Why is untreated dna viscous

In the case of Type II systems, the primary sequence of the restriction endonuclease and methyltransferase specificity domains demonstrate little, if any, homology. However, specificity may be relaxed and cleavage rates significantly decreased. Despite the lack of primary sequence homology, three-dimensional structure among Type II homodimers is similar for those enzymes where crystallography data is available.

In general, the holoenzyme dimer resembles a "U" shape, with each side constituting a monomer containing both recognition and catalytic domains with an overlapping bridging domain at the bottom. The DNA is bound between the two subunits.

FokI, the most studied Type IIs enzyme, appears to exist primarily as a monomer but transiently forms a similar dimer at the recognition site 3. Restriction endonucleases bind dsDNA both specifically and non-specifically. After binding at a non-cognate sequence, several enzymes have been shown to locate their targets through linear diffusion.

During this process a large number of water molecules appear to fill the spaces between the enzyme and the DNA. Once the cognate recognition sequence is found, much of the water is excluded as a highly redundant number of contacts evolve between the enzyme and the bases and phosphodiester backbone of the DNA.

In the case of EcoRI, 50 water molecules are excluded at the cognate site 6. Generally, non-specific bases on either side of the target sequence are required for proper recognition. Conformational changes occur in both the enzyme and DNA as the specific complex forms. The resulting induced fit positions the catalytic center in reactive proximity to the substrate. Using the known co-crystal structures of enzymes bound to their cognate sequences and substitution experiments in the enzyme or DNA for a limited number of additional enzymes, a mechanism for DNA cleavage has been postulated.

Evidence for most enzymes studied to date supports a substrate assisted catalysis model 7. Hydrolysis begins by in-line nucleophilic attack of an activated water molecule. Although all restriction enzymes bind DNA nonspecifically, under optimal conditions the difference in cleavage rates at the cognate site and the next best site single base substitution is very high.

However, under non-optimal conditions, the differences in cleavage rates between cognate and next-best sites change dramatically for many enzymes. This loss of fidelity or increase in cleavage at sites similar to the cognate site is commonly referred to as star activity. A number of reaction parameters can increase the rate of cleavage at star sites relative to cognate sites.

In conjunction with this increase in star activity, cleavage rates at the cognate site generally decrease. Several plausible explanations for star activity are based on the proposed mechanisms for target site identification and hydrolysis see Structure and Mechanism of Action for more information. During nonspecific binding, a large number of water molecules are present at the protein-DNA interface.

When tighter binding and positioning of the catalytic site occurs upon recognition of the target sequence, the number of these interface water molecules is significantly reduced. The higher osmotic pressure caused by volume excluders results in the same reduction in the amount of interface water molecules and allows easier active complex formation at star sites 3. At alkaline pH, higher OH - concentrations may reduce the need for an activated water molecule, which normally initiates nucleophilic attack on the scissile phosphorous.

Although all restriction enzymes probably exhibit some decrease in the cleavage rate difference between cognate and near-cognate sites under such extreme conditions as 4M ethylene glycol, most are not significantly affected under common usage conditions. Those that are susceptible to star activity are induced to different degrees by variations in reaction conditions or by combinations of the conditions listed above. The Table below lists the enzymes sold by Promega that may exhibit star activity, especially under reaction conditions that deviate from those recommended.

In multiple enzyme digests or multiple step applications, it is advisable to stay at or near the optimal conditions for these enzymes whenever possible. When presented with multiple recognition sites that differ in their flanking sequences, most restriction enzymes exhibit slight preferences and cleave the sites at different rates. These rate differences are such that the addition of a small excess of enzyme will avoid any problems due to incomplete digestion.

As always, however, one must be aware of the experimental molar concentration of recognition sites and digest conditions relative to that of the unit definition. See Substrate Considerations for further information. A few restriction enzymes have considerably greater difficulty in cleaving some of their recognition sites. Original experiments with these enzymes led to designation of their site preferences as shown:. Enzymes that have cleavable, slow, and resistant sites in the same or different DNAs have been designated Type IIe restriction enzymes.

