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A Stimulus-Responsive Peptide for Effective Delivery and Release of DNA in Plants Jo-Ann Chuah, and Keiji Numata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00016 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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A Stimulus-Responsive Peptide for Effective Delivery and Release of DNA in Plants Jo-Ann Chuah and Keiji Numata* Enzyme Research Team, Biomass Engineering Research Division, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

E-mail: [email protected]

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ABSTRACT

For efficient gene delivery in plant systems, non-viral vector and DNA complexes require extracellular stability, cell wall/membrane translocation capability, and the ability to mediate both endosomal escape and intracellular DNA release. Peptides make appealing gene delivery vectors due to their DNA-binding, cell-penetrating, and endosome escape properties. However, DNA release within cells has so far been inefficient, which results in poor and/or delayed gene expression, while the lack of understanding of both internalization and trafficking mechanisms is a further obstacle to the design of efficient peptide gene delivery vectors. Here, we report successful gene delivery into plants using a cellular environment-responsive vector, BPCH7, which is an efficient cell-penetrating peptide with a cyclic DNA-binding domain that is formed by a disulfide bond between two cysteines. The cyclic structure of BPCH7 confers high avidity attachment to DNA in vitro. Following endocytosis into cells, disulfide bond cleavage facilitated by intracellular glutathione induces structural changes within BPCH7 that enable the release of the associated DNA cargo. Comparative studies with BPKH, a cell-penetrating peptide with a linear DNA-binding domain, show that BPCH7 maximized and expedited gene transfer in cells, and unveil for the first time the crucial role of plant stomata in the internalization of peptideDNA complexes.


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INTRODUCTION Peptides – biodegradable molecules with tailorable sequences and functionality – have garnered much attention as non-viral tools for nucleic acid delivery into plant and animal cells.1-5 The clearest distinguishing feature between plant and animal cells is the presence of a cellulosic cell wall in plants, which bestows rigidity and hinders the penetration of foreign particles. The cell wall presents not only a physical barrier but also a chemical barrier; its negatively charged surface can form hydrogen bonds with cationic molecules and deter their passage through the cell membrane.6, 7 Hence, existing approaches for delivering functional, exogenous genes into plant cells are significantly limited in terms of plant type and efficiency compared to the multiple well-developed systems that are available for delivering genes into animal cells (especially mammalian cells). Transporting plasmid DNA (pDNA; circular8-10 and linearized11 forms), double-stranded RNA (dsRNA),12 double-stranded DNA (dsDNA),13 and transfer DNA14 into various types of plant cell has been accomplished using a plant-infecting virus capsid protein12 as well as conventional cell-penetrating peptides (CPPs), such as Tat2,9, 11, 13, 14 Pep-1,13 and arginine-rich CPPs.8, 10, 15 More recently, our research group developed dual-domain peptides with segregated cell-penetrating, endosomal escape, and DNA-binding functions for use as gene delivery vectors in plants. We conjugated the BP100 peptide (hereafter abbreviated as BP), a membrane-active antimicrobial peptide with efficient cell-penetrating ability16, with a short peptide of alternating lysine and histidine residues, and the resultant peptide (BPKH, amino acid sequence: KKLFKKILKYLKHKHKHKHKHKHKHKHKH, 3,810 Da) enables the delivery of pDNA,17 dsRNA,18 and dsDNA19 into leaf cells. Meanwhile, other well-known CPPs, such as transportan,


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TP10, penetratin, and pVEC, have shown potential for further evaluation as gene delivery agents in plants.20 Despite these advances, the biochemical and biophysical mechanisms underlying peptidecargo association and dissociation are unresolved, and a clear insight into the mechanism of cellular uptake and internalization of these complexes in plants has yet to emerge. For effective DNA delivery in plants, peptide-based carriers must be capable of binding and subsequently releasing DNA cargo and overcoming several cellular barriers, including plasma and endosomal membranes.1, 3 Although each step is critical in determining the final gene delivery efficiency, a major bottleneck is the timely intracellular unpacking of peptide-DNA complexes to enable gene transfer and expression.21-23 A bioresponsive peptide may therefore satisfy the requirements for DNA association in vitro and rapid dissociation within intracellular environments, i.e., the cytoplasm or nucleus. Despite the importance of vector unpacking to free bound DNA cargo inside the plant cell, this process was neither understood nor controlled in any of our previous studies using the BPKH peptide. Herein, we designed a stimulus-responsive peptide, BPCH7 (amino acid sequence: KKLFKKILKYLHHCRGHTVHSHHHCIR, 3,358 Da), derived from the conjugation of BP and CH7, a reducible pDNA-binding domain for a cellular environment-responsive DNA release.24 We evaluated BPCH7, which is cyclic due to a disulfide bond between two cysteines, for its ability to bind pDNA and respond to endogenous glutathione (GSH) as a stimulus to release the associated cargo in plant cells (Scheme 1). BPKH, a functional cell-penetrating peptide without a tunable DNA release function, and BPLH7, the linear analogue of BPCH7, were studied in comparison. We also elucidated the mechanism underlying BPCH7-DNA complex dissociation,


