Enhanced enzymatic degradation resistance of plasmid DNA in ionic liquids

Abstract

Plasmid deoxyribonucleic acid (pDNA) is a promising therapeutic in gene therapy and as a DNA vaccine, but it is susceptible to degradation by nucleases in the extracellular environment. In this study, we report on the enhanced enzymatic degradation resistance of pDNA, when stored in a hydrated buffered ionic liquid (BIL) based on choline dihydrogen phosphate (CDHP). The stability of pDNA stored in buffered CDHP (bCDHP) in the presence of Turbo DNase was studied using agarose gel electrophoresis, which showed a prolonged shelf life at room temperature over a period of 28 days in bCDHP compared to within 10 minutes for phosphate buffered saline (PBS). In addition, the biological activity of pDNA was maintained under such conditions, as demonstrated by the expression of yellow fluorescent protein (YFP).

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Introduction

Plasmid DNA (pDNA) is a powerful tool in genetics for multiplying or expressing particular genes or therapeutic agents. However, pDNA is susceptible to nucleases particularly in vivo and during long term storage.¹ It has been reported that the half-life of pDNA in vivo can be as short as 5–15 min.^1,2 It would also be advantageous to store plasmid DNA at room temperature in non-aseptic conditions, which would enable ease of global transportation and ease of administration. Presently, the ideal storage conditions for pDNA are in a dry solid state at room temperature, at −20 °C, −80 °C or cryogenic preservation at −196 °C for prolonged storage and stability of stock solutions.³

One strategy that has been used for improving the stability of DNA is by storage in BILs.⁴ One type of proton buffered ILs is achieved by introduction of a conjugate acid/base pair such as H₂PO₄⁻/HPO₄²⁻ ion components. These materials are typically water miscible and can be formulated at various water contents from low water levels (when they are often referred to as hydrated ionic liquids) to relatively high water contents where their properties approach those of a traditional aqueous salt solution. ILs, particularly the systems based on choline dihydrogen phosphate (CDHP) and its buffered version (bCDHP), have demonstrated their great potential as storage media for several biomolecules to date, including siRNA,⁵ linear DNA,^6–11 and several enzymes/proteins, which were found to display enhanced stability and activity in the presence of the IL.^12–18 Despite this, storage of pDNA in BILs has not yet demonstrated to the best of our knowledge.

Here, we thoroughly investigated the biological, thermal and structural activity of pDNA in bCDHP. The pDNA studied consists of 5 kbps with the yellow fluorescent protein (YFP) as the reporter gene. It is representative of a typical plasmid that may be used for DNA vaccines or gene therapy (see ESI Fig. S1†). When stored at 25 °C or 37 °C in the presence of Turbo DNase (TD), we found a significant improvement in the thermal and structural stability of pDNA using bCDHP as a storage buffer in comparison to PBS. In addition, the biological activity of the plasmid was maintained, as expression of YFP was observed in TD treated pDNA stored in bCDHP for up to one month at 25 °C or up to one week at 37 °C, highlighting the benefit of storing therapeutic plasmids in a BIL.

Results & discussion

Structural stability of pDNA in bCDHP

We first investigated the structural stability of YFP-pDNA stored in PBS, 20% (w/w) bCDHP or 50% (w/w) bCDHP in the absence and presence of TD using agarose gel electrophoresis (Fig. 1). TD offers 50 times more activity and 350% greater catalytic activity than DNase I. It is also capable of maintaining its activity in the presence of high salt environments and cleaves double stranded DNA (dsDNA) and single stranded DNA (ssDNA) non-specifically.^19,20 It can be seen in Fig. 1 that the plasmid (in PBS) displays the typical three distinct forms of pDNA: relaxed circular (20 kbps); linear (7 kbps); and supercoiled, which made up the majority of pDNA present. In both 20% (w/w) bCDHP and 50% (w/w) bCDHP however, only two forms of pDNA were distinct – the relaxed circular and supercoiled forms, again with the majority present in the supercoiled form. This was compared to TD treated YFP-pDNA in PBS, 20% (w/w) bCDHP and 50% (w/w) bCDHP whereby it can be seen (Fig. 1b) that PBS no longer illustrates any bands indicating complete degradation. YFP-pDNA in bCDHP on the other hand does not display any change in comparison to untreated YFP-pDNA.