There is evidence to suggest that Eco57I also belongs to this group 2. Investigation revealed that binding of a second recognition sequence, in cis or trans , to a distal, non-catalytic site on the enzyme allows slow and resistant sites to become cleavable.

This e ffector sequence alters the kinetics in one of two ways. In the V class NaeI, BspMI , binding of activator DNA increases the V max without changing the K m , indicating that the increased catalytic activity is not related to the affinity of the enzyme for its substrate.

It is assumed that the flanking sequences of a recognition site influence the kinetics of cleavage at that site, but at this time the interaction is not understood.

Considerable differences also exist in the ability of effector sequences to stimulate cleavage. Generally, a recognition site flanked by the sequence from a site that is cleaved easily is a useful starting point for designing good effector sequences. Each restriction enzyme has optimal reaction assay conditions and different conditions for long term storage.

The recommended assay and storage conditions are both determined by the manufacturer to provide the user with the highest activity, best fidelity and greatest stability for each enzyme.

Factors that must be considered include temperature, pH, enzyme cofactors, salt composition, ionic strength and stabilizers. Promega restriction enzyme Reaction Buffers are designed to provide the best balance of optimal activity and convenience. All enzyme storage conditions are validated through our Quality Assurance re-assay program to maximize long term stability.

Setting up digests with a single restriction enzyme is relatively straightforward. However, digests using multiple enzymes that have different buffer requirements may demand the use of alternative buffers and may require adjustments in the number of units of enzyme used. If no compatible buffer can be found a sequential reaction may be performed in which additional buffer or salt is added to the reaction before the second enzyme, or each digest may be performed sequentially using the optimal buffers.

The latter option will require either a DNA precipitation or purification step after the first digest. Regardless of the type of digest performed, the addition of BSA is recommended to stabilize the enzyme and enhance activity 1 2.

Salt Concentration: Restriction enzymes are diverse in their response to ionic strength. A few enzymes prefer acetate to chloride anions. Suboptimal ionic strength or type of ion may lead to star activity.

BSA: Bovine Serum Albumin is used in restriction enzyme storage buffers and is added to digestion reactions to stabilize the enzyme. BSA can protect restriction enzymes from proteases, non- specific adsorption and harmful environmental factors such as heat, surface tension and interfering substances. Typically, the addition of 0. The Acetylated BSA provided with Promega's restriction enzymes has been modified and extensively tested to ensure that no degrading activities are present.

A few enzymes require higher or lower temperatures for optimal activity e. For incubations greater than 1 hour with high temperature enzymes, cover the reactions with a drop of mineral oil to prevent evaporation.

Generally, the incubation temperature for the enzyme reflects the growth temperature of the bacterial strain from which it is derived. This type of information is particularly useful when performing double digests. Volume: Viscous DNA solutions inhibit enzyme diffusion and can reduce enzyme activity.

DNA concentrations that are too dilute can fall below the K m of the restriction enzyme and also affect enzyme activity. Use of an unusually large volume of DNA or enzyme may give aberrant results. Caution should be exercised to prevent higher than normal concentrations of EDTA and glycerol.

The following is an example of a typical analytical single restriction enzyme digestion:. Larger scale restriction enzyme digestions can be accomplished by scaling this basic reaction proportionately. If all of the restriction enzymes in a multiple digest have the same optimal buffer, setting up the digest is straightforward.

However, when this is not the case, several options are available. Note: Perform each digest sequentially using the optimal buffers. This will require either a DNA precipitation or purification step after the first digest. Although this procedure involves more steps than those listed above, in situations where options are not satisfactory, it may be the best alternative.

Some common controls used for restriction enzyme digestion and gel analysis are given in the Table below. Restriction enzymes differ in their reaction kinetics. Variations in the number of enzyme units used and the reaction incubation times were tested.

