Investigation of the physicochemical and functional properties of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated aptamers

Abstract

Polymer conjugation is a common strategy to improve the pharmacokinetics of aptamers, yet its effects on aptamer properties are incompletely understood. Poly(ethylene glycol) (PEG) is the most widely used polymer for this purpose, but concerns about anti-PEG immune responses have prompted interest in alternative polymers. We previously reported that conjugation with the zwitterionic polymer poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) significantly prolongs the circulation time of a DNA aptamer while avoiding anti-PEG antibody recognition. In this study, we evaluated the physicochemical and functional consequences of PMPC conjugation of aptamers. Biophysical analyses suggested that the secondary structure and target-binding affinity of the aptamer were preserved, while functional consequences upon PMPC conjugation varied with the targets. The activity of a membrane receptor-targeting aptamer partially decreased, likely due to spatial constraints around the cell membrane, while RB005, targeting soluble activated coagulation factor IX, retained its full activity. In addition, PMPC conjugation significantly prolonged the in vivo plasma retention of RB005. By elucidating the effects of PMPC on aptamer properties and introducing another example that further supports the general applicability of PMPC conjugation in enhancing aptamer pharmacokinetics, these findings support PMPC as a promising alternative to PEG.

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Introduction

Aptamers are single-stranded oligonucleotides that fold into defined structures and bind to a variety of targets with high specificity and affinity. While aptamers recognize and bind to disease-related targets similarly to antibodies, they offer advantages over them such as low immunogenicity, batch-to-batch consistency, and cost-effective chemical synthesis, which make them attractive candidates for therapeutic applications. However, the clinical application of aptamers is limited by their short half-lives in vivo. Due to their small sizes below the renal filtration threshold, aptamers are readily cleared from the circulation unless chemically modified, and poly(ethylene glycol) (PEG) has been widely used to increase the aptamer size and prolong their circulation time.¹

Although PEG has long been considered biologically inert and widely used since it was devised in the late 1960s, there are growing concerns about the immunological side effects associated with PEG.^2,3 The prevalence of pre-existing anti-PEG antibodies in healthy individuals has recently been reported to have increased to 43.1–65.3%, up from 0.2% reported in 1984, likely due to the ubiquity of PEG in consumer products and improved detection techniques.^4–6 These pre-existing anti-PEG antibodies have been implicated in allergic reactions during the clinical trials of PEGylated aptamer and peptide drug candidates.^7,8 As a result, alternative strategies to PEG for aptamer modification are being actively explored.

We focus on zwitterionic polymers, which contain both positively and negatively charged groups within each repeating unit and have been studied not only for use in medical devices and implants but also as modifiers of small molecule drugs and proteins.^9–12 They exhibit stronger hydration than PEG through electrostatic interactions^13,14 and have chemical structures that resemble naturally occurring biomolecules, both contributing to high biocompatibility and low immunogenicity.¹¹ These properties make zwitterionic polymers promising candidates for aptamer modification.

We recently reported a zwitterionic polymer poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as a potential PEG alternative for aptamer modification. Conjugation of PMPC improved the pharmacokinetics of an anti-inflammatory aptamer to a degree comparable to PEG and enhanced its therapeutic efficacy, while exhibiting significantly lower reactivity to anti-PEG antibody.¹⁵ Despite these promising results, the impact of zwitterionic polymer modification on the structural and functional properties of aptamers, as well as its applicability to other aptamers, has not been addressed and thus remains unclear.

A major concern in modifying middle-sized therapeutics such as aptamers is that conjugation with bulky polymers may hinder target binding. PEGylation often slightly reduces the binding affinity of therapeutic molecules, possibly due to steric hindrance or conformational changes, and this effect has also been reported for aptamers. For example, PEGylation of Pegaptanib increased its dissociation constant (K_d) to VEGF from 49 to 200 pM, compensating for the prolonged half-life and improved in vivo efficacy.¹⁶ Such trade-offs are commonly observed in polymer-modified therapeutics, and zwitterionic polymer modification has also been reported to affect the conformation and function of protein or peptide therapeutics.^17,18 However, its effect on aptamer behavior has not been investigated and further studies on zwitterionic polymers as aptamer modifiers are needed to expand their applicability in aptamer therapeutics. Therefore, in this study, we examine the effects of zwitterionic polymer modification on the physicochemical and functional properties of aptamers.