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and present microscopy data that provides important insights into the translocation of peptidebased complexes across the complex layer intrinsic to plant cells.

Scheme 1. Schematic representation of the glutathione-reducible peptide (BPCH7) and the proposed mechanism for intracellular delivery and subsequent pDNA release. BPCH7 (KKLFKKILKYLHHCRGHTVHSHHHCIR) can form sufficiently stable complex with plasmid DNA extracellularly and once delivered into the plant cell (endocytosis), the reductive intracellular environment, mediated mainly by GSH, induces cleavage of the intramolecular disulfide bond within the cyclic CH7 domain, thereby causing complex dissociation and subsequent release of pDNA in the cell for expression in the nucleus.

EXPERIMENTAL SECTION Materials. Peptides were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis.25 The amino acid sequences and molecular weights are as follows: BPCH7

and

BPLH7

(KKLFKKILKYLHHCRGHTVHSHHHCIR,

3,358

Da);

BPKH


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(KKLFKKILKYLKHKHKHKHKHKHKHKHKH, 3,810 Da); BP (KKLFKKILKYL, 1,422 Da); CH7 (HHCRGHTVHSHHHCIR, 1,954 Da). The purities of these peptides were characterized by HPLC with an Inertsil ODS-3 column (GL Sciences, Tokyo, Japan) at 25°C (Fig. S1). The mobile phase comprised 15–45% CH3CN containing 0.1% TFA. The flow rate was 1.0 mL/min. The pDNA used encoded either Renilla Luciferase (RLuc) or green fluorescent protein (GFP) genes expressed under the control of the constitutive cauliflower mosaic virus 35S promoter (p35S-RLuc-tNOS and p35S-GFP-tNOS, respectively).17 Glutathione (reduced form) was purchased from Wako Chemical Co. (Osaka, Japan). The DiaEasyTM Dialyzer (1 kDa Molecular Weight Cut-off) was purchased from BioVision, Inc. (Milpitas, CA, USA). The Renilla Luciferase Assay System was purchased from Promega (Madison, WI, USA). The Label IT® Nucleic Acid Labeling Kit, Cy3 was purchased from Mirus Bio, LLC (Madison, WI, USA). Hoechst 33258 and BCECF-AM were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Circular Dichroism (CD) Spectroscopy. The CD spectra of peptides in various solvents were acquired using a Jasco J-820 CD spectropolarimeter. Peptides (10 µM) were dissolved in water, acetate buffer (20 mM; pH 4, 5, or 6), phosphate buffer (20 mM; pH 7 or 8), or Tris buffer (20 mM; pH 9) with and without GSH (10 mM). Background scans were obtained for the individual solvents. Measurements were acquired using a quartz cuvette with a 0.1 cm pathlength. Each spectrum represents the average of ten scans from 190 to 240 nm with a 1 nm resolution, obtained at 200 nm min-1 with a bandwidth of 1 nm. The secondary structure contents of each peptide were calculated by the DichroWeb online CD analysis server using the CONTIN algorithm in combination with reference dataset 4 optimized for data ranging from 190–240 nm.