Incubation time for the unit definition assay is one hour. The concentration of the DNA sample can influence the success of a restriction digestion. Viscous DNA solutions, resulting from large amounts of DNA in too small of a volume, can inhibit diffusion and can significantly reduce enzyme activity 1.

DNA concentrations that are too low also may inhibit enzyme activity see Substrate Quality. Typical K m values for restriction enzymes are between 1nM and 10nM, and are template-dependent 2. Recommended final DNA concentrations for digestion range from 0.

Substrate structural variations, concentration and special considerations are discussed below according to DNA type. In lambda DNA, the cos ends, base, complementary, single-stranded overhangs at the end of each molecule may re-anneal during digestion.

This can give the appearance that digestion is incomplete. Compared to linear DNA, plasmids often require more units of restriction enzyme for complete cleavage due to the supercoiling 1 or the total number of sites to be digested see Recognition Site Density.

See Digestion of Supercoiled Plasmid DNA for information on the relative units needed for complete cleavage of a typical plasmid vector with common cloning enzymes. If a supercoiled plasmid is first linearized with another restriction enzyme or relaxed with topoisomerase, less enzyme may be needed for digestion.

Viscosity can be adjusted by increasing the reaction volume. Addition of spermidine to final concentration of mM also has been reported to increase enzyme activity in the digestion of genomic DNA 4. Addition of BSA to restriction digests at a final concentration of 0. The number of enzyme units needed must be balanced with the total number of sites to assure complete cleavage. Longer incubation times may be required to ensure complete digestion.

Consult the Promega Product Information sheet for the overdigestion value of the enzyme. For many common restriction enzymes, acceptable activity is seen in PCR buffer, although digestion after amplification may not result in the expected compatible ends due to residual polymerase activity 5. Digestion near the end of a PCR product may also present problems.

If an oligonucleotide primer is designed with a cut site that is too close to the end of the DNA, the site may cut poorly or not at all. Since it is very difficult to assay for cutting near the end of DNA, the effectiveness of compensation with extra enzyme units or increased incubation time is difficult to determine. Another reason for incomplete digestion of PCR fragments may be primer dimers.

If the restriction site is built into the primer, primer dimers will contain a double-stranded version of the site, usually in vast molar excess over that of the desired target PCR fragment. Double-Stranded Oligonucleotides: Many of the same considerations for PCR products apply to the digestion of double-stranded oligonucleotides.

In this case high densities of recognition sites per unit of mass can be present and the site may also be near the end of the DNA molecule. Studies have shown, however, that several restriction enzymes that appear to cleave single-stranded DNA actually recognize folded-back duplex regions within the single-stranded genomes e. Therefore, these enzymes are not digesting single-stranded DNA, rather individual sites that are in the duplex form. Digestion required 20 to fold higher enzyme levels than those needed for duplex DNA.

It is possible but not proven that the RNA was also cleaved with large excesses of enzyme. Influence of Flanking Sequence: The sequences flanking the restriction enzyme recognition sequence can influence the cleavage rate of many restriction enzymes although the differences are usually less than fold. A small number of enzymes e. Methylation: Methylation of nucleotides within restriction enzyme recognition sequences can affect digestion.

Methylation may occur as 4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine or 6-methyladenine in DNA from bacteria including plasmids , eukaryotes and their viruses. The sensitivity, or lack thereof, to site-specific methylation, is known for many restriction enzymes Often, isoschizomers differ in their methylation sensitivity. Refer to Cat. A provide an easy and effective way to isolate and purify DNA, free of salt or macromolecular contaminants.

Genomic DNA purified by traditional techniques can contain double-stranded breaks due to mechanical shear forces. Such breaks can be a source of background in megabase mapping of fragments of kb. To avoid this, mammalian, bacterial and yeast cells can be embedded in agarose strips and the cells lysed and treated with proteinase K in situ Most restriction enzymes can cut DNA embedded in agarose provided that more enzyme and longer incubation times are used.

The anti-coagulant used during blood collection can affect the ability of restriction enzymes to completely digest DNA. Use EDTA as an anti-coagulant rather than Heparin, which can bind tightly to the enzyme and interfere with digestion. A number of rapid DNA purification protocols have been written that do not require separation of white cells from red cells 12 These techniques can yield good quality DNA from small volumes of blood, but the DNA obtained after scale-up may be of poorer quality.

DNA purified with this system is suitable for digestion with restriction enzymes. A provides a reliable method for purification of double-stranded PCR-amplified DNA from any salts or macromolecular contaminants. When digesting other substrates, adjustments may be needed based on the amount of substrate, the number of recognition sites per molecule and the incubation time.

The following table illustrates the effect of differences in substrate recognition sites per molecule for EcoRI while keeping the substrate mass and incubation time constant.

A 2 3 or by embedding the cells of interest in blocks or beads of agarose and enzymatically digesting the cell membranes and proteins 4. Large DNA is quite susceptible to mechanical shearing and it is difficult to obtain DNA of 50kb or more unless it is embedded in agarose. Regardless of the preparation method, genomic DNA is frequently less pure than plasmid or other smaller DNA that can be treated more harshly during isolation. In addition, genomic DNA, especially that of higher organisms, may contain more modifications such as methylation.

The methylation sensitivity of potential restriction enzymes may need to be considered for genomic digests. Excess restriction enzyme units and extended incubation times are standard for genomic digestions.

For long incubations, especially at elevated temperatures, evaporation of water from the buffer can concentrate components of the reaction and cause star activity. The reaction can be overlaid with mineral oil or the digestion performed in an incubator to avoid evaporation. Addition of spermidine to a final concentration of mM has also been shown to be helpful for genomic digests 1 5. Incubation time is typically 4 hours to overnight.

A general protocol for embedding and digesting mammalian cells in agarose is provided below. Conditions will differ significantly for other cell types. The conditions required for digestion of agarose-embedded DNA differ from those required for digestion of DNA in solution. In general, much more restriction enzyme is needed. We have tested a number of enzymes for their ability to digest DNA embedded in agarose see Table below. The exact amount of enzyme needed varies depending on the DNA type and preparation.

A general protocol for digestion of agarose embedded DNA is provided below. It is possible to calculate the expected average fragment size for a given genomic DNA if the percent GC content of the DNA and the recognition sequence of the restriction enzyme are known. The probability of cutting any given 6 base sequence is 0. The equation can be refined if there is a known bias in the frequency of dinucleotide and trinucleotide repeats in the DNA being digested 5.

For a sequence N 1 N 2 N 3 N 4 N 5 N 6 where N 1 through N 6 are the bases in the restriction enzyme recognition sequence , the expected frequency of digestion can be calculated as. Where p N is the frequency of N in the genome and p N a N b is the dinucleotide repeat frequency. Where p N a N b is the dinucleotide repeat frequency and p N a N b N c is the trinucleotide repeat frequency.

The GC content and dinucleotide frequencies of many organisms have been determined 6. Because the sequences of many organisms have been elucidated it is now possible to generate complete restriction maps of entire genomes. Larger DNAs re-orient more slowly and thus have slower net migration rates. Restriction enzyme units are usually defined using linear DNA substrates containing multiple recognition sites as these tend to give more reproducible results. Lambda and Adenovirus are the two substrates used most frequently because of their commercial availability and high quality.

Molecular biology applications frequently involve cutting a supercoiled plasmid at a single site within the multiple cloning sequence. There are several reasons why this is the case. For example, there are 0. HindIII cleaves this substrate 7 times or 0. For a 3, base pair plasmid with a single recognition site, there are 0.

The ability of a restriction enzyme to find a single site by linear diffusion in the supercoiled plasmid is also presumed to be different than for any of the sites on a linear substrate. Although it is not common, some enzymes exhibit differences in their ability to cut supercoiled DNA depending on the buffer conditions used. For example, SacII exhibits a pronounced difference in its ability to cut supercoiled plasmids depending on buffer conditions, but this sensitivity is not seen nearly as dramatically with linear substrates.

In order to recognize and cleave their recognition sequence, most restriction enzymes need some flanking DNA. Because of this it can be difficult to achieve complete digestion of PCR products that have restriction sites engineered near the end of a primer or to perform double digests using two enzymes that cut at sites close to each other in a polylinker region.

Such digestions may be improved by using long hour incubation times. When performing multiple digests within a polylinker region, it is important to determine if the sites overlap such that cleavage at one site will destroy another. Alternatively, if the DNA is first digested with SmaI, it will leave the sequence shown below, which can be digested with KpnI, although there may be problems due to a lack of flanking bases.

Studies by Kaufman and Evans 1 , and Moreira and Noren 2 show the efficiency of digestion of polylinker regions with a variety of enzymes. This data can be used to help determine the order in which two enzymes should be used for the most efficient multiple digests, or to predict whether enzymes will work effectively in a double-digest.

In general, the addition of extra bases upstream of an engineered restriction site in a PCR primer will greatly increase the efficiency of digestion of the amplification product, but this is dependent on the enzyme used.

Table 2. PCR products in which the first base pair of the restriction site was flush with 0 , or 1, 2, or 3 base pairs away from the end of the fragment were tested with a variety of enzymes. Purified PCR fragments ng were digested at least twice with 0. Table reproduced by permission of Eaton Publishing. The addition of upstream bases to PCR primers is not the only method used to improve digestion efficiency. A number of protocols have been proposed to improve digestion including proteinase K treatment to remove any thermostable polymerase that may be blocking the DNA, end-polishing with Klenow or T4 DNA Polymerase and the addition of spermidine.

However, none of these methods have been shown to improve cloning efficiency significantly 4 5. An additional drawback to the incorporation of restriction enzyme sites in PCR primers is that it can be quite difficult to resolve digested PCR products from those that remain uncut. This allows identification of products that have been cut successfully because the label is lost upon digestion 6.

An alternative method that has been used successfully to improve digestion of PCR products is to concatemerize the fragments after amplification 1 5. This is achieved by first treating the cleaned up PCR products with T4 Polynucleotide Kinase if the primers have not already been phosphorylated.

The ends will already be blunt if a proofreading thermostable polymerase such as Pfu was used or may be treated with T4 DNA Polymerase to polish the ends if a non-proofreading polymerase such as Taq was used.

This effectively moves the restriction enzyme sites away from the ends of the fragments and allows efficient digestion. This troubleshooting guide addresses common problems that may be encountered while using restriction enzymes. If problems persist contact Promega Technical Services at techserv promega.

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Username Username not found. Send Email. A password reset email has been sent to the primary email address associated with your account. The nature of the PPC chemotype means that it is a discrete and modular DNA binding device with high potential as a drug-design scaffold. Binding studies of AH44 to DNA polymers of varying base composition—or topology—revealed high affinity and limited discrimination in terms of binding constant and mode 11 , Both TriplatinNC and AH44 are significantly more effective nucleic acid condensing agents compared to spermine 11 , A further modification to affect DNA binding interactions is variation of alkanediamine chain length in the trinuclear structures of Scheme 1.

In principle, modifying distances between phosphate clamp coordination units In this contribution we describe the effects of TriplatinNC congeners, Scheme 1 , on DNA binding affinity and inhibition of endonuclease enzyme recognition by various biophysical and molecular biological methods. A general question in all of this work is: how do the solution binding properties reflect the crystallographically determined modes of groove spanning and backbone tracking as shown in Figure 1?

Chemicals and deoxyribonucleic acid sodium salt from salmon testes were purchased from Sigma Aldrich Ireland and used without further purification. HPLC Purity Experiments were conducted in a similar manner to the high-throughput method reported by Kellett et al.

A working solution of Fluorescence measurements were recorded using a Bio-Tek synergy HT multi-mode microplate reader with excitation and emission wavelengths being set to and nm.

Kinetic studies were analyzed over a 1-h period at room temperature with measurements taken at s intervals. Experiments were conducted in a similar manner to the high-throughput method reported previously Thermal melting measurements were recorded at nm at 0.

Temperature was calibrated for each measurement using a temperature probe placed in an identical black-walled cuvette containing 50 mM of phosphate buffer. The temperature was ramped at 3. The spectral bandwidth SBW was set to 1. All samples were degassed prior to measurement. Subsequently the samples were divided into two equal parts. One mixture was used as control and the other one was dialyzed against the same buffer for 24 h. The buffer solution was exchanged every 8 h.

The experiment was conducted thrice and the determined platinum concentrations were averaged. Varying amounts of mithramycin A were added in the r range of 0. Fluorescence spectra were recorded with a Cary Eclipse spectrofluorometer. To avoid photodegradation the fluorescence excitation wavelength was set to nm. The absorbance of the samples at this wavelength was less than 0. Spectra were recorded in the range of — nm.

All samples were prepared in The assignments of the resonances for the oligomers and in the presence of TriplatinNC were conducted in the standard manner as described in previous reports 19 , Supercoiled plasmid pUC19 bp was linearized by the AatI endonuclease according to the standard methods. Reactions were then subjected to electrophoresis in 1. Hypochromicity and a bathochromic shift to nm with isosbestic points at nm for ctDNA was observed, and a shift to nm with an isosbestic point at nm for tRNA was observed in the binding analysis.

Stock solutions of metal complexes and metal salt were prepared in nuclease-free H 2 O Ambion, AM The plate was allowed to incubate at room temperature for 1 h before analysis using a Bio-Tek synergy HT multi-mode microplate reader with excitation and emission wavelengths being set to and nm, respectively. Stock solutions of metal complexes were prepared in nuclease-free H 2 O. An aliquot of test reagent was then added to each cuvette such that an r value of 0.

Temperature was calibrated for each measurement using a temperature probe placed in an identical black-walled cuvette containing 50 mM of phosphate buffer and 1-M NaCl.

The SBW was set to 1. To identify DNA binding properties of this series, ctDNA and stDNA were examined through a variety of biophysical methods including EtBr fluorescent competition studies, fluorescence quenching of limited bound EtBr 23 and Hoechst dye 24 , thermal melting and viscosity analysis.

Triplatin complexes all stabilized ctDNA melting temperature to a similar degree as netropsin e. Competitive fluorescence binding to EtBr-saturated ctDNA solutions was performed in triplicate using a high-throughput binding method 14 and results are shown in Supplementary Figure S3A and Table 1. The intrinsic binding constant K b of EtBr was identified as 8.

The K app values for netropsin and pentamidine were identified as 2. As expected, EtBr exhibited classical intercalative binding with concentration-dependent relative viscosity enhancement 26 while the groove-binding agent, netropsin, had negligible influence only.

Indeed, viscosity by Triplatin molecules decreases in linear fashion with the overall trend almost dwarfing the relative viscosity changes of the standard tested agents. To mitigate the effects of long-range coiling, which can significantly impact viscosity 28 , high molecular weight stDNA fibers were vigorously sheared see Supplementary S4 29 before being examined under identical conditions described above.

We postulate that shorter DNA fragments aggregate more efficiently due to enhanced intramolecular phosphate clamping interactions driven by greater accessibility to smaller nucleotide fragments. The minor-groove binding agents, netropsin and pentamidine, and the cationic cobalt III complex—which has surface binding properties on nucleic acids 30 —displaced the minor-groove fluorogen Hoechst with greater specificity than intercalated EtBr.