Results and discussion

Synthesis of zwitterionic polymers

Zwitterionic polymers to be studied here are PMPC and poly(sulfobetaine methacrylate) (PSBMA). To investigate whether the previously reported effectiveness of PMPC as an aptamer modifier is a general feature of zwitterionic polymers or results from the specific properties of PMPC, among a variety of zwitterionic polymers with chemical complexity, we utilized PSBMA. PSBMA shares the same backbone structure as PMPC and differs only in the side-chain structure, allowing us to discuss the effect of the chemical group. PEG_{40 kDa}, a widely used standard for aptamer modification, was also included as a reference.

In our previous report, PMPC, with a degree of polymerization (DP) of around 200 and a hydrodynamic size comparable to that of PEG_{40 kDa}, was shown to be similarly effective in prolonging aptamer circulation in vivo.¹⁵ Based on this, PMPC and PSBMA with a DP of 200 (PMPC₂₀₀ and PSBMA₂₀₀) were synthesized by atom-transfer radical polymerization (Fig. 1). After polymerization, the terminal tert-butoxycarbonyl protecting group was deprotected by trifluoroacetic acid treatment to expose the terminal amino group. The synthesized polymers were then characterized by dynamic light scattering (DLS) in phosphate buffer containing NaCl, revealing that the hydrodynamic diameter (D_h) of PSBMA₂₀₀ was 7.6 nm, which was smaller than those of both PMPC₂₀₀ and PEG_{40 kDa} (9.9 and 9.4 nm, respectively), despite identical backbone lengths and DP values (Table 1), which may be attributed to a difference in side-chain behavior.^19,20 Also, the zeta potentials of PMPC₂₀₀ and PSBMA₂₀₀ were found to be close to neutral despite the presence of charged distal groups on each side chain (Table 1), suggesting that electrostatic interactions between the polymers and aptamers may not be significant.

Fig. 1 Synthetic scheme of polymers with an amino terminal group. MPC: 2-methacryloyloxyethyl phosphorylcholine; SBMA: sulfobetaine methacrylate; Bipy: 2,2′-bipyridyl; and TFA: trifluoroacetic acid.

Table 1 Surface charge and mass-averaged hydrodynamic diameter of the synthesized polymers (2 mg mL⁻¹ in 20 mM phosphate buffer (pH 7.4) containing 100 mM NaCl), determined by zeta potential and dynamic light scattering measurements at 25 °C

	Zeta potential (mV)	Hydrodynamic diameter (nm)
Data obtained under varying conditions are presented in Fig. S1.
PEG40kDa	−0.9 ± 3.7	9.4 ± 0.1
PMPC200	2.3 ± 1.7	9.9 ± 0.6
PSBMA200	0.6 ± 0.5	7.6 ± 0.1

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Synthesis of zwitterionic polymer–aptamer conjugates

A 50-nucleotide DNA aptamer named SL1, which specifically binds to c-Met, was selected as a model aptamer (Fig. 2A). A receptor tyrosine kinase c-Met is involved in cellular activities, such as proliferation and migration, and is therefore a biologically significant target.²¹ Also, SL1 has been extensively characterized in previous studies, with a well-defined sequence, structure, and binding profile,^22–27 making it a suitable model for evaluating the consequences of aptamer modification.

