, 2004).
Typically the system can be operated in three different modes: non-contact, contact and tapping mode. In non-contact mode, the cantilever is approached away from the sample whilst oscillating at or near its natural resonance frequency. This allows the extraction of topographical information from measuring the shift from its natural resonance. In contact mode, sample measurements are made by detecting interaction forces between the sample and the cantilever tip whilst the tip is contact with the sample. The main drawback with contact mode is the damage occurring to the sample from the movement of the tip.
Tapping mode has proven to be a major advance in AFM technology, combining both non-contact and contact mode. The mode is based on oscillation of the cantilever at or near its resonance frequency combined with contact of the surface for a minimum time. Tapping mode allows the extrapolation of high resolution images from samples that are soft (Jalili and Laxminarayana, 2004). Detailed topographic images of potential drug targets have been produced and the ability of the AFM to provide single molecule resolutions make it a suitable tool to use in the examination of how small molecules bind to DNA.
Figure. 2. Bisintercalation dimers, from left to right; LH4.40, LH4.42 & LH4.44.
For this study, DNA and the bisintercalation with three dimers (Figure. 1) will be studied using AFM to examine the binding.
The LH4.40 can be described as 2-acridine-4-carboxamides linked through a triazole ring. The other two structures are acridine-4-carboxamide and acriding-3-carboxamide linked via a triazole respectively. It is important to note the different substitutions on the benzene ring. The LH4.40 and LH4.44 are ortho substituted whereas the LH4.42 is meta. The pBR-322 DNA is approximately 4361 base pairs in length and will be cut using a variety of restriction enzymes, which cut the DNA by making two incisions, through each sugar-phosphate backbone of the double helix (Watson, 1988). Our aim is to observe the effect of the three dimers on the pBR-322 DNA structure. Evidence of conformational effects on DNA will be observed quantitatively by determining contour lengths and qualitatively by providing visual indication. The length of the DNA is expected to increase; as a result, measurement of the individual strands will give us an indication of intercalation.
2.1. DNA fragments
Plasmid pBR-322 DNA was isolated from E.coli. The DNA was fragmented initially by EcoR1 and then further in a
mixture containing restriction enzymes NDE1 and PST1 with Buffer H and deionised water. The resultant mixture was purified using a QIAquick PCR purification kit. This involved the addition of buffers PB, PE and EB, to
elute the DNA each followed by a series of centrifugations. The DNA products were identified using agarose gel electrophoresis giving DNA fragments of approximately 2296, 1316 and 749 base pairs. Following gel extraction using the QIAquick kit, only the 749-bp DNA fragment was used for imaging. Prior to imaging the sample preparation included the addition of 3ul of DNA to 97ul of 10mmol HEPES & 10mmol MgCl. A 20ul droplet was deposited onto mica and was left to dry for 4 minutes. The mica was then washed with 3ml and dried using an airduster.
2.2. Sample preparation
The 3 dimers were given by Dr. M. Searcey; LH4.40, LH4.42 and LH4.44. The samples were prepared by mixing the dimers with DNA. The mixtures consisted of 3ul of DNA, 10mmol HEPES & 10mmol MgCl and 6ul of the dimer. These were prepared in the same manner as before but compressed air was used to dry the sample, ready to be used for imaging.
2.3. Atomic force microscopy
Imaging performed using atomic force microscope. Images were captured in height mode at a scan rate of … Hz in 512 x 512 pixel format. (Need to get information from afm room)
2.4. Data analysis
Prior to analysis, the nanoscope software function was used to flatten all images to remove tilt. Additionally colour contrast was changed to produce clear images to be used for contour length and volume analysis. Contour lengths and volume analysis of DNA molecules were investigated using the image analysis program Scion Image written at the National Institutes of Heath. The program extracted DNA contour length measurements using the free-hand tool and particle analysis was performed on DNA complexes which couldn’t be measured for length. The data was then exported into Microsoft Excel to make any necessary calculation and construct the relevant graphs. Values are expressed as means of n observations, where n only represents the DNA fragments used for analysis and not excluded fragments.
AFM images were used to study the interaction between the 3 different bisintercalators and DNA, where contour length was used to measure intercalation. Intercalation is characterized by an increase in length by 0.34nm, thus bisintercalation of the 3 different molecules into DNA should give rise to 0.68nm (Gale et al. 1982). Where contour length couldn’t be measured, the volume of the DNA-dimer complex was calculated using the particle analysis tool. The volume will give us a comparison between LH4.40 and LH4.42, with differences in volume highlighting an extent of bisintercalation. DNA contour lengths were analyzed using Scion Image, where individual lengths of DNA were measured. Particle analysis using a density slice was performed on DNA molecules, allowing the individual volumes of the fragments to be calculated. For both analysis tools, any molecules touching the edges were excluded from analysis. Figure 2 depicts a representation of sample images used in analysis.
Figure. 2. AFM images showing measurable 749-bp DNA molecules on mica for analysis. Image A shows sample to be used in contour length analysis. Image B shows sample to be used for volume analysis. Scale bar: 1.0µm.