In contrast to these standards, the Triplatin series exhibited unique profiles. The cooperative binding of Hoechst can be understood from the minor-groove width opening caused by the phosphate clamps as structurally characterized in the Dickerson—Drew dodecamer 1 , 2 , The quenching of EtBr fluorescence, in contrast, may be connected to a conformational change, or condensation effect, on the tertiary structure of DNA that disfavors intercalative penetration.

In the case of the platinum compounds studied here it is of fundamental interest to examine how the two canonical modes of binding elucidated by crystallography are reflected in base-specific interactions. This finding is in contrast to complex AH44 Scheme 1 that displays some slight preference for A-T sequences when compared to nucleic acids of varying base composition Are there limited but high-affinity binding sites on A-T-rich tracts, and are the distinctive binding modes—groove spanning and backbone tracking—linked to base-specificity or tertiary helical topology, and if so, which of these modes are the drivers behind binding affinity and condensation?

The platinum compound significantly alters the secondary structure of poly[d A-T 2 ] with ellipticity of the band at Control experiments confirmed that TriplatinNC did not interfere with the intrinsic fluorescence of mithramycin A itself results not shown. The changes in the proton resonances for the A-T-rich dodecamer in the presence of TriplatinNC confirm that for both strands mainly the thymines and adenosines are affected by platinum drug binding Supplementary Figures S9A and B.

A preference of binding toward one strand over the other is not observed. These latter sugar connectivities in the proximity of the backbone phosphates point toward backbone binding. Contacts to the imino protons of the nucleobases are also not detected. This could reflect the narrowing of the minor groove as a consequence of the binding of the platinum drug. In summary the results strongly suggest that TriplatinNC spans the minor groove.

These shift variations arise most probably from induced structural changes since the spectra showed only a few contacts in the minor groove. The fact that no connections with G-NH 2 or C-NH 2 are detected implies that binding via major or minor-groove spanning can be excluded. In summary, elucidation of solution binding by TriplatinNC and congeners to DNA clearly display two modes consistent with backbone tracking and groove-spanning interactions.

We suggest that backbone tracking is the predominant binding mode of TriplatinNC-type complexes in duplexes containing G-C nucleobases. Conversely, groove-spanning interactions are localized to the minor groove and are specifically dependent of A-T content. Furthermore, the groove-spanning motif has far greater impact on tertiary nucleic acid structure and is the main driver of DNA condensation and cooperative interactions at the minor groove.

To characterize Triplatin interactions with ribonucleic acid, and thus potentially differentiate binding activity with DNA, yeast tRNA was examined through fluorescence quenching experiments and thermal melting analysis.

We initially characterized the intrinsic binding constant K b of EtBr to tRNA using direct spectrophotometry at nm as 6. The titration processes were repeated until there was no change in the spectra indicating that binding saturation had been achieved. The changes in the metal complex concentration due to dilution at the end of each titration were negligible.

Emission measurements were carried out by using a HitachiF Fluorescence Spectrometer. Tris-buffer was used as a blank to make preliminary adjustments. The excitation wavelength was fixed and the emission range was adjusted before measurements. Viscosity experiments were carried out using an Ostwald viscometer maintained at a constant temperature Calf thymus DNA samples, approximately base pairs in average length, were prepared by sonicating in order to minimize complexities arising from DNA flexibility [ 29 ].

The samples were analyzed by electrophoresis for 2. Molecular structures of the complexes are given in Figure 1. The IR spectral data for the complexes are given. In the 1 H-NMR spectra of the Co III complexes, the peaks due to various protons of pyridine shifted downfield compared to the free ligand suggesting complexation.

As expected the signal for pyridine appeared in the range between 6. All cell culture reagents and media were purchased from Sigma-Aldrich and used without further purification unless otherwise noted. Cytotoxicity assay were performed using Chinese hamster ovarian CHO in order to assess the cancer chemotherapeutic potential of the cells.

Cytotoxicity was assessed using MTT assay. Test compounds were dissolved in culture media. A miniaturized viability assay using 3- 4,5-dimethylthiazolyl -2,5-diphenyl tetrazolium bromide MTT was carried out according to method described by Mosmann [ 31 ].

Each assay was carried out using five replicates and repeated on at least three separate occasions. Viability was calculated as a percentage of solvent-treated control cells, and expressed as a percentage of the control. The significance of any reduction in cellular viability was determined using one-way ANOVA analysis of variance. A probability of. Absorption titration experiments of Co III complexes in buffer were performed by using fixed cobalt complex concentration to which increments of the DNA stock solution were added.

Cobalt solutions were allowed to incubate for 10 minutes before the absorption spectra were recorded see Figures 2 a and 2 b. Based on the observations of complexes, we presume that there are some interactions between complexes and DNA.

K is given by the ratio of slope to intercept. Intrinsic binding constants K obtained about 2. Absorption spectra of complexes: a complex 1, b complex 2, in tris-HCl buffer.

Isosbestic points at , for complex 1. Isosbestic points at , for complex 2. This implies that complexes can strongly interact with DNA and be protected by DNA efficiently, since the hydrophobic environment inside the DNA helix reduces the accessibility of solvent water molecules to the duplex and the complexes mobility is restricted at the binding site, lead to decrease the vibrational modes of relaxation.

Fluorescence emission spectra of complexes: a complex 1, b complex 2 in tris-HCl buffer. Obviously, complex 2 inserts into DNA much deeper than 1. The absorption and fluorescence spectroscopy studies determine the binding of complexes with DNA. Mode of interaction between the metal complexes and DNA was clarified by viscosity measurements.

Optical photophysical probes are necessary, but not sufficient to support a binding model. Hydrodynamic measurements are sensitive to length change i. A classical intercalation model results in unwinding of the DNA helix, which would lead to an increase in viscosity. Effect of the complexes on the viscosity of rod-like DNA is shown in Figure 6.

Based on the viscosity results, it was observed that these complexes bind with DNA through groove binding, result from DNA melting experiment further supported the above result. As intercalation of the complexes into DNA base pairs causes stabilization of base stacking and hence raises the melting temperature of the double-stranded DNA, the DNA melting experiment is useful in establishing the extent of intercalation [ 34 ].

The presence of monophasic melting curves with no change in pH. There has been considerable interest in DNA endonucleolytic cleavage reactions which are activated by metal ions [ 37 ]. The delivery of high concentrations of metal ion to the helix, in locally generating oxygen or hydroxide radicals, yields an efficient DNA cleavage reaction.

When circular plasimd DNA is subjected to electrophoresis, relatively fast migration will be observed for the supercoiled form form I. If scission occurs on one strand nicking , the supercoils will relax to generate a slower-moving open circular form form II [ 38 ].

That incubation with Co III without light yields significant strand scission. Further study required to find out the path of reaction mechanism. The ability of the cobalt complexes 1 and 2 to kill human-derived cancer cells was investigated using CHO cells and a standard bioassay, MTT.

Cells were continuously exposed to test agent for 72 hours, and their effects on cellular viability was evaluated. It was intended that the results from these studies would allow the identification of those derivatives with cancer chemotherapeutic potential. Therefore, profiles of cell viability against complex concentration were established Figure 9 and were used to calculate the IC 50 values for each derivative see Table 2.

Comparison of IC 50 values allowed the relative potency of each of the test complexes to be determined and ranked. Photographs of treated and untreated CHO cells are presented in Figure Both complexes screened displayed a concentration dependent cytotoxic profile. The order of the observed cytotoxicity was seen as complex 2 appearing as the potent. Effects of complex 2 [C], 1[B], and control [A] on the viability of CHO cells human hepatocellular , following continuous incubation for 72 hours, with increasing drug concentration 0.

The morphological effects exerted by complexes on CHO cells 24 hours after treatment. Photographs were taken using a Nikon inverted light microscope 20X objective. The binding behavior of complexes with DNA was characterized by absorption titration, fluorescence, and fluorescence quenching and viscosity measurements.