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Formation and Stability of Peptide-pDNA Complexes. Peptide-pDNA complexes were prepared by adding different amounts of each peptide to pDNA at various N/P ratios (0.5, 1, and 2) and autoclaved Milli-Q water to obtain the final volumes required for each experiment. The solution was thoroughly mixed by repeated pipetting and allowed to stabilize for 1, 5, 10, or 24 h at 25°C. Electrophoretic mobility shift assays were performed to detect the stabilities of complexes formed between the peptide and pDNA as previously described.17 Each peptide was added to pDNA (0.2 µg) at various N/P ratios, adjusted to a final volume of 20 µL, and electrophoresed on a 1% (w/v) agarose gel for 30 min at 100 V. Dynamic Light Scattering (DLS) and Zeta Potential Analyses. Each peptide was mixed with pDNA (20 µg) at various N/P ratios and adjusted to a final volume of 800 µL with autoclaved Milli-Q water. The solutions were thoroughly mixed by repeated pipetting and allowed to stabilize for 1, 5, 10, or 24 h at 25°C. DLS and zeta potential analyses were carried out using a zeta potentiometer (Zetasizer Nano-ZS; Malvern Instruments, Ltd., Worcestershire, UK) as previously described.17 Atomic Force Microscopy (AFM). Naked pDNA (3.7 nM) or peptide-pDNA complexes at various N/P ratios were prepared in water and allowed to stabilize for 1 h at 25°C before being deposited onto freshly cleaved mica. Following a 10-min incubation period, excess solution was washed with water and dried with a gentle stream of nitrogen. The morphologies and distributions of the pDNA and peptide-pDNA complexes were visualized by atomic force microscopy (AFM5300E, Hitachi High-Technologies Corporation, Tokyo, Japan) as previously described.17 The cantilever had a spring constant of 20 N/m. Triggered Destabilization of Peptide-pDNA Complexes. Peptide-pDNA complexes were prepared by adding the peptide to pDNA (0.2 µg) at various N/P ratios in water, acetate


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buffer (20 mM; pH 4, 5, or 6), phosphate buffer (20 mM; pH 7 or 8), or Tris buffer (20 mM; pH 9) with and without GSH (10 mM) or KCl (100 mM)/NaCl (30 mM). Complex solutions were adjusted to a final volume of 20 µL, allowed to stabilize for 1 h at 25°C, and electrophoresed under the conditions described above. Raman Spectroscopy. Lyophilized BPCH7 was measured in the solid state without further preparation. To induce the cleavage of intramolecular disulfide bonds within BPCH7, the peptide (2.98 mM) was dissolved in phosphate buffer (20 mM, pH 8) supplemented with GSH (10 mM) and incubated for 1 h at 25°C. Glutathione (GSH/GSSG) was removed by dialysis against Milli-Q water for 30 h at 25°C. The purified peptide (BPLH7) was collected, lyophilized, and subjected to analysis. Data were collected using a Jasco NRS-4100 laser Raman spectrometer (JASCO, Tokyo, Japan) in the spectral range of 400–2700 cm-1 at a resolution of 1 cm-1. For each spectrum, the acquisition time was 60 s, and the number of averaged spectra was two. Extra-/Intracellular pDNA Release and Gene Expression. A time-course study of pDNA release from peptide-pDNA complexes was performed by preparing the complexes at various N/P ratios in Milli-Q water, and then incubating them in phosphate buffer (20 mM, pH 8) supplemented with GSH (10 mM). At 3, 6, 9, 12, 24, and 36 h, complex solutions were loaded on to a 1% (w/v) agarose gel and electrophoresed under the conditions described above. The intensities of selected DNA bands were quantified by densitometry analysis of the gel images using ImageJ software (version 1.48, National Institutes of Health, MD, USA). Released DNA was expressed as the fraction of DNA with restored mobility relative to pDNA alone, based on the relative intensities of the corresponding bands. For intracellular studies, wild-type and transgenic (YFP) A. thaliana plants, which served as model systems in this study, were grown


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under previously described conditions.18 Leaves were infiltrated with the complexes by syringe as described, and sampled at the same time points as those used for the time course study of extracellular DNA release. Intracellular expression of the RLuc gene was evaluated quantitatively by the RLuc assay as previously detailed.17 GFP fluorescence in leaf cells infiltrated with pDNA alone and in complexes with BPLH7 or BPCH7 (N/P 0.5) were observed by CLSM as previously described.17 Subcellular Localization of Peptide-pDNA Complexes. p35S-GFP-tNOS was labeled with a Nucleic Acid Labeling Kit according to the manufacturer's instructions. Leaves of wildtype or transgenic A. thaliana plants were infiltrated with Cy3pDNA (5 µg or 15 µg) in complex with BPCH7 or BPKH at N/P 0.5 and incubated for 1 h (except for time-lapse experiments). Epidermal and mesophyll cells from the adaxial side were observed and imaged using a CLSM as previously described.17 Colocalization analysis of micrographs was performed using Zen 2011 operating software. Leaves were stained with 5 µg/ml Hoechst 33258 solution or 10 µM BCECFAM when needed. Image stacks of transgenic A. thaliana leaves infiltrated with BPCH7Cy3pDNA or BPKH-Cy3pDNA complexes were collected in the z-direction at 0.3 µm increments for 19.8 µm. 3D reconstructions and digital processing of images and movies were performed using ImageJ software. Statistical Analysis. SPSS 22.0 for Mac (IBM, Armonk, NY) was employed for statistical analysis. Tukey’s Honest Significant Difference (HSD) test was used for pairwise comparisons among means. Differences between two means were considered statistically significant at P < 0.05 or P < 0.01 and are indicated with asterisks (* or **, respectively). Experimental data are expressed as the means ± standard deviation.