Fig. 1 Degradation profile of YFP-pDNA in PBS, 20% (w/w) bCDHP and 50% (w/w) bCDHP for day 0 (a) YFP-pDNA and (b) YFP-pDNA treated with TD for 10 minutes at 37 °C.

We further investigated the degradation kinetics of TD treated YFP-pDNA in bCDHP (Fig. 2). When TD is added to the storage solution (initial degradation at 37 °C for 10 min then stored at 25 °C), the absence of bands indicates complete digestion or degradation of the plasmid stored in PBS, whereas YFP-pDNA stored in TD treated BIL remained intact (Fig. 2) for up to 42 days. Previous studies of linear DNA in CDHP suggests that the stabilization of YFP-pDNA may be a consequence of the groove binding mechanism, particularly in the A–T rich sequence which forms the narrow minor grooves of DNA^21–23 whereby choline ions interact providing the added stability to the generally more weaker A–T base pairs.^24,25 The degradation kinetics of TD was observed to be very rapid, as within 10 minutes YFP-pDNA in PBS was completely digested (Fig. 2a). The degradation was considerably slower in 20% (w/w) bCDHP and 50% (w/w) bCDHP, which may be due to the choline ions binding to the minor groove of the DNA.²³

Fig. 2 Degradation profile of YFP-pDNA in PBS, 20% (w/w) bCDHP and 50% (w/w) bCDHP in the presence of TD for day 0, (b) day 14, (c) day 28 and (d) day 42.

TD plays a similar role to DNase I in regards to the mechanism of degradation. The degradation mechanism of DNase I has been previously reported to be a multi-step process^26–31 where: nicks are inserted at various random points in each of the strands of pDNA, but not opposite each other. pDNA is only completely degraded when the incurred nicks are directly opposite in the strands. Therefore, pDNA in PBS is rapidly degraded, but when stored in the bCDHP the pDNA has a measure of extra stability as the double strands are protected with choline in the minor grooves preventing the rapid break down of nitrogenous bases.

YFP-pDNA in bCDHP also demonstrated enhanced structural stability using circular dichroism (Fig. 3). There are three possible conformations DNA: A-form, B-form and Z-form, with the A-form most common for DNA–RNA duplexes or RNA, the B form, which is common for DNA (right handed structure) and the Z-form that is a left handed structure. B-form structure of pDNA was retained in both 20% (w/w) bCDHP and 50% (w/w) bCDHP, similar to PBS.^32,33 This is illustrated by the positive maxima at ∼273 nm and a negative maxima at ∼243 nm, with a crossover occurring at ∼252 nm.^33,34

Fig. 3 Circular dichroism spectra of YFP-pDNA in the presence and absence of TD for (a) day 0, (b) day 14 and (c) day 28.

It can be seen in Fig. 3 that YFP-pDNA demonstrates small to negligible changes in its structure over the duration of 28 days for both untreated YFP-pDNA and those treated with TD, initially at 37 °C for 10 minutes and subsequently stored at room temperature, in 20% (w/w) bCDHP and 50% (w/w) bCDHP. Any slight decrease in the circular dichroism signal under these conditions may be the result of the choline ions interacting with the DNA.³⁵ On the other hand, YFP-pDNA in PBS displays a significant change in its structural integrity over the 28 day period, particularly for TD treated YFP-pDNA. YFP-pDNA stored in PBS displayed limited structural stability and integrity, exhibiting a reduction in its structural stability as the peak at 273 nm is reduced for the control samples after fourteen days and further by 28 days. Similarly, those treated with TD showed pronounced loss of structural integrity, with an immediate decrease at 273 nm that further decreased at day 14 and 28, and a concurrent positive increase of the negative maxima, which indicates unwinding of the DNA helix.³⁶

