Enantioselective cleavage of supercoiled plasmid DNA catalyzed by chiral macrocyclic lanthanide(III) complexes

Journal of Inorganic Biochemistry 107 (2012) 1–5

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Enantioselective cleavage of supercoiled plasmid DNA catalyzed by chiral macrocyclic lanthanide(III) complexes Artur Krężel a,⁎, Jerzy Lisowski b,⁎⁎ a b

Department of Protein Engineering, Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland Department of Chemistry, University of Wrocław, 14 F. Joliot-Curie, 50-383 Wrocław, Poland

a r t i c l e

i n f o

Article history: Received 12 July 2011 Received in revised form 2 October 2011 Accepted 26 October 2011 Available online 3 November 2011 Keywords: DNA cleavage Dinuclear lanthanide complex Enantioselectivity Chiral macrocycle

a b s t r a c t The enantiomers of the Sm (III), Eu (III) and Yb (III) complexes [LnL(NO3)2](NO3) of a chiral hexaazamacrocycle were tested as catalysts for the hydrolytic cleavage of supercoiled plasmid DNA. The catalytic activity was remarkably enantioselective; while the [LnL SSSS(NO3)2](NO3) enantiomers promoted the cleavage of plasmid pBR322 from the supercoiled form (SC) to the nicked form (NC), the [LnL RRRR(NO3)2](NO3) enantiomers were inactive. Kinetics of plasmid DNA hydrolysis was also investigated by agarose electrophoresis and it indicated typical single-exponential cleavage reaction. The hydrolytic mechanism of DNA cleavage was confirmed by the successful ligation of hydrolysis product by T4 ligase. The NMR study of the solutions of the complexes in various buffers indicated that the complexes exist as monomeric cationic complexes [LnL (H2O)3]3 + in slightly acidic solutions and as dimeric cationic complexes [Ln2L2(μ-OH)2(H2O)2] 4 + in slightly basic 8 mM solutions, with the latter form being a possible catalyst for hydrolysis of phosphodiester bonds. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Due to a Lewis acidity, high coordination numbers and labile character, the lanthanide(III) complexes are exceptionally active catalyst for hydrolysis of phosphodiester bonds, including that of DNA and RNA chains [1-5]. In particular, the macrocyclic [6-12] and dinuclear [6-8,11,13-16] lanthanide complexes have been shown to be very effective catalysts for the cleavage of the phosphate ester bonds. These complexes are promising candidates for artificial nucleases. It has been shown that lanthanide hydroxo derivatives play a pivotal role in this hydrolytic reaction. The hydroxo-bridged dinuclear lanthanide complexes proved to be very active catalysts, which are often more effective than corresponding mononuclear species. Since DNA double helix is inherently chiral, its interactions with chiral molecules should be, in principal enantioselective [17]. This is indeed observed e.g. in the case of chiral drugs [18] and metal complexes [19-26], in particular chiral propeller-shape complexes of ruthenium [27-32]. There are also reports of enantioselective interactions of chiral transition metal complexes with DNA leading to preferred cleavage of DNA by one of the enantiomers over the other [22,26,28,29]. In particular an enantioselective photocleavage of DNA has been reported for propeller-shape ruthenium complexes. The above examples of enantioselective cleavage of DNA chain involve oxidative cleavage mechanism related to the presence of radicals and active oxygen species. For the potential applications as ⁎ Corresponding author. Tel.: + 48 71 3752 765; fax: + 48 71 3752 608. ⁎⁎ Corresponding author. Tel.: + 48 71 3757 252; fax: + 48 71 3282 348. E-mail addresses: [email protected] (A. Krężel), [email protected] (J. Lisowski). 0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.10.011

artificial nucleases, however, the hydrolytic cleavage is preferred, since it results in fragments that can be used in further ligation. Here we present, to the best of our knowledge, the first example of enantioselective cleavage of DNA chain involving hydrolytic mechanism. We have studied the interactions of lanthanide(III) complexes of chiral macrocycle L (Scheme 1) with plasmid pBR322. The racemic forms of the dimeric Y(III) and Nd(III) complexes of the macrocycle L, [Ln2L2(μ-OH)2(H2O)2](NO3)4, have been previously reported to be effective catalysts for the hydrolytic cleavage of DNA [33]. In this report we have used the enantiopure forms of the representative Sm(III), Eu(III) and Yb(III) complexes of L. The lanthanide(III) complexes with this macrocycle can exist both in monomeric [34-38] and dimeric [33,39,40] forms, in particular, in the water solution they exist as complex cations [LnL(H2O)3] 3 + and [Ln2L2(μ-OH)2(H2O)2] 4 +. The dependence of dimer formation on the concentration and the effective pH of solution is also discussed.

2. Materials and methods/experimental section 2.1. General methods Tris base, boric acid, EDTA (ethylenediaminetetracetic acid disodium salt dehydrate), MES (2-(N-morpholino)ethanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), and agarose were purchased from Sigma-Aldrich. D2O (99.9%) and DCl (35% solution) were from Cambridge Isotope Laboratories. The monomeric [LnL(NO3)2](NO3) complexes have been synthesized as previously described [34-37].

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30 pH = 5.5

pH = 7.0 pH = 8.5

20

Δε [M-1cm-1]

10 0 -10 -20 Scheme 1. Mononuclear and dinuclear Ln(III) complexes of macrocycle L (RRRR enantiomer, axial ligands and charges omitted for simplicity).

2.2. NMR and CD spectroscopy The NMR spectra of solutions of the starting [LnL(NO3)2](NO3) monomeric complexes in various buffers were recorded on Bruker Avance 500 spectrometer either at 27 or 45 °C. Solutions of 0.2–8 mM of lanthanide complex in 30 mM or 50 mM were prepared in appropriate buffer. pH⁎ readings in D2O calibrated with standard buffers in H2O were corrected to pH values according to Krężel and Bal formula: pH = pH* × 0.929 + 0.42 [41]. The CD spectra were recorded at 25 °C on Jasco J-715 spectropolarimetre, over the range of 205–360 nm, using 1 mm quartz cuvette. The spectra are expressed in terms of Δε = εl − εr, where εl and εr are molar absorption coefficients for left and right circularly polarized light, respectively. 2.3. DNA cleavage assay The ability of [LnL(NO3)2](NO3) enantiomers to induce strand cleavage in DNA chain was tested with the use of pBR322 plasmid. To minimize relaxation and breaking of supercoiled DNA plasmid during a storage, fresh plasmid sample was prepared directly before cleavage experiments. DH10B Escherichia coli cells transformed with commercial pBR322 from Promega were cultivated overnight in standard LB medium. Plasmid was purified using QIAfilter Plasmid Midi Kit (Qiagen GmbH, Hilden, Germany) and stored at 4 °C no longer than a week. pBR322 DNA plasmid was incubated with 0.05–2.0 mM of particular enantiomer complex in 50 mM Tris HCl buffer, pH= 7.5 at 45 °C over the period of 0–600 min. In case of pH dependent cleavage additionally 50 mM MES buffer was applied. After reaction 20 μl of the sample was mixed with 4 μl of loading buffer (bromophenol blue in 30% glycerol) and load on 1% agarose gel, containing ethidium bromide, in Tris/ borate/EDTA buffer, pH 8.0. Samples collected for kinetic studies were kept at 4 °C to slow down hydrolytic reaction and load on the agarose gel when all samples were collected. To prove hydrolytic mechanism of DNA damage sample of pBR322 plasmid was cleaved by 1 mM [EuLSSSS(NO3)2](NO3) in 50 mM Tris HCl buffer, pH= 7.5 at 45 °C. Product of the reaction was ligated by T4 DNA ligase in appropriate ligase buffer (Fermentas) at room temperature. Progress of the ligation was analyzed by agarose gel electrophoresis after 2, 4 and 12 h.

-30 210

240

270

300

330

360

λ [nm] Fig. 1. The CD spectra of the enantiomers [EuLRRRR(NO3)2](NO3) (dot dashed lines) and [EuLSSSS(NO3)2](NO3) (solid lines) at different pH (30 mM MES, HEPES and Tris⋅HCl buffer) at 22 °C. Spectra were recorded for 0.2 mM complexes.

[LnL SSSS(NO3)2](NO3) complexes with the representative Sm(III), Eu(III) and Yb(III) lanthanide ions. In the conditions used (50 mM Tris HCl, pH = 7.5, T = 45 °C) we have observed a strong dependence of the catalytic activity on the chirality of the complexes and the kind of lanthanide ion (Fig. 2). For the Sm(III) and Eu(III) complexes the [LnLSSSS(NO3)2](NO3) isomers with all-S configuration at the cyclohexane carbon atoms showed clear catalytic activity in the cleavage of the supercoiled form (SC) of the plasmid pBR322 to the nicked form (NC). On the other hand the [LnLRRRR(NO3)2](NO3) enantiomers are practically inactive (Fig. 2). This demonstrates a profound difference in the interactions of the two isomers with DNA backbone. The pH profiles of the [EuLSSSS(NO3)2](NO3) catalyzed cleavage indicate the hydroxo species as the active species. While this complex is inactive at pH = 5.5, the catalytic activity is already observed at pH= 6.5 and increases as the pH is raised to 8.5 (Supporting Fig. S1). The percentage of cleaved supercoiled plasmid DNA is also increasing as the concentration of [EuLSSSS(NO3)2] (NO3) increases (Fig. 3). On the contrary, the [EuL RRRR(NO3)2](NO3) enantiomer is inactive under all studied conditions. Remarkably, at pH= 8.5 and 0.2 mM concentration of complexes, the electrophoresis results indicate 100% conversion of linear to nicked form of the plasmid for the all –S enantiomer and 0% conversion for the all –R enantiomer. Similarly at pH= 7.5 and c = 1 mM 100% and 0% conversions are observed for the all –S and all –R enantiomers of the Eu(III) complex, respectively.

3. Results and discussion 3.1. The catalytic activity of the [LnL(NO3)2](NO3) complexes The chiral enantiomeric complexes can be easily obtained in the enantiopure form starting from (1R,2R)-1,2-diaminocyclohexane or (1S,2S)-1,2-diaminocyclohexane, respectively. The chiral nature of these complexes is reflected in their strong CD spectra (Fig. 1). In order to establish the role of chirality of the complex on its catalytic activity in DNA chain cleavage we have investigated the cleavage of DNA plasmid pBR322 in the presence of the [LnLRRRR(NO3)2](NO3) and

Fig. 2. Agarose gel electrophoresis of pBR322 DNA plasmid cleavage by enantiomers of [LnL(NO3)2](NO3) complexes. The samples containing 0.2 mM of each enantiomer complex were incubated for 4 h at 45 °C in Tris⋅HCl, pH = 7.5.

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Fig. 3. Agarose gel electrophoresis of pBR322 DNA plasmid incubated with increasing concentration of [EuLSSSS(NO3)2](NO3) enantiomer in 50 mM Tris⋅HCl buffer, pH = 7.5 for 4 h at 45 °C; 0 (lane 1), 0.05 (lane 2), 0.1 (lane 3), 0.2 (lane 4), 0.5 (lane 5), 1.0 (lane 6) and 2.0 mM complex (lane 7). For a comparison DNA plasmid incubated with 1.0 mM [EuLRRRR(NO3)2](NO3) enantiomer is presented (lane 8).

Since the Sm(III) and Eu(III) are practically redox-inactive, the mechanism of the DNA cleavage is most likely hydrolytic, as observed previously for other Ln(III) complexes [1,4]. This mechanism is confirmed by the successful ligation of the reaction products. The supercoiled plasmid is quantitatively recovered from the nicked form obtained from the [EuL SSSS(NO3)2](NO3) catalyzed cleavage by using the T4 ligase, in accord with the hydrolytic cleavage of P―O3′ bond in the catalyzed process (Fig. 4). Time dependent pBR322 DNA plasmid cleavage performed for Sm(III) and Eu(III) enantiomers (Fig. 5) confirms observation that only SSSS enantiomers are hydrolytic active and nicked form is only one topological DNA product. Both the decrease of the supercoiled form and the increase of the nicked form fit well to a single-exponential decay curves (Fig. 6) and follow pseudo first order kinetic behavior. The determined rate constants are equal to 8(2) × 10 − 5 s − 1 and 7(1) × 10 − 5 s − 1 for the [SmL SSSS (NO3)2](NO3) and [EuL SSSS(NO3)2](NO3) complexes, respectively (50 mM Tris⋅HCl, pH 7.5, T = 45 °C) (Supporting Fig. S2). These values indicate a moderate catalytic activity, much lesser than that of the best dinuclear Ln(III) complexes (Supporting Table S1). Although the mode of binding of the investigated complexes with DNA and the catalytic mechanism have yet to be established, the shape of the molecules, that is the sense of helical twist of the macrocycle L is clearly determining the catalytic efficiency. This effect most likely is caused by the various spatial disposition of the OH − nucleophile, rather than the various affinities of the two enantiomers for DNA. For instance, if the dinuclear species are the active catalyst form, the availability of the bridging OH group, which is hidden in a kind of a cleft in the helical dimeric [Ln2L2(μ-OH)2(H2O)2] 4 + complex (Fig. 7) may be a key factor. This group is a likely nucleophile attacking the phosphodiester bond; possibly in the [Ln2L SSSS2(μ-OH)2 (H2O)2] 4 + enantiomer it is better disposed for the approach to the DNA backbone. Similarly, if the active form is a monomeric hydroxo complex resulting form the dissociation of the dimer, the helicity of the macrocycle may dictate favorable or unfavorable orientation of the nucleophile in the HO―Ln(III)L―O―P intermediate. In contrast to the Eu(III) and Sm(III) [LnL(NO3)2](NO3) complexes, the interaction of both isomers of the ytterbium complex [YbL(NO3)2] (NO3) results in a smeared traces in gel electrophoresis (Fig. 2). This results likely from the aggregation of DNA, caused by the Yb(III) complex or its decomposition products (the [YbL(NO3)2](NO3) complex is more prone to decomposition of Schiff base macrocycle in comparison with the [EuL(NO3)2](NO3) and [SmL(NO3)2](NO3) complexes).

Fig. 4. Agarose gel electrophoresis of pBR322 DNA plasmid cleavage by stereospecific 0.2 mM [EuLSSSS(NO3)2](NO3) enantiomer and DNA ligation product. Plasmid DNA incubated without (lane 1) and with 1 mM complex for 4 h at 45 °C in 50 mM Tris⋅HCl, pH = 7.5 (lane 2). Cleaved product was than ligated with DNA T4 ligase and checked for ligation progress after 2 (lane 3), 4 (lane 4) and 12 h (lane 5).

Fig. 5. Agarose gels electrophoresis of pBR322 DNA plasmid incubated with both 0.2 mM RRRR and SSSS enantiomers of [SmL(NO3)2](NO3) and [EuL(NO3)2](NO3) in Tris HCl, pH = 7.5 for 0 (lane 1), 25 (lane 2), 60 (lane 3), 120 (lane 4), 180 (lane 5), 250 (lane 6), 360 (lane 7) and 600 min (lane 8), 45 °C, pH = 7.5.

3.2. The dependence of the monomer vs. dimer formation on the buffer The lability of lanthanide(III) complexes results in an easy exchange of ligands in the coordination sphere, including those coming form the used buffer. In the present case it raises the question

Fig. 6. Kinetics of pBR322 DNA plasmid cleavage by 0.2 mM [SmLSSSS(NO3)2](NO3) enantiomer in 50 mM Tris HCl, pH = 7.5 at 45 °C. Fraction values of DNA forms were received by densitometry analysis of agarose gel image. A) Decrease and increase of supercoiled (■) and nicked (□) plasmid forms, respectively; B) logarithmic plots of DNA fractions indicating pseudo-first order kinetics of DNA cleavage.

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pH=9.5

pH=8.5

pH=7.5

pH=7.0

pH=6.5

pH=6.0 Fig. 7. Shapes of the [Ln2LSSSS2(μ-OH)2(H2O)2]4 + (upper) and [Ln2LRRRR2(μ-OH)2 (H2O)2]4 + (lower) enantiomers (based on deposited cif files from Refs.[33] and [39]). B—DNA double helix shown for comparison.

pH=5.5 10 1

whether under the used conditions the lanthanide(III) complexes of macrocycle L exist as monomeric units with axial water molecules or hydroxo-bridged dimers. Since both forms give rise to distinct NMR spectra [39,40], the solutions of these complexes in different 1 buffers were monitored by using H NMR spectroscopy (Figs. 8 and 9) and formation of dinuclear complexes was evident for 8 mM solutions at neutral or basic pH. For instance, the starting monomeric [SmLSSSS (H2O)3] 3 + form present at slightly acidic pH gives rise to characteristic three signals in the aromatic region: a triplet and doublet of the pyridine protons at 8.92 and 8.73 ppm, respectively, and a singlet of the azomethine proton at 8.50 ppm. On the other hand, under basic conditions the triplet at 8.30 ppm, the doublet at 8.63 ppm and the singlet at 9.43 ppm are observed in the same region (Fig. 8). This change corresponds to formation of a dinuclear cationic [Sm2LSSSS2(μ-OH)2

pH=9.5

pH=8.5

pH=7.5

pH=7.0

pH=6.5

pH=6.0

pH=5.5 9.2

9.4 1

9.0

8.8

8.6

[ppm]

Fig. 8. Region of the H NMR spectra (27 °C) of the [SmLSSSS(NO3)2](NO3) complex (8 mM) measured in MES (pH = 6.5, 6, 6.5) HEPES (pH = 7) and Tris⋅HCl (pH = 7.5, 8.5 and 9.5) buffers.

0

- 10

- 20 [ppm]

SSSS

Fig. 9. The H NMR spectra (27°) of the [EuL (NO3)2](NO3) complex (8 mM) measured in MES (pH = 6.5, 6, 6.5) HEPES (pH = 7) and Tris⋅HCl (pH = 7.5, 8.5 and 9.5) buffers.

(H2O)2] 4 + complex. The differences between the monomeric and dimeric complexes are even more evident in the case of Eu(III) complex, due to the sensitivity of the paramagnetic shift to the change of the coordination sphere (Fig. 9). For instance, the azomethine signals are observed at −22.2 and −18.2 ppm for the [EuLSSSS(H2O)3] 3 + and [Eu2LSSSS2(μ-OH)2(H2O)2]4 + complexes, respectively. The formation of dinuclear species is also evident form doubling of the number of resonances (Fig. 9, Supporting Figs. S3, S4), which reflects the lowering of symmetry from D2 to C2. The NMR spectra indicate that the dimeric hydroxo forms start to form already at pH= 6.5. At pH= 7.5 used for the DNA cleavage this form is prevailing although the monomeric form is still present. The chemical exchange between the monomeric and dimeric complex, related to dissociation of hydroxide anions, is slow on the NMR scale; however at pH= 6.5, 7 and 7.5 additional subtle shift and broadening of the signals of the monomeric form are observed (Figs. 8 and 9). This shift reflects the presence of small amount of additional transient complex form, which is in a fast (on the NMR time scale) exchange with the monomeric [LnLSSSS(H2O)3] 3 + complex cation. This additional form may correspond to a monomeric hydroxo derivative [LnLSSSS(OH) (H2O)2] 2 + or a form with amine group of the buffer molecule coordinated in the axial position. These averaged signals of various monomeric forms grow in intensity and move to lower frequencies as the Sm(III) complex is diluted from 8 mM to 0.2 mM at pH = 7.5, while the signals of dinuclear species gradually disappear (Supporting Fig. S4). These changes most likely reflect dissociation of the dinuclear [Sm2L SSSS2(μ-OH)2(H2O)2] 4 + form to at least two new complex forms. These new forms are likely to be the mononuclear [SmL SSSS (OH)(H2O)2] 2 + and [SmL SSSS(H2O)3] 3 +, but other species such as [LnL SSSS(OH)2(H2O)] +, [Sm2L SSSS2(μ-OH)(H2O)2] 5 + or complex with amine group of the buffer molecule may also contribute to the averaged signals. This kind of signals averaged by chemical exchange is sole signal of diluted 0.2 mM samples of Sm(III) complex at all the investigated pH values (Supporting Fig. S5); the shift of the signals as pH changes from 5.5 to 9.5 reflects predominantly the gradual formation of monomeric hydroxo forms. For the Sm(III) complex the NMR spectra measured at various concentration and pH values reflect at least two equilibria:

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[Sm2L SSSS2(μ-OH)2(H2O)2] 4 + ⇌ [SmLSSSS(OH)(H2O)2] 2 + (slow on the NMR time scale) and [SmLSSSS(OH)(H2O)2]2 + ⇌ [SmLSSSS(H2O)3]3 + (fast on the NMR time scale). In contrast, as the Eu(III) complex is diluted from 8 mM to 0.2 mM at pH = 7.5 the signals of dinuclear form still dominate the spectrum for all concentrations (Supporting Fig. S3). This observation reflects higher stability of the Eu(III) dinuclear species towards dissociation; however the averaged signals of dissociation products may also be present, but simply broadened beyond detection due to additional paramagnetic effects. In summary, the NMR spectra indicate that the presence of dinuclear vs. mononuclear species is determined by the pH and concentration and not by the form of the starting complex. Bligh and coworkers have previously observed cleavage of pSP72 plasmid DNA by the racemic dinuclear Y(III) and Nd(III) complexes, while the racemic mononuclear Gd(III), Y(III) and Dy(III) complexes were not active catalysts for hydrolysis of double-stranded DNA [33]. These experiments were performed in slightly different conditions (Tris–HCl buffer pH 7.6 at 55 °C, mixed water/DMSO solvent) than the one used in this study. While we observe the catalytic activity of starting monomeric complexes, the active species may be the cationic dimeric complexes [Sm2L SSSS2(μ-OH)2(H2O)2] 4 + and [Eu2L SSSS2(μ-OH)2(H2O)2] 4 + prevailing under the used conditions. The discrepancy regarding the activity of the starting monomeric complexes results most likely from the form of the complexes used. The racemic form [LnL rac(NO3)2](NO3) used in the previous studies may be less soluble hence less active than the enantiopure form [LnL SSSS(NO3)2](NO3), which we used in this study.

Acknowledgement This work was supported by the Polish Ministry of Science and Higher Education, grant no. NN204 017135 and in part by the Polish Foundation for Science, grant F1/2010. We thank Hanna Krężel for help in plasmid purification.

Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.jinorgbio.2011.10.011.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

4. Conclusions The catalytic activity of the [LnL(NO3)2](NO3) complexes in the hydrolytic cleavage of DNA strongly depends on the kind of the lanthanide(III) ion and the helicity of the complex. The [SmLSSSS(NO3)2] (NO3) and [EuLSSSS(NO3)2](NO3) enantiomers promoted the cleavage of the supercoiled form of plasmid pBR322 to the nicked form, while the [LnLRRRR(NO3)2](NO3) enantiomers of opposite sense of the helical twist of the macrocycle L were inactive. In particular at pH =8.5 and c =0.2 mM or pH= 7.5 and c =1 mM, the conversion of linear to nicked plasmid DNA is close to 100% for the all –S enantiomer and 0% for the all – R enantiomer. On the other hand, the analogous Yb(III) complex triggered aggregation of DNA. The NMR spectra of the solutions of [LnL (NO3)2](NO3) complexes in various buffers indicate that the monomeric cationic complex [LnL(H2O)3]3 + is a sole form present at pH= 5.5 and the dimeric hydroxo-bridged cationic complex [Ln2L2(μ-OH)2 (H2O)2] 4 + is a sole form at pH = 9.5 and c = 8 mM. At pH values and concentrations used for cleavage experiments, both the dinuclear form and monomeric hydroxo derivatives may be present. While the mechanism and active catalytic form have yet to be established, the catalytic activity of the studied complexes in the hydrolytic cleavage of DNA is clearly governed by the chirality of the macrocyclic ligand.

[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]

Abbreviations SC DNA supercoiled DNA plasmid NC DNA nicked DNA plasmid pBR322 commonly used E. coli cloning vector having 4361base pairs DH10B E. coli chemically competent E. coli cells used for plasmid DNA transformation LB medium Luria–Bertani medium

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