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RESULTS AND DISCUSSION Structural and DNA-binding properties of BPCH7. BPCH7 was stable and highly water-soluble at ambient conditions. To analyze its structure in aqueous solution, we acquired circular dichroism (CD) measurements. Constituent domains of the cyclic BPCH7 peptide, BP and CH7, exhibited no defined secondary structures in water, while BPCH7 presented a helical structure (Fig. 1a). We subsequently examined the ability of BPCH7 to interact with pDNA using an electrophoretic mobility shift assay, and compared its DNA-binding properties with those of BP, CH7, and BPKH, our previously designed bipartite peptide with a linear pDNAbinding domain.17 Both the cyclic BPCH7 and linear BPKH peptides were capable of forming complexes with pDNA at various N/P ratios, although different pDNA migration patterns were observed (Fig. 1b). The N/P ratio is defined as the number of amine groups from the peptide versus the number of phosphate groups from pDNA. Higher pDNA-binding stabilities were observed for BPCH7 than for BPKH, regardless of the N/P ratio or the incubation duration for complex formation. In comparison, BP did not show noticeable DNA-binding ability in any of the conditions, while CH7 retarded DNA mobility only slightly with prolonged incubation (up to 10 or 24 h). Both the helical structure and longer backbone length of BPCH7 cooperatively contribute to its ability to bind pDNA, an observation found to be true for other peptides with similar properties,26 which also explains the inability of the shorter as well as unstructured BP and CH7 domains to bind pDNA. These findings indicate that the cyclic BPCH7 peptide could interact favorably with pDNA without any disruptions and sufficiently pack the pDNA molecules in resultant complexes. In contrast, the linear BPKH has higher chain flexibility that contributes to a high conformational entropy, which consequently compromises the pDNA packing efficiency. The


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enhanced stability of BPCH7-pDNA complexes in vitro may be important for improving gene delivery efficiency.27 The current results reveal that in combination, unstructured BP and CH7 domains produced BPCH7 with a helical structure (which promotes cellular uptake).28-31 These data also reveal that unlike the other peptides tested, BPCH7 has the ability to form stable complexes with pDNA in vitro.

(a) [θ] × 10-3 deg cm 2 dmol-1

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40 20 0 -20 -40 190

210 230 Wavelength (nm)

(b) N/P 0 N/P 0.5 N/P 2 N/P 1 time (h): 0 5 10 24 0 5 10 24 0 5 10 24

BP

N/P 0 N/P 0.5 N/P 2 N/P 1 time (h): 0 5 10 24 0 5 10 24 0 5 10 24

BPCH7

N/P 0 N/P 0.5 N/P 1 N/P 2 time (h): 0 5 10 24 0 5 10 24 0 5 10 24

CH7

N/P 0 N/P 0.5 N/P 2 N/P 1 time (h): 0 5 10 24 0 5 10 24 0 5 10 24

BPKH

Figure 1. Secondary structure and DNA binding characteristics of peptides. (a) CD spectra of BPCH7 (solid line) and its individual domains, BP (dotted line) and CH7 (dashed line) in water. (b) Electrophoretic mobility of pDNA in complexes with BP, CH7, BPCH7 or BPKH at various N/P ratios ranging from 0 (pDNA alone) to 2.

Size, surface charge, stability and morphology of BPCH7-pDNA complexes. The biophysical characteristics of complexes – particularly their size, zeta potential, homogeneity,