Fig. 2 Synthesis and characterization of SL1–polymer conjugates. (A) Sequence of SL1. (B) Synthetic scheme of polymer–aptamer conjugates. DBCO-NHS: dibenzocyclooctyne-N-hydroxysuccinimidyl ester; DIPEA: N,N-diisopropylethylamine; DMSO: dimethyl sulfoxide; IEX: ion exchange chromatography; and SEC: size exclusion chromatography. (C) SEC chromatograms during the purification of polymer–aptamer conjugates after the conjugation reactions. Full chromatograms are provided in Fig. S2A.

Polymer–aptamer conjugates were synthesized via copper-free strain-promoted alkyne–azide cycloaddition (SPAAC) (Fig. 2B). A dibenzocyclooctyne (DBCO) group was introduced into amino-terminated polymers, and aptamers were functionalized with an azide group. The SPAAC reaction between DBCO and azide groups was then carried out with gradual freeze–thaw treatment to enhance reaction efficiency.²⁸ Unreacted polymers were removed by anion exchange chromatography, and the conjugates were further purified from unreacted aptamers using size exclusion chromatography (SEC) (Fig. 2C and S2A).

In SEC chromatograms (Fig. 2C), all SL1–polymer conjugates eluted earlier than unmodified SL1, indicating the increase in size. Of note, SL1-PSBMA₂₀₀ eluted later than SL1-PMPC₂₀₀ and SL1-PEG_{40 kDa}, suggesting a smaller hydrodynamic volume. This result is consistent with the DLS results of polymers (Table 1), where PSBMA₂₀₀ showed a smaller D_h than PEG_{40 kDa} and PMPC₂₀₀, indicating that the size-enhancing effect of PMPC is not a general property of zwitterionic polymers but stems from its strong hydration. In contrast, PSBMA exhibits weaker hydration and a more compact conformation.^19,20 Accordingly, PMPC is a more suitable choice for aptamer modification when an effective increase in molecular size is required, and subsequent evaluation focused on PMPC₂₀₀ and PEG_{40 kDa} to examine their effects on the structural and functional properties of aptamers.

Effects of PMPC modification on the structure of the SL1 aptamer

SL1 forms a parallel G-quadruplex (G4) and a stem-loop structure (Fig. 2A) and disruption of either structure, by substituting a specific G in the loop with A or deleting bases from the stem sequence, reduces binding affinity for c-Met.^22,23,29 To assess the impact of polymer conjugation on the secondary structure of SL1 in PBS, circular dichroism (CD) spectroscopy and UV melting curve analysis were performed.

Circular dichroism analysis. A DNA duplex typically shows a positive peak at around 275–280 nm and a negative peak at around 245 nm, while a parallel G-quadruplex exhibits a positive peak at around 260 nm and a negative peak near 240 nm.³⁰ CD spectra of SL1, SL1-PEG_{40 kDa}, and SL1-PMPC₂₀₀ feature a positive peak at around 270 nm and a negative peak at around 240 nm (Fig. 3A), consistent with a stem-loop-containing structure and indicative of a parallel G4 conformation. The similarity in CD spectra suggests that conjugation with PMPC₂₀₀ or PEG_{40 kDa} does not substantially disrupt the structure of SL1.

Fig. 3 Effect of polymer modification on the secondary structure of SL1. (A) CD spectra and (B and C) UV-melting profiles with derived thermodynamic parameters for 5 µM SL1, SL1-PEG_40kDa and SL1-PMPC₂₀₀ in PBS, monitored at (B) 260 nm and (C) 295 nm. (B) and (C) display both the original melting curves (lighter lines) and smoothed curves (bold lines) processed using the Savitzky–Golay method.

UV melting analysis. To evaluate the effect of polymer conjugation on the structural stability of the SL1 aptamer, UV melting analyses were performed. Thermal denaturation profiles revealed transitions at 260 and 295 nm, corresponding to the melting of the duplex and G4 domains, respectively (Fig. 3B and C).³¹ At 260 nm (Fig. 3B), the value increased slightly from −2.4 kcal mol⁻¹ in SL1 to −1.9 kcal mol⁻¹ in both SL1-PEG_40kDa and SL1-PMPC₂₀₀. The observed values showed little variation and were within experimental variability, suggesting that the destabilizing effect is limited. However, shifts of ΔH° toward less negative values were observed, indicating that the duplex region in the polymer-conjugated forms may experience weaker base pairing, consistent with previously reported effects of molecular crowding with MPC or PEG on a DNA duplex.^32,33 Polymer conjugation also increased ΔS° values, suggesting reduced conformational and solvation freedom in the unfolded state.³⁴

At 295 nm (Fig. 3C), the values remained constant across all samples, indicating that the thermodynamic stability of the G4 motif is preserved upon polymer conjugation. However, conjugation with PEG_{40 kDa} or PMPC₂₀₀ both resulted in decreases in ΔH° and ΔS°. These changes may reflect structural constraints or local environmental effects by the conjugated polymers,³⁵ and this reduction in enthalpy and entropy changes also aligns with the changes observed with MPC or PEG as molecular crowders.^33,36

Collectively, structural analyses indicate that polymer conjugation preserves the structural integrity of the aptamer, while altering the folding thermodynamics of the aptamer with a similar trend to the polymers as molecular crowders.³⁶

Function of the PMPC-modified SL1 aptamer in cultured cells

To assess whether polymer conjugation affects the biological function of SL1, cellular assays were performed using A549 cells, which express c-Met. c-Met signaling is initiated by hepatocyte growth factor (HGF)-induced c-Met dimerization and autophosphorylation at tyrosine residues Y1234 and Y1235. SL1 inhibits this process by competitively blocking HGF binding to c-Met, thereby preventing receptor activation (Fig. 4A).²²

Fig. 4 Function of polymer–aptamer conjugates in c-Met-expressing cells. (A) Schematic representation of HGF-induced c-Met activation and its inhibition by SL1. (B) Binding of Alexa Fluor 647-labeled aptamers (5′ terminus) with or without polymer modification at the 3′ terminus to A549 cells. Cells were incubated with aptamers at 37 °C for 20 min, washed, and fixed before analysis using flow cytometry. Median fluorescence intensity (MFI, arbitrary unit) was plotted against aptamer concentration. The binding curve shown represents one of the two independent experiments, each performed in triplicate, and K_d values reflect the average of both experiments. The full plot is provided in Fig. S3. (C) Inhibition of c-Met activation in A549 cells. Cells were incubated with the aptamer and its conjugates for 20 min, stimulated by HGF (0.3 nM) for 15 min, washed, fixed, permeabilized, and immunostained to measure the c-Met(Tyr1234/1235) phosphorylation level by flow cytometry. Each data point represents the average of mean values from three independent experiments, each performed in duplicate or triplicate and normalized to PBS-treated controls. Error bars indicate the standard deviation. (D) Inhibition of proliferation of U87MG cells with HGF autocrine signaling. SL1 and its polymer conjugates (300 nM in medium containing 1% FBS) were incubated for 5 days at 37 °C. Error bars represent standard deviations (N = 7). Relative proliferation is determined by dividing the absorbance of each well (measured by the CCK-8 assay) by that of PBS-treated control wells. Statistical significance was examined by one-way ANOVA followed by post hoc Tukey's multiple comparisons test. Not significant (ns) p > 0.05, ***p ≤ 0.001, and ****p ≤ 0.0001.

Binding to the c-Met-expressing cell surface. Binding analyses by flow cytometry revealed that both SL1-PEG_{40 kDa} and SL1-PMPC₂₀₀ retained nanomolar-range K_d values comparable to that of unmodified SL1 (Fig. 4B), suggesting that the folded conformation responsible for target binding was not significantly altered by polymer conjugation. While minor differences were observed in thermodynamic profiles, the consistency in CD spectra and retained binding affinities indicate that the folded conformation required for binding remained intact. However, the total fluorescence intensity notably decreased in the polymer-modified aptamers, indicating a smaller number of aptamers bound to the cell surface. This reduction is likely attributable to steric hindrance from the polