Depth-Profiling the Nuclease Stability and the Gene Silencing Efficacy

Jul 24, 2017 - PEGylation of an oligonucleotide using a brush polymer can improve its biopharmaceutical characteristics, including enzymatic stability...
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Depth-Profiling the Nuclease Stability and the Gene Silencing Efficacy of Brush-Architectured Poly(ethylene glycol)−DNA Conjugates Fei Jia, Xueguang Lu, Dali Wang, Xueyan Cao, Xuyu Tan, Hao Lu, and Ke Zhang* Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States S Supporting Information *

to side effects that arise from protein−DNA interactions, such as enzymatic degradation, immunostimulation (toll-like receptor 9-DNA interaction), coagulopathy (DNA binding to thrombin), and hepatic capture. The pacDNA enters cells via endocytosis despite having a “stealthy” PEG corona, and can effectively regulate cellular gene expression through an antisense mechanism.6c The discovery of protein inhibition for pacDNA is in line with the recognition that brush polymers are capable of sterically excluding other macromolecules.8 On the other hand, the extent of steric protection of pacDNA is sensitive to the relative length between the oligonucleotide and the PEG.6a As the sequence becomes longer, it extends beyond the protective radius of the brush, leading to a rapid drop-off in the degree of protein shielding. Therefore, while we and others have demonstrated the presence of a depth effect,8a so far the picture is a rough one. Furthermore, with the current design, the size of the pacDNA and its protein accessibility are interrelated and cannot be separately tuned. To use pacDNA as a biopharmaceutical, it is desirable to separate the steric shielding character from the overall size of the conjugate, at least to some degree, and to obtain a higher-resolution “depth profile” of nuclease accessibility of the brush polymer, which can provide predictive capability with respect to pacDNA stability prior to synthesis. To achieve these improvements, we have designed a series of 21-mer DNA probes (Table S1), each with 5′ Cy5 and 3′ fluorescein modifications, and an anchor point (a dibenzocyclooctyne (DBCO)-functionalized thymine base, FT) within the sequence for conjugation with the brush polymer. By moving the anchor point along the sequence, for example from 3′ to 5′, the 3′ becomes more exposed and loses nuclease stability, as the 5′ becomes increasingly hindered by the brush and gains stability (Scheme 1). Therefore, a sequence attached via a midchain base to the brush should have identical protection as a half-length sequence attached at 3′ or 5′, in effect allowing a small brush to protect a long oligonucleotide without increasing the overall conjugate size. By comparing different pacDNAs in this series, a detailed depth profile can be generated. A highly repetitive sequence consisting of seven GTG repeating units is chosen for all probes to minimize the effect of local sequence preference of the enzyme (DNase I). The overall length of the probe (21 b) is consistent with that of typical antisense or siRNA strands.

ABSTRACT: PEGylation of an oligonucleotide using a brush polymer can improve its biopharmaceutical characteristics, including enzymatic stability and biodistribution. Herein, we quantitatively explore the nuclease accessibility of the nucleic acid as a function of “depth” toward the backbone of the brush polymer. It is found that protein accessibility decreases as the nucleotide is located closer to the backbone. Thus, by moving the conjugation point from the terminus of the nucleic acid strand to an internal position, much smaller brushes can be used to achieve the same level of steric shielding. This finding also makes it possible to assess antisense gene regulation efficiency of these brush−DNA conjugates as a function of their nuclease stability.

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ligonucleotide therapy has seen remarkable progress in recent years, with three new drugs approved since 2010 (reaching a total of five), and over a hundred candidates currently under clinical trial.1 Despite these achievements, several long-lasting challenges remain serious obstacles for oligonucleotides to be used as biopharmaceuticals, including poor delivery efficiency and off-target side effects, such as coagulopathy and activation of the innate immune system.2 Efforts to address these problems have been spent mainly on two approaches: chemical modification of oligonucleotides,3 and utilization of polycationic carrier systems.4 Still, not all negative aspects associated with oligonucleotides have been adequately addressed chemically, and carriers oftentimes introduce new challenges in delivery and safety.5 Recently, we have shown that covalent conjugation of oligonucleotides to brush-architectured poly(ethylene glycol) (PEG) can be a new route to improving the biopharmaceutical properties of nucleic acid-based drugs. Termed pacDNA (polymer-assisted compaction of DNA), these structures consist of nucleic acid strands covalently linked to the backbone of a brush polymer having PEG side chains.6 PEG is generally considered as safe for foods and pharmaceuticals, and is expected to significantly elevate the pharmacokinetics and safety profiles of oligonucleotides.7 The brush architecture provides oligonucleotides with much higher local PEG densities than linear or slightly branched counterparts, whereby protein access to the DNA component is substantially more hindered.6a Interestingly, hybridization of the pacDNA to a target sequence is nearly unaffected, both kinetically and thermodynamically.6b This steric-based selectivity renders pacDNA much less prone © 2017 American Chemical Society

Received: May 16, 2017 Published: July 24, 2017 10605

DOI: 10.1021/jacs.7b05064 J. Am. Chem. Soc. 2017, 139, 10605−10608

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Journal of the American Chemical Society Scheme 1. High- and Low-Density PEG−DNA Probes for Investigating Enzyme Accessibility

The pacDNAs are prepared by conjugating DBCO-modified DNA via copper-free “click” chemistry with azide-functionalized diblock brush polymers, brush5k and brush10k (side chain Mn 5 and 10 kDa, overall Mn 134 and 204 kDa, respectively) following previously published protocols (Scheme S1).6a The number of DNA strands per brush is determined to be ∼1.4− 1.9 by gel permeation chromatography (GPC) peak integration (Figure 1a and Table S2). Agarose gel electrophoresis indicates

Figure 2. (a) Schematics for FRET-based nuclease stability assay. (b) Representative degradation kinetics of pacDNA5k (FT1, FT4, and FT7). (c) Nuclease half-life as a function of DNA conjugation position. Solid lines: first-order approximation.

the fluorescence of both fluorophores, the relative stabilities of the two termini of the DNA can be estimated. It is found that the initial degradation rate of the 3′ increases as the attachment point moves toward the 5′. Using the nuclease half-life (t0.5) of naked, free DNA probe (DNA-1) as reference, the relative stability, defined as t0.5(pacDNA)/t0.5(DNA-1), can be plotted as a function of conjugation position (Figure 2c). Indeed, the relative stability increases as the oligonucleotide is further inserted toward the brush backbone, with an increment of ∼1 t0.5(DNA-1) per nucleotide (∼0.33 nm) for the brush5k, as revealed by first order approximation (RCy5 > 0.98). For brush10k, the rate is higher, at ∼1.5 t0.5(DNA-1) per nucleotide, implying that the thicker shell of the larger brush makes it more difficult for the enzyme to access the DNA. In addition, for brush10k, the relative stability trend is less linear (RCy5 > 0.90), suggesting that the PEG density distribution within the brush’s hydrodynamic sphere is less homogeneous. The difference between brush-architectured PEG and linear or slightly branched PEG, which are often used for biopharmaceutical conjugation, is evident when one compares the relative nuclease stability of pacDNA with a series of Yshaped PEG−DNA conjugates (YPEG-DNA) bearing the same DNA sequences. The YPEG herein has a Mn of 40 kDa, and is used in Pegaptanib (brand name Macugen), a PEG-modified aptamer that has been on the market since 2004.10 Remarkably, despite having longer PEG arms than the pacDNAs (20 kDa each arm vs 5 or 10 kDa), YPEG-DNA conjugates only show baseline-level nuclease stabilities, irrespective of the conjugation site. These results support our hypothesis that the steric congestion associated with the brush architecture makes it more effective in the context of protein shielding than linear or slightly branched architectures (Scheme 1). The backbone number-average degree of polymerization (DPn) may also affect the protein shielding capability of the brush, because a small DPn cannot create the necessary steric congestion. To demonstrate, we compare the t0.5 of several