Thermal stability of pDNA in bCDHP

As further investigation into the stability of YFP-pDNA, we also tested the potential thermal stability provided by bCDHP. TD treated and non TD treated YFP-pDNA in PBS, 20% (w/w) bCDHP and 50% (w/w) bCDHP were measured for thermal stability. The presence of the BIL enhanced the thermal stability of YFP-pDNA relative to PBS, with up to 7 °C greater in melting temperature in 20% (w/w) bCDHP and 17 °C in 50% (w/w) bCDHP (Table 1 and see ESI Fig. S2†). Upon thermodynamic analysis using the two state van't Hoff model as an approximation of the pDNA denaturation, we also demonstrate enhanced binding stability in bCDHP represented by the Gibbs free energy (ΔG⁰₂₅). YFP-pDNA binding stability stored in bCDHP increases by almost 2 fold and 3 fold for 20% (w/w) bCDHP and 50% (w/w) bCDHP respectively. This suggests much greater thermal and thermodynamic stability of YFP-pDNA in bCDHP.

Table 1 Thermodynamic parameters for the melting temperature of YFP-pDNA in PBS, 20% (w/w) bCDHP and 50% (w/w) bCDHP^a

	ΔH^0 [kcal mol^−1]	TΔS^0 [kcal mol^−1]	ΔG^025 [kcal mol^−1]	Tm^b [°C]
a All experiments were conducted in 0.01 M PBS and 20% (w/w) bCDHP and 50% (w/w) bCDHP. Thermodynamic parameters were evaluated from Cary UV Thermal software using van't Hoff modelling.b Melting temperature was calculated at a strand concentration of 0.2 μM.c n.d denotes that melting temperature was not able to be determined due to complete degradation.
PBS
pDNA	−34.79 ± 0.8	−21.13 ± 2.4	−13.66 ± 0.9	72.54
pDNA + TD	n.d^c	n.d^c	n.d^c	n.d^c
20%(w/w) bCDHP
pDNA	−98.71 ± 2.9	−73.26 ± 8.0	−25.41 ± 2.4	80.18
pDNA + TD	−40.88 ± 0.9	−22.32 ± 3.4	−18.57 ± 0.5	81.10
50% (w/w) bCDHP
pDNA	−142.0 ± 2.9	−101.1 ± 18	−35.38 ± 2.0	89.48
pDNA + TD	−41.86 ± 0.9	−23.43 ± 2.5	−18.45 ± 0.8	87.15

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Following treatment with TD, the melting temperature of YFP-pDNA in PBS was not possible to determine, indicating degradation of the plasmid, which supports the gel electrophoresis data seen in Fig. 1 and 2. YFP-pDNA in the bCDHP remains very much intact, as the presence of TD only changes the T_m by a degree or two (Table 1 and see ESI Fig. S3†). In addition, we also investigated the thermodynamic parameters of TD treated YFP-pDNA in bCDHP. The Gibbs free energy (ΔG⁰₂₅) gives an indication of the stability of the plasmid, and the greater the negative value, the greater the stability. The ΔG⁰₂₅ of TD treated YFP-pDNA in bCDHP decreases in both 20% (w/w) bCDHP and 50% (w/w) bCDHP. Such an occurrence was expected as the binding stability is affected by the presence of TD as it attempts to disrupt the choline cation binding in the minor grooves. Thus despite a loss in binding stability in the presence of TD is seen in bCDHP, the melting temperature is retained.