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morphology, and distribution – are known to influence internalization, intracellular trafficking, and transfection efficiency.1, 32 We determined complex sizes and zeta potentials by dynamic light scattering (DLS) and zeta potential analyses, respectively, while morphological characteristics and homogeneity were observed by atomic force microscopy (AFM). The diameters of BPCH7-pDNA complexes (produced at an N/P of 0.5 and 1) were approximately 170–200 nm, suitable for cell internalization via clathrin-mediated endocytosis,33, 34 and did not differ significantly over a 10-h incubation period, suggesting good complex stability against premature dissociation or aggregation (Fig. 2a). BPCH7 was also capable of condensing pDNA into spherical particles 7). The high stringency in conditions enabling BPCH7 reduction renders the pDNA release target-specific, i.e., in the cytosol or nucleus with GSH present at millimolar levels in combination with pH values of approximately 7.539 (or higher, in actively dividing cells).41 More importantly, pDNA release in acidic intracellular compartments (pH 4–6)38, such as vacuoles or endosomes/lysosomes, prior to escape (assuming that complex uptake into cells occurs via the endocytic pathway) can be circumvented. We envision future modifications of this system (e.g., incorporation of sequences targeting mitochondria or chloroplasts as reported previously40) to enable stimuli-responsive gene delivery to organelles with high GSH contents and an alkaline pH,38, 42 for which efficient gene delivery systems are currently unavailable. Meanwhile, marginal changes in the migration patterns of pDNA bound to linear BPLH7 or BPKH peptides can be attributed to pH-induced conformational changes in the pDNA-binding domains of both peptides containing several titratable histidine residues.40

Molecular mechanism underlying BPCH7-pDNA complex dissociation. The stimulus-responsive behavior of BPCH7 and subsequent rapid complex dissociation prompted us to investigate the underlying mechanism. By CD analysis, we acquired secondary structures of BPCH7 at all conditions inducing the destabilization of BPCH7-pDNA complexes (Fig. S4).


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BPCH7 adopted a predominantly helical structure from pH 4 to 7 (50–60% helical content), and a transition from a helix to an disordered structure (50–70%) was observed at pH 8 and 9 (Fig. S4 and Table S2). The cleavage of intramolecular disulfide bonds within BPCH7, mediated by GSH at alkaline pH, presumably caused the peptide to linearize, which subsequently disrupted its inherent helical conformation in water (approximately 41% helical content; Table S2). We infer that this structural transition in conditions consistent with the cytoplasm and nucleus perturbs the stability of extracellularly formed complexes, eventually leading to complete dissociation and release of the entrapped pDNA cargo. To obtain further structural evidence, the secondary structures of BPLH7 and BPKH were examined in water and at pH 8 with and without GSH, and compared to that of BPCH7 (Fig. S5 and Table S3). For BPCH7, the loss of pDNA-binding stability at pH 8 with and without GSH relative to its stability in water can be explained by the helix-to-disordered transition, as discussed above. BPLH7, whose pDNA-binding stabilities did not differ among the tested conditions, assumed a largely disordered structure (60–70%) that remained unchanged throughout the experiment. Similarly, the pDNA-binding tendencies of BPKH were constant at the different conditions, and no significant variations were observed in the secondary structures of the peptide at the corresponding conditions (almost equal contents of helical and disordered structures). Taken together, our structural data substantiate our proposed mechanism for BPCH7 peptide-mediated gene delivery.

Intracellular gene expression. Having established the ability of BPCH7-pDNA complexes to be destabilized by GSH-induced reduction, we proceeded to evaluate the


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transfection efficiencies of these complexes compared to BPKH-pDNA complexes, whose gene delivery efficiency we have evaluated in a previous study.17 To create a correlation between complex dissociation and transgene expression, we studied the pDNA release profile in vitro. As noted in experiments above, the release of attached pDNA from BPCH7 occurred rapidly (within 1 h) and almost completely even at the earliest time point of 3 h for complexes prepared at N/P 0.5 (Fig. 4a, based on the relative intensities of corresponding DNA bands in the gel images shown in Fig. S6). For evaluation of in vivo gene expression, we infiltrated the leaves of Arabidopsis thaliana with peptide-DNA complexes and quantified expression using a Renilla luciferase (RLuc) assay, sampling leaves intermittently during an incubation duration of up to 36 h. The delay in the increase in transgene expression levels from 3 h (~90% of released pDNA) to a maximum at 12 h (Fig. 4b and Table S4) following intracellular pDNA release is accounted for as the time required from transcription initiation to synthesize detectable amounts of the transgene product. As with all transient expression systems, transgene expression then diminished gradually as pDNA is cleared or degraded over time. BPCH7-pDNA complexes formed at higher N/P ratios (1 and 2) were more stable and thus required more time for pDNA release, consequently giving rise to lower transgene expression levels. Nevertheless, the role of the cyclic bioreducible pDNA-binding domain in BPCH7 in activating pDNA release is evidently effective, as the eventual amounts of released pDNA surpassed 90% at all N/P ratios, even if longer durations were required with increasing N/P ratios (Fig. 4a). While BPKH-pDNA complexes exhibited similar pDNA release profiles to BPCH7pDNA complexes, pDNA release from the former occurred at a slower pace, and the total fraction of released pDNA was lower (~80% at N/P 1 and

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