Figure 1. (a) Aqueous GPC chromatograms of the reaction mixture containing pacDNA and unreacted free DNA, showing baseline separation. (b) Agarose gel electrophoresis of purified pacDNA5k-FT7 and free DNA-1. (c) Aqueous GPC chromatograms of all PEG−DNA probes in this study (nonpeak region removed for clarity).

the successful synthesis of pacDNAs (Figures 1b and S1). Both brushes and their DNA conjugates have unimodal, narrow size distributions (Figure 1c). Transmission electron microscopy (TEM) reveals that the pacDNA exhibits a spehrical morphology with a dry-state diameter of ∼16 nm (pacDNA5k) or ∼27 nm (pacDNA10k) (Figure S2). These values are consistent with dynamic light scattering (DLS) hydrodynamic size measurements (Figure S3 and Table S3). The ζ-potential of pacDNA is ∼−11 mV (pacDNA5k) or nearly neutral (pacDNA10k), less negative than that of free DNA (∼−26 mV), indicating successful conjugation (Table S4). To study the depth effect of the brush, a fluorescence resonance energy transfer (FRET)-based assay is adopted (Figure 2a). 9 All pacDNAs (five pacDNA 5k and five pacDNA10k) are each prehybridized with two equivalents of a quencher-labeled complementary sequence (dabcyl moieties at both 3′ and 5′, Figure S4). When the quencher and fluorophore are in close proximity, fluorescence is greatly reduced. Upon introduction of 0.1 unit/mL bovine pancreas DNase I, dsDNA is cleaved, which leads to liberation of the fluorophore and a corresponding increase in fluorescence signals. By monitoring 10606

DOI: 10.1021/jacs.7b05064 J. Am. Chem. Soc. 2017, 139, 10605−10608

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Journal of the American Chemical Society

We next study gene silencing activities toward Bcl-2 in SKOV3 cells. Strikingly, Western blotting shows that cells treated with internally anchored pacDNAs lead to generally greater knockdown at an intermediate dosage (1 μM DNA), with the best being FC3 and FC4 conjugates (∼40% by band densitometry analysis), which have the anchor point located near the middle of the sequence. A similar level of efficiency is observed for Lipofectamine-complexed DNA (46%). However, Lipofectamine proves to be significantly more cytotoxic than the PEG-based pacDNA (Figure 4e). In contrast, anchor points

pacDNA5k conjugates with increasing brush DPn (1, 9, 14, 26, 50, Figure 3a and Table S5). The protein shielding capacity

Figure 3. (a) DMF GPC chromatograms of diblock brush5k of varying backbone lengths. (b) Nuclease stability of pacDNA5k as a function of brush backbone DPn.

reaches a limit when DPn = 26 (Figure 3b), suggesting steric congestion is maximized, and further increases of the DPn only reduces DNA loading and does not yield additional protective benefits. Interestingly, this DPn value coincides with the structural transition of the brush from spherical to cylindrical.11 For brush10k, the highest DPn achievable is 20 due to limitations of the polymerization reaction. Therefore, for depth profiling, a DPn of 20 is used although it may not represent the best possible protection that can be achieved with 10 kDa PEG side chains. Interestingly, pacDNA5k-FT4 shows an overall stability enhancement (determined by the least stable terminus) similar to that achieved with the brush10k at the FT3 position (indicated with a red dashed line in Figure 2c). This result implies that, by adjusting the oligonucleotide conjugation position, 1) a small brush (brush5k) can yield better shielding than a large one (brush10k), and 2) nuclease stability and pacDNA size can be independently accessed. The latter is important to elucidating certain biological observations associated with the pacDNA. Previously, we have discovered that larger pacDNAs bearing antisense sequences show higher gene silencing efficacy.6c These pacDNAs also exhibit greater cell endocytosis, especially in macrophage cells.6d Therefore, it is unclear whether the increased nuclease stability is a contributing factor for the high antisense activity for large pacDNAs. To investigate the contribution of protein shielding toward antisense gene regulation, we choose an anti-B-cell lymphoma 2 (Bcl-2) antisense sequence as a model system for pacDNA synthesis (TCT CCC AGC GTG CGC CAT). Analyzing the sequence, we find that several cytosine bases (bold and italicized) are spaced within the sequence such that they can give varying levels of enzyme stability once conjugated to a brush. We synthesize those sequences (FC1-FC5, Table S1) with a 5′ Cy3 modification for fluorescent tracking and each C in turn being a DBCO-modified anchor point, and form pacDNAs using brush5k. We first test the cellular uptake for these pacDNAs in SKOV3 cells, a human ovarian cancer line, using naked Cy3-labeled DNA as control. Following 4 h of incubation at 1 μM DNA concentration, flow cytometry reveals little to no difference in cell uptake among the pacDNAs, all of which being 10−12× higher than naked DNA, consistent with our previous studies. Confocal microscopy confirms that the fluorescence originates from intracellular compartments (Figure 3a, b, Figure S5), except for naked DNA, for which signals are undetectable under identical imaging settings.

Figure 4. (a−c) Confocal microscopy of SKOV3 cell treated with 1 μM Cy3-labeled DNA or pacDNA. Cell nuclei are stained with DAPI (blue). (d) Flow cytometry of SKOV3 cells incubated with 1 μM Cy3labeled DNA or pacDNA. (e) MTT cytotoxicity for pacDNAs and Lipofectamine-complexed DNA.

near the termini and a scrambled sequence result in nearly no antisense activity (Figure 5b). The same experimental

Figure 5. (a) Western blot of cells treated with anti-Bcl2 pacDNA and controls (1 μM DNA). (b) Gene silencing efficiency for pacDNA5k of varying DNA anchoring locations.

procedures are carried out using a different cell line (SKBr3), which yield similar results (Figure S6). Collectively, these data provide evidence that antisense gene regulation efficiency is strongly correlated with DNA protein accessibility. In summary, we have obtained a “depth profile” of nuclease accessibility for pacDNAs, which shows that, for each nucleotide’s distance toward the backbone of the brush, a gain of 1−1.5× the half-life of the naked strand can be expected. We demonstrate that anchoring the nucleic acid via an internally modified base to the brush polymer can further enhance the shielding property of the brush, which is strongly correlated with the antisense activity of the nucleic acid. 10607

DOI: 10.1021/jacs.7b05064 J. Am. Chem. Soc. 2017, 139, 10605−10608

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(9) (a) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K. R.; Han, M. S.; Mirkin, C. A. Science 2006, 312, 1027. (b) Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano Lett. 2009, 9, 308. (c) Xu, X.; Han, M. S.; Mirkin, C. A. Angew. Chem. 2007, 119, 3538. (10) Ng, E. W. M.; Shima, D. T.; Calias, P.; Cunningham, E. T.; Guyer, D. R.; Adamis, A. P. Nat. Rev. Drug Discovery 2006, 5, 123. (11) (a) Jha, S.; Dutta, S.; Bowden, N. B. Macromolecules 2004, 37, 4365. (b) Liao, L.; Liu, J.; Dreaden, E. C.; Morton, S. W.; Shopsowitz, K. E.; Hammond, P. T.; Johnson, J. A. J. J. Am. Chem. Soc. 2014, 136, 5896.

Collectively, our work suggests that the architectural and structural details of a PEG−DNA conjugate can have substantial impact on its properties and potential applications, and this rule should also be applicable to other PEGylated molecules.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b05064. Materials, experimental procedures, instrumentation, and supplemental figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Ke Zhang: 0000-0002-8142-6702 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from Northeastern University start-up, NEUDFCI seed grant, and NSF CAREER award (1453255) is gratefully acknowledged.

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DOI: 10.1021/jacs.7b05064 J. Am. Chem. Soc. 2017, 139, 10605−10608