Biological stability of pDNA in bCDHP

While the data above has demonstrated enhanced structural and thermal stability of pDNA in bCDHP, a key requirement for pDNA is its ability to still be biological active and confer gene expression in host cells. In this study, we have used pDNA with an inserted YFP reporter gene, but it is a typical plasmid that may be applicable as a pDNA vaccine by replacing the YFP gene with another gene of interest.³⁷

The transfection with treated or non-treated YFP-pDNA was tested in cultured HEK 293T cells, using polyethylenimine (PEI) as the transfection agent. An increase in the fluorescence intensity of 293T cells, which is a result of the production of YFP, is indicative of successful transfection. Typically, untreated 293T cells are non-fluorescent and the fluorescence intensity of these cells was used as the base value. As expected, transfection from YFP-pDNA stored in PBS, resulted in substantial expression of YFP (see ESI Fig. S4a†), with the majority of cells (65%) increasing in fluorescence intensity. Similarly, successful transfection was achieved from YFP-pDNA stored in the bCDHP mixtures, with transfection efficiencies of 31% and 21%, respectively, at day 0 (see Table 2 and ESI Fig. S4b and c†).

Table 2 Transfection efficiency of FACS data for aged transfections (see ESI Fig. S4)^a

	Transfection efficiency [%]
	Day 0	Day 14	Day 28
a Each sample was conducted in triplicates.
PBS
pDNA	64.5 ± 1.5	54 ± 2.2	39.7 ± 5.0
pDNA + TD	3.4 ± 2.4	1 ± 0.2	0.1 ± 0.5
20% (w/w) bCDHP
pDNA	31.3 ± 0.5	42.6 ± 0.5	42.5 ± 1.7
pDNA + TD	29.4 ± 0.8	40 ± 4.3	1.1 ± 0.9
50% (w/w) bCDHP
pDNA	20.9 ± 0.5	39.3 ± 5.6	42 ± 0.9
pDNA + TD	20.6 ± 0.4	42.6 ± 0.2	40.1 ± 0.9

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In the presence of TD, however, there is no longer YFP gene expression from YFP-pDNA stored in PBS. Meanwhile, YFP-pDNA stored in bCDHP maintained YFP gene expression levels similar to that of untreated plasmid for both 20% (w/w) bCDHP and 50% (w/w) bCDHP up to day 14 (see ESI Fig. S4(i and ii)†). Storage in latter mixture appeared to be superior to the former as the gene expression of TD treated YFP-pDNA was similar to that of untreated YFP-DNA for up to 28 days, whereas in 20% (w/w) bCDHP, there is a gradual decrease in transfection efficiency with incubation time and by day 28, the TD treated YFP-pDNA is no longer expressing the YFP gene indicating loss of biological activity.

The expression of YFP-pDNA stored in various buffers was also tested over a seven day period with continual storage temperature of 37 °C. Aliquots were taken at 0 hours (no TD treatment) and then TD treated samples at 10 minutes, 4 hours, 12 hours, 24 hours, 48 hours and 7 days (Fig. 4). Similar to the trend seen in Fig. S4,† it can be seen in Fig. 4 that TD treated YFP-pDNA stored in PBS is degraded rapidly within 10 minutes (Fig. 4) and no transfection is observed. TD treated pDNA in 20% (w/w) bCDHP and 50% (w/w) bCDHP continue to increase their level of gene expression or up to 12 hours at 37 °C, after which the YFP expression from 20% (w/w) bCDHP begins to decrease while for 50% (w/w) bCDHP the pDNA maintains a similar level of YFP production continually for 7 days (see ESI Fig. S5† for transfection efficiencies).

Fig. 4 Flow cytometry analysis of PEI-facilitated YFP-pDNA transfection from plasmid stored in (a) PBS, (b) 20% (w/w) bCDHP and (c) 50% (w/w) bCDHP, over a period of 7 days at 37 °C storage. The black line in the histogram represents fluorescence intensity of cells treated with buffer only, blue line represents the fluorescence intensity of untreated YFP-pDNA and the red line represents YFP-pDNA treated with TD for 10 min followed by storage at 37 °C.

Cytotoxicity of bCDHP

As a means of testing the toxicity of the bCDHP we diluted the stock concentrations of pDNA in PBS and bCDHP, and conducted cell viability tests. The cell viability tests were carried out using flow cytometry at the same concentrations used for transfections. The viability of cells in the presence of bCDHP normalized against PBS treated HEK 293T cells (see ESI Fig. S6†). Under the conditions used for transfection of pDNA, there was insignificant difference in the cell viability levels between cells treated with PBS and those treated with bCDHP. Our results are supported by previous findings on the cytotoxicity levels and the biocompatibility of bCDHP,⁵ which exhibits minimal cytotoxicity in comparison to PBS. There was also very little difference in the toxicity levels between 20% (w/w) bCDHP and 50% (w/w) bCDHP. Thus the negligible cytotoxicity allows for the elimination of removing bCDHP from pDNA prior to biological testing and illustrates its potential as the new generation of storage buffers for biological material.

Confocal microscopy

As another means of confirming YFP gene expression, confocal laser scanning microscopy was also used. Fig. 5 shows the resulting transfected cells with the blue indicating the cell nuclei and green representing the YFP. Blank 293T cells in the absence of any transfections were also imaged to confirm there was no green fluorescence seen (data not shown). Fig. 5 confirms our flow cytometry data seen in see ESI Fig. S4† and were prepared on the same time scale as per Fig. 1 and S4.† Fig. S4a–c† corresponds to day 0, it can be seen that PBS, 20% (w/w) bCDHP and 50% (w/w) bCDHP all illustrate YFP gene expression (Fig. S4a–c(i)†) in HEK 293T cells, however once treated with TD at 37 °C for 10 minutes the YFP-pDNA in PBS has lost its biological activity and is unable to express the YFP gene with no presence of any green in Fig. S4a(ii).† 20% (w/w) bCDHP and 50% (w/w) bCDHP seen in Fig. 4b and c(ii) are both able to produce a considerable amount of YFP protein. This was again repeated on day 28 of storage and whilst all untreated YFP-pDNA was still able to produce YFP protein, YFP-pDNA treated with TD illustrates consistent result with Fig. S4a and b(iii)† corresponding to Fig. S7d and e(ii)† which is for PBS and 20% (w/w) bCDHP and no expression of YFP is detected although YFP-pDNA in 50% (w/w) bCDHP (Fig. S7f(ii)†) was still able to express YFP. Thus confocal microscopy is in agreement with our flow cytometry data for the transfections conducted.

Fig. 5 Confocal microscopy images for transfection at day 0: (a) YFP-pDNA in PBS (b) YFP-pDNA in 20% (w/w) bCDHP (c) YFP-pDNA in 50% (w/w) bCDHP (d) YFP-pDNA in PBS + DNase (e) YFP-pDNA in 20% (w/w) bCDHP + DNase (f) YFP-pDNA in 50% (w/w) bCDHP + DNase. For each image (i) blue stain of nuclei of the HEK 293T cells present and (ii) green represents YFP that have been successfully transfected and expressed in HEK 293T cells.

Conclusions

In summary, we have systematically investigated the stability of YFP-pDNA in bCDHP under various conditions. Our results demonstrate that pDNA is able to retain its native B conformation in the presence of bCDHP, and prolong the stability of YFP-pDNA in the presence of nucleases. Enhanced thermal stability was also seen, with a further emphasis on the nuclease treated samples in bCDHP able to maintain their thermal stability, even increasing the overall melting temperature of the plasmid. Moreover the YFP-pDNA stored in bCDHP was able to remain biologically active and elicit gene expression. The gene expression from plasmid stored in 50% (w/w) bCDHP was maintained for over a period of 28 days, even if pre-incubated with degrading nucleases. This study leads the way to the applicability of bCDHP in the storage of pDNA at room temperature under non aseptic conditions, without loss of stability and activity. It also serves to eliminate the need for lyophilization or refrigeration for the storage and transportation of pDNA, particularly if the pDNA is being applied as vaccines.

Acknowledgements

Australia Discovery Projects [120100170]. CCJ is funded through an Australian Research Council (ARC) Super Science Fellowship through grant FS100100073. CCJ acknowledges the Melbourne Centre for Nanofabrication for the Technology Fellowship. DRM is grateful to the ARC for his Laureate Fellowship funding. R.R.M acknowledges the Monash ChemEng Departmental scholarship. This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra05518k

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