3.1. DNA Length
Images of naked DNA showed a lot of smaller molecules, some dot like in appearance. This could have been due to either the deposition of an unknown, maybe an impurity. Any DNA fragments touching other fragments were excluded from the contour analysis.
Figure. 3. Histograms of DNA contour lengths. Image A represemts DNA only, Image B represents DNA-LH4.44.
The 749-bp DNA fragment measured 254.1nm (n = 54). After bisintercalation, involving titration of the LH4.44 dimer into DNA, an average of 320.4nm (n= 24) was observed. A significant increase in the contour length is prevalent in the addition of the dimer (Figure. 3). These images showed the presence of some longer fragments and some shorter fragments. The shorter fragments from the DNA-LH4.44 are suggestive of incomplete or no binding to the DNA.
3.2. Volume analysis
Volume analysis was completed for DNA molecules that presented ‘blob’ like in appearance. Surface height and background height were needed to calculate the volume. The density slice allowed the selection of the pixels above the surface which represented the molecules to be analyzed. The major and minor axes allowed values to be removed from the data set.
Figure. 4. Histograms of DNA volume analysis. Image A; DNA-LH4.40; Image B; DNA-LH4.42.
The DNA-LH4.40 sample measured 241.8nm3 (n = 227). In contrast, the DNA-LH4.42 sample measured more then twice at 538.6nm3 (n = 175). Assumptions suggest bisintercalation of the LH4.42 dimer causes more of an increase in the DNA length as the volume is significantly higher.
Contour lengths were analyzed for molecules displaying a simple structure, where molecules displayed a complex structure, they were excluded from contour length analysis. Simple molecules included single isolated strands and strands that were not in contact with another. Some molecules were curved in nature; these were also included in contour length analysis. Complex molecules were those ring like in structure and had visible cross linking. Furthermore cross linking and complex structures have been associated with intercalative binding. Primary or tertiary structural changes in DNA have been made apparent in previous studies (Pope et al, 2000). Morphology of the structures was only investigated for DNA & DNA-LH4.44 samples. The other DNA-dimer samples were difficult to ascertain the extent of the morphology as images produced, required analysis of the volume as structures were all ‘blob’ like in appearance.
Although there was an increase in contour length, the prevalence of molecules displaying simple and complex morphology was the same (Figure. 4).
Figure. 4. The proportion of molecules displaying simple and complex morphology.
The interactions between the 3 different dimers and DNA have been investigated using AFM. Preparation of samples to be used for AFM, involved imaging at a 2-dimensional conformation on a surface. Deposition of the DNA was crucial, as it was necessary for the adsorbed molecules to equilibrate on the surface. Although the use of Mg2+ in the deposition buffer supported the 2-dimensional conditions, deposition proved to be a problem, as on occasions samples imaged produced no DNA fragments (Rivetti et al, 1996). Primarily this could have been due to the amount of time left for the sample to dry.
Using pBR-322 DNA, a 749-bp fragment was used to titrate in the dimers. Increase in length of the DNA has been observed from the DNA-LH4.44 complex analysis. This finding correlated with the intercalation theory of the unwinding of the DNA helix, resulting in an increase of the length. A comparison with the other 2 dimers cannot initially be made as results from
Figure. 5. AFM images of DNA and DNA-LH4.44 complex using 749-bp DNA fragments. Image A of DNA shows single strands. Image B of DNA-LH4.44 shows single strands and a few ring structures. Scale bar: 1.0 µm.
the data could only inference information on volume and not length. Additionally analysis was based on a small proportion of molecules, and only one concentration of DNA-dimer complex was used.
For future studies, a larger sample population and increase in concentrations may give a better representation of the effects of the dimers on DNA. I was limited to use only the one concentration, due to time restraints, however it would be interesting to see the effect of increasing concentration on bisintercalation of the dimers.
To my knowledge, there aren’t many studies that have analyzed the volume of DNA with bisintercalation interaction. Generally studies using the AFM analyze DNA lengths, bend angles and the morphology. This makes it difficult to make any sort of comparison with previous literature. Additionally the comparison in volume between DNA and DNA-dimer was not possible, as images for DNA only produced strands and not ‘blobs’ thus rendering them suitable for measurement of the contour length.
The measurement of contour lengths was based on a free-hand tool, thus negating
the sample to user inconsistency and errors. Analysis for DNA-LH4.44 complexes doesn’t take into account any unbound drug molecules. Along with incomplete binding of the dimer to DNA, this could certainly contribute to an inaccurate estimate. At the concentration used, images frequently showed single DNA molecules. Additionally there were few tendencies for the interactions to produce complex structures, ring like in structure (Figure. 5). Images of DNA produced single strands, with limited complexity in the structures. Images of DNA-LH4.44 complexes showed less single strands, but similarly complexity of strands were limited.
This study presents direct evidence on the conformational effects of DNA bisintercalation with different dimers. Moreover, results demonstrate the ability of the AFM to obtain quantitative data using statistical analysis as well as qualitative data at single molecule resolution. The AFM’s ability to provide direct evidence of DNA-dimer interactions makes it an increasingly useful tool in this area of research.
Acknowledges the support of Lesley Howell, Rosemary and Jonathan Mofatt.
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