Chapter 7 Department of Chemistry (SPU) 192 7 DNA CLEAVAGE STUDY BY GEL ELECTROPHORESIS TECHNIQUE 7.1 General A sensitive and easy-to-use assay for studying drug-DNA interactions is the measurements of drug-induced changes in a closed circular DNA molecule using agarose gel electrophoresis. This technique uses a naturally-occurring or modified closed circular DNA, usually a plasmid, as a drug binding substrate and measures how fast the DNA molecule with bound drug moves through the gel when current is applied and electrophoresis is carried out. Since the DNA in the gel can be stained with the dye ethidium bromide (EtBr), which brightly fluoresces under UV light when it intercalates between the base pairs of DNA, the location of DNA in the gel can easily be seen. However, in order for the assay to work with a DNA-binding drug, two important conditions must be met. Since the time required for electrophoresis is relatively long and the drug needs to be bound to the DNA molecule during migration in the gel, the ‘off’ rate constant of the drug from DNA must be small. A second important requirement for the technique is that the drug, when it is bound to DNA, must cause a structural change in the DNA that alters the mobility of the DNA in the gel. The migration of charged colloidal particles in an electric field was originally given the name cataphoresis or electrophoresis. Because there has been some diversity of opinion about the definition of a colloid, and thus about the distinction between colloidal and molecular systems, there has also been some difference of opinion as to how widely the term ‘electrophoresis’ should be used. Some authors prefer the term ionophoresis to describe the movement of relatively small molecules or ions under such conditions. The applications of methods making use of the migration of particles in an electric field were developed in 1940 to 1950. These applications covered the whole range of particle sizes from the largest protein molecules to small molecules like amino acids, sugars (at high pH) and even simple inorganic ions, using the simple types of procedures and apparatus. Although it is not a form of chromatography, the differences in the rates of migration of the charged particles provide a powerful means of separating biocolloids such as proteins, polysaccharides and nucleic acids, as well as for the characterization of their components. For these reasons, and also for
Transcript
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7 DNA CLEAVAGE STUDY BY GEL ELECTROPHORESIS TECHNIQUE
7.1 General
A sensitive and easy-to-use assay for studying drug-DNA interactions is the
measurements of drug-induced changes in a closed circular DNA molecule
using agarose gel electrophoresis. This technique uses a naturally-occurring
or modified closed circular DNA, usually a plasmid, as a drug binding
substrate and measures how fast the DNA molecule with bound drug moves
through the gel when current is applied and electrophoresis is carried out.
Since the DNA in the gel can be stained with the dye ethidium bromide
(EtBr), which brightly fluoresces under UV light when it intercalates between
the base pairs of DNA, the location of DNA in the gel can easily be seen.
However, in order for the assay to work with a DNA-binding drug, two
important conditions must be met. Since the time required for
electrophoresis is relatively long and the drug needs to be bound to the DNA
molecule during migration in the gel, the ‘off’ rate constant of the drug from
DNA must be small. A second important requirement for the technique is
that the drug, when it is bound to DNA, must cause a structural change in
the DNA that alters the mobility of the DNA in the gel.
The migration of charged colloidal particles in an electric field was originally
given the name cataphoresis or electrophoresis. Because there has been
some diversity of opinion about the definition of a colloid, and thus about
the distinction between colloidal and molecular systems, there has also been
some difference of opinion as to how widely the term ‘electrophoresis’ should
be used. Some authors prefer the term ionophoresis to describe the
movement of relatively small molecules or ions under such conditions. The
applications of methods making use of the migration of particles in an
electric field were developed in 1940 to 1950. These applications covered the
whole range of particle sizes from the largest protein molecules to small
molecules like amino acids, sugars (at high pH) and even simple inorganic
ions, using the simple types of procedures and apparatus. Although it is not
a form of chromatography, the differences in the rates of migration of the
charged particles provide a powerful means of separating biocolloids such as
proteins, polysaccharides and nucleic acids, as well as for the
characterization of their components. For these reasons, and also for
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historical reasons, it is now general practice to use the term ‘electrophoresis’
to refer all these procedures. Electrophoresis pertains to the transport of
electrically charged particles/ions, colloids, macromolecular ions or
particulate matter in an electric field.
Electrophoresis is a useful separation technique which involves the
separation of charged species (molecules) on the basis of their movement
under the influence of an applied electric field. Electrophoresis experiments
are usually carried out to obtain information on the electrical double layers
surrounding the mobile particles, to analyze a mixture, or to separate it into
components.
Agarose gel electrophoresis is a method used in biochemistry and molecular
biology to separate DNA, or RNA molecules by size. This is achieved by
moving negatively charged nucleic acid molecules through an agarose matrix
with an electric field (electrophoresis). Shorter molecules move faster and
migrate farther than longer ones [1].
The advantages are that the gel is easily poured, does not denature the
samples. The samples can also be recovered. The disadvantages are that gels
can melt during electrophoresis, the buffer can become exhausted, and
different forms of genetic material may run in unpredictable forms. After the
experiment is finished, the resulting gel can be stored in a plastic bag in a
refrigerator.
7.1.1 Factors affecting migration
The most important factor is the length of the DNA molecule, smaller
molecules travel farther. But conformation of the DNA molecule is also a
factor. To avoid this problem linear molecules are usually separated, usually
DNA fragments from a restriction digest, linear DNA [PCR] products, or
RNAs.
Increasing the agarose concentration of a gel reduces the migration speed
and enables separation of smaller DNA molecules. The higher the voltage,
the faster the DNA moves. But voltage is limited by the fact that it heats and
ultimately causes the gel to melt. High voltages also decrease the resolution
(above about 5 to 8 V/cm). Conformations of a DNA plasmid that has not
been cut with a restriction enzyme or by any chemical agent will move with
different speeds (slowest to fastest): nicked or open circular, linearized, or
molecules are covalently linked end-to-end to form a circular DNA which has
no ‘ends’ [5]. since these DNA molecules are quite long, the DNA double helix
can be gently ‘bent’ so that the two ends of the strands can be joined
together to form circular DNA. However, before covalently linking the ends
together, the enzymatic ‘machinery’ in the cell uses energy to slightly alter
the linear DNA molecule by taking out some of its turns; that is, the spiral
that is characteristic of the double helix. This reduces the angle between
individual base pairs of DNA, called the twist angle, from the optimal value
of ~36°. Since the cell needs to do work on the DNA to reduce the twist angle
and seal the ends, the closed circular structure which results is a high-
energy form of DNA. In order for the closed circular DNA to return the twist
angle to the original value of ~36°, the DNA distorts, introducing super
helical turns where in the Watson-Crick double helical stands, which remain
intact, pass over one another in a left-hand sense to form a second higher-
order helix called a super helix. This DNA, which is called supercoiled DNA
or form I DNA, looks like a rubber band that one has twisted by rolling it
between fingers [5].
If an agent, such as a drug molecule, binds to form I DNA reduces the twist
angle between the two base pairs at the adduct site. The amount by which
the drug reduces the normal twist angle of the closed circular DNA is called
the unwinding angle. This reduction in the twist angle at each site makes
the DNA more open or doughnut-like in shape, which slows the migration
rate of the DNA in a gel relative to control DNA without bound.
While closed circular DNA is convenient substrate for investigating the
binding of drugs to DNA, it is also useful for studying drugs that can cleave
the sugar-phosphate backbone of DNA. If an agent breaks the backbone at
any point along either strand, either by hydrolyzing the phosphodiester
linkage of the backbone or by chemically damaging the deoxyribose sugar,
thus breaking the carbon chain of the backbone, all of the energy stored in
supercoiling is immediately released and the DNA adopts an open-circular
structure with no supercoiling. This form of closed circular DNA is called
nicked circular DNA, or relaxed DNA or form II DNA. If the cutting agent has
low or no sequence specificity, i.e. if it randomly cuts at all possible
nucleotide positions of the DNA and if it is allowed to cut for an extended
period of time, a break in the backbone will eventually occur on one strand
near an existing break on the opposing strand. When this occurs the short
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segment of the duplex DNA between the two breaks will melt, that is, the
Watson-Crick base pairs will separate and the DNA will alter its form again
to produce linear DNA or form III DNA, this DNA usually has many breaks in
its sugar-phosphate backbone, but since it has significant Watson-Crick
regions it is basically a linear rod-like molecule which moves in the gel at a
migration rate that is different from either form I or form II DNA.
In our study we used pUC19 plasmid, which is a plasmid cloning vector
created by Messing and co-workers in the University of California. ‘p’ in the
name stands for plasmid and ‘UC’ represents the University of California. It
is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of
the most widely used vector molecules as the recombinants, or the cells into
which foreign DNA has been introduced, can be easily distinguished from
the non-recombinants based on color differences of colonies on growth
media[6]. pUC19 and pUC18 vectors are small, high copy number, E.coli
plasmids, 2686 bp in length. They are identical except that they contain
multiple cloning sites (MCS) arranged in opposite orientations.
7.3 Experimental
7.3.1 Preparation of pUC19 DNA
Many methods have been developed to isolate and purify plasmids from
bacteria. These methods invariably involve three steps:
• growth of the bacterial culture
• harvesting and lysis of the bacteria
• purification of the plasmid DNA
Growth of the bacterial culture:
Wherever possible, plasmids should be purified from bacterial cultures that
have been inoculated with a single transformed colony picked from an agar
plate. Usually, the colony is transferred to a small starter culture, which is
grown to late log phase. Aliquots of this culture can be used to prepare small
amounts of the plasmid DNA (mini-preparation) for analysis and/or as the
inoculums for a large-scale culture. The conditions of growth of the large-
scale culture depend chiefly on the copy number of the plasmid and whether
it replicates in a stringent or relaxed fashion. At all times, the transformed
bacteria should be grown in selective conditions, i.e., in the presence of the
appropriate antibiotic.
Harvesting and lysis of the culture:
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Bacteria are recovered by centrifugation and lysed by anyone of a large
number of methods, including treatment with nonionic or ionic detergents,
organic solvents, alkali, and heat. The choice among these methods is
dictated by three factors: the size of the plasmid, the strain of E.coli, and the
technique used subsequently to purify the plasmid DNA.
Purification of the plasmid DNA:
All three methods of lysis yield preparations of plasmid DNA that are always
contaminated with considerable quantities of RNA and variable amounts of
E. coli chromosomal DNA. Crude preparations of plasmid DNA can be readily
visualized in agarose gels and can be used as templates and substrates for
most restriction enzymes and DNA polymerases.
Isolation of pUC19 plasmid DNA from pure culture of E. coli was carried out
by alkaline lysis with SDS “midi-preparation method” [1, 7-8].
7.3.2 DNA cleavage assay
All the experiments involving interaction of the complex with DNA were
conducted in duplicate using TAE buffer (pH-8.0). The ratio of absorption of
DNA in buffer at 260 and 280 nm was found to be 1.68 which indicates that
the DNA was sufficiently free from protein. The DNA concentration per
nucleotide was determined by absorption spectroscopy using the molar
absorption coefficient (12858 M–1cm–1) at 260 nm [9].
The plasmid pUC19 (4,363 base pairs in length, density of supercoiling, r = -
0.065), was prepared by transformation of pUC19 into safe competent cells
(Escherichia coli strain), amplification of a clone [1, 10]. After concentration
by ethanol precipitation, DNA was stored in TE buffer (pH 8.0) at -20 °C. The
relative amount of the supercoiled (SC) form was checked by gel
electrophoresis on agarose. The preparations contained about 100% of the
SC form and 0% of the open circular (OC) form. Electrophoresis was carried
out in a Submarine Mini-gel Electrophoresis Unit. Supercoiled pUC19 DNA
(200 ng) in Tris–HCl buffer (50 mM) containing 50 mM NaCl (pH 7.4) was
treated with prepared complexes to yield a total volume of 10 µL and then
incubated in dark for 1.5 h at 37 °C. The reaction was quenched by the
addition of 3 µL loading buffer, and then the resulting solutions were loaded
on a 1.5% agarose gel. Electrophoresis was carried out at 50 V for 2 h in
TAE buffer (pH 8.0). DNA bands were visualized under UV light and
photographed. The quantification of each form of DNA was made by
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densitometric analysis of ethidium bromide containing agarose gel, by using
the volume quantization AlphaDigiDocTM RT. Version V.4.1.0 PC-Image
software. Note that small differences in staining make exact quantitative gel-
to-gel comparisons difficult. Densitometry is uncorrected for differential
uptake of ethidium bromide by SC and non-SC DNA. A previous study with
pUC19 plasmid under similar conditions showed this factor was small [10].
7.4 Literature survey
Gel electrophoresis is one of the most important methods to probe the
nucleic acids and binding with small molecules. Patel et al. studied the effect
of mixed-ligand complexes of oxovanadium(IV) [10-11], cobalt(II) [12-13],
Zn(II) [14], Cu(II) [15-16] and Fe(II)/(III) [15, 17-19]with fluoroquinolones by
means of gel-electrophoresis. Hernandez-Gil et al. studied the DNA cleavage
activity of some binary and ternary complexes of copper(II) with ciprofloxacin
and 1,10 phenanthroline [20]. Using the same technique Kulkarni et al.
studied the DNA cleavage by Co(II), Ni(II), and Cu(II) complexes of ONNO
donor Schiff bases [21]. DNA binding, cleavage activity of some
heterometallic macromolecules was studied by Tabassum et al. [22]. DNA
nuclease activity of two cytotoxic copper terpyridine complexes has been
reported by Shi et al. [23].
7.5 Results and discussion
7.5.1 Interaction of the Ni(II) complexes with pUC19 DNA
The interaction of pUC19 DNA in the presence of the complexes was studied
to determine the efficiency with which it sensitizes DNA cleavage. This can
be achieved by, monitoring the transition from the naturally occurring,
covalently closed circular form (Form I) to the open circular relaxed form
(Form II). This occurs when one of the strands of the plasmid is nicked, and
can be determined by gel electrophoresis of the plasmid. Extended
interaction results in a buildup of nicks on both strands of the plasmid,
which eventually results in its opening to the linear form (Form III). When
circular plasmid DNA is subjected to gel electrophoresis, relatively fast
migration will be observed for the supercoiled form (Form I). Form (II) will
migrate slowly and Form III will migrate between Form II and Form I [24-25].
Figures 7.1 and 7.3 show gel electrophoresis separation of pUC19 DNA after
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incubation with complexes 1-18. Control experiments suggest that untreated
DNA alone did not show any significant DNA cleavage (Figure 7.1 lane 1).
However, in presence of metal salt and ciprofloxacin (Figure 7.1 lanes 2–3) as
well as all the complexes were found to exhibit good nuclease activity. In the
presence of 25 µM of complexes the plasmid DNA was nicked as evident from
the formation of Form II and gradual disappearance of the supercoiled form
in the electrophoretic experiment (Figure7.1, lane 4-12; Figure7.3, lane 2-10).
Figure 7.1 Gel electrophoretogram of pUC19 DNA with Ni(II) complexes. Lane 1. DNA alone; 2. DNA + Ni(II); 3. DNA + HCip.; 4. DNA + complex I; 5. DNA + complex II; 6. DNA + complex III; 7. DNA + complex IV; 8. DNA + complex V; 9. DNA + complex VI; 10. DNA + complex VII; 11. DNA + complex VIII; 12. DNA + complex IX.
Figure 7.2 Quantification of gel electrophoresis bands originating from SC (I), NC (II) and OC (III) DNA in cleavage experiments. The sum of intensities of bands is standardized to 100% for each individual lane.
Figure 7.3 Gel electrophoretogram of pUC19 DNA with Ni(II) complexes. Lane 1. DNA alone; 2. DNA + complex X; 3. DNA + complex XI; 4. DNA + complex XII; 5. DNA + complex XIII; 6. DNA + complex XIV; 7. DNA + complex XV; 8. DNA + complex XVI; 9. DNA + complex XVII; 10. DNA + complex XVIII.
Figure 7.4 Quantification of gel electrophoresis bands originating from SC (I), NC (II) and OC (III) DNA in cleavage experiments. The sum of intensities of bands is standardized to 100% for each individual lane.
7.5.2 Interaction of the Cd(II) complexes with pUC19 DNA
As mentioned above the mixed-ligand complexes of ciprofloxacin can cleave
the supercoiled DNA. However, in the presence of cadmium salt and
ciprofloxacin (Figure 7.5, lanes 2–3) as well as all the complexes were found
to exhibit good nuclease activity. In the presence of 25 µM of complexes the
plasmid DNA was nicked as evident from the formation of Form II and
decrease in percentage of the supercoiled form in the electrophoresis
experiment (Figure7.5, lane 4-11; Figure 7.7, lane 2-11).
Figure 7.5 Gel electrophoretogram of pUC19 DNA with Cd(II) complexes. Lane 1. DNA alone; 2. DNA + Cd(II); 3. DNA + HCip.; 4. DNA + complex I; 5. DNA + complex II; 6. DNA + complex III; 7. DNA + complex IV; 8.DNA + complex V; 9.DNA + complex VI; 10.DNA + complex VII; 11.DNA + complex VIII.
Figure7.6 Quantification of gel electrophoresis bands originating from SC (I), NC (II) and OC (III) DNA in cleavage experiments. The sum of intensities of bands is standardized to 100% for each individual lane.
Figure 7.7 Gel electrophoretogram of pUC19 DNA with Cd(II) complexes. Lane 1. DNA alone; 2. DNA + complex IX; 3. DNA + complex X; 4. DNA + complex XI; 5. DNA + complex XII; 6. DNA + complex XIII; 7. DNA + complex XIV; 8. DNA + complex XV; 9. DNA + complex XVI; 10. DNA + complex XVII; 11.DNA + complex XVIII.
Figure 7.8Quantification of gel electrophoresis bands originating from SC (I), NC (II) and OC (III) DNA in cleavage experiments. The sum of intensities of bands is standardized to 100% for each individual lane.
7.5.3 Interaction of the VO(IV) complexes with pUC19 DNA
The ability of the VO(IV) complexes in effecting DNA cleavage has been
studied by gel electrophoresis using pUC19 DNA. Figure 7.9 shows the gel
electrophoretic separations of plasmid pUC19 DNA after 1 h incubation in
the presence of VO(IV) complexes (50 µM) and Figure 7.10 shows the relative
% intensity of three forms produced due to the reaction of pUC19 DNA and
compounds.
Figure 7.9 Agarose Gel electrophoresis of pUC19 DNA with VO(IV) complexes. Lane 1: pUC19 (Control); 2: pUC19 + cip.; 3: pUC19 + VOSO4; 4: pUC19 + I; 5: pUC19 + II; 6: pUC19 + III; 7: pUC19 + IV; 8: pUC19 + V; 9: pUC19 + VI; 10: pUC19 + VII; 11: pUC19 + VIII; 12: pUC19 + IX.
Figure 7.10 Quantification of gel electrophoresis bands originating from SC (I), NC (II) and OC (III) DNA in cleavage experiments. The sum of intensities of bands is standardized to 100% for each individual lane.
Interaction of pUC19 DNA to the complexes is typical example of
intercalative mode [10]. From the experiment, it was observed that the
complexes make conformational changes on plasmid DNA by making single
strand nicking (NC) or by unwinding the super coiled (SC) plasmid DNA to
open circular (OC) forms. The electrophoresis experiment showed that the
interaction of the complexes with DNA induce strand breakages. In addition,
it was also observed that change in intrinsic viscosity provide absolute proof
of intercalative binding.
7.6 References
[1]. Sambrook, J.; Russell, D.W. Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.
[2]. Lederberg, J. Cell genetics and hereditary symbiosis, Physiol. Rev. 1952, 32, (4), 403-430. [3]. Lipps, G. Plasmids: current research and future trends, Caister Academic Press, Norfolk,
Lucas, I.; Becker, A.; Cowie, A.; Gouzy, J.; Golding, B.; Puhler, A. The complete sequence of the 1,683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti, Proc. Natl. Acad. Sci. U. S. A. 2001, 98, (17), 9889-9894.
[5]. Dabrowiak, J.C. Metals in medicine, Wiley, Hoboken, 2009. [6]. Lengeler, J.W.; Drews, G.; Schlegel, H.G. Biology of the prokaryotes, Thieme, Stuttgart; New
York Malden, MA, 1999. [7]. Birnboim, H.C.; Doly, J. A rapid alkaline extraction procedure for screening recombinant
[12]. Patel, M.N.; Chhasatia, M.R.; Gandhi, D.S. DNA-interaction and in vitro antimicrobial studies of some mixed-ligand complexes of cobalt(II) with fluoroquinolone antibacterial agent ciprofloxacin and some neutral bidentate ligands, Bioorg. Med. Chem. Lett. 2009, 19, (10), 2870-2873.
[13]. Patel, M.N.; Chhasatia, M.R.; Gandhi, D.S. Interaction of drug based binuclear mixed-ligand complexes with DNA, Bioorg. Med. Chem. 2009, 17, (15), 5648-5655.
[14]. Patel, M.N.; Chhasatia, M.R.; Parmar, P.A. Antibacterial and DNA interaction studies of zinc(II) complexes with quinolone family member, ciprofloxacin, Eur. J. Med. Chem. 2010, 45, (2), 439-446.
[15]. Pansuriya, P.B.; Patel, M.N. Dicoumarol complexes of Cu(II), Fe(II) and Fe(III): Preparation, characterization, in-vitro antibacterial and DNA binding activity, Appl. Organomet. Chem. 2007, 21, (9), 719-727.
[20]. Hernández-Gil, J.; Perelló, L.; Ortiz, R.; Alzuet, G.; González-Álvarez, M.; Liu-González, M. Synthesis, structure and biological properties of several binary and ternary complexes of copper(II) with ciprofloxacin and 1,10 phenanthroline, Polyhedron 2009, 28, (1), 138-144.
[21]. Kulkarni, A.; Patil, S.A.; Badami, P.S. DNA cleavage and in vitro antimicrobial studies of Co(II), Ni(II), and Cu(II) complexes with ONNO donor Schiff bases: synthesis, spectral characterization, and electrochemical studies, J. Enzyme Inhib. Med. Chem. 2010, 25, (1), 87-96.
[22]. Tabassum, S.; Bhat, I.U.; Arjmand, F. Synthesis of new heterometallic macromolecules: their DNA binding, cleavage activity and in vitro model electrochemotherapy study, Spectrochim. Acta A Mol. Biomol. Spectrosc. 2009, 74, (5), 1152-1159.
[23]. Shi, P.; Lin, M.; Zhu, J.; Zhang, Y.; Jiang, Q. DNA-binding affinity and nuclease activity of two cytotoxic copper terpyridine complexes, J. Biochem. Mol. Toxicol. 2009, 23, (4), 295-302.
[24]. Shi, S.; Liu, J.; Li, J.; Zheng, K.C.; Huang, X.M.; Tan, C.P.; Chen, L.M.; Ji, L.N. Synthesis, characterization and DNA-binding of novel chiral complexes delta- and lambda-[Ru(bpy)2L]2+ (L = o-mopip and p-mopip), J. Inorg. Biochem. 2006, 100, (3), 385-395.
[25]. Chao, H.; Mei, W.J.; Huang, Q.W.; Ji, L.N. DNA binding studies of ruthenium(II) complexes containing asymmetric tridentate ligands, J. Inorg. Biochem. 2002, 92, (3-4), 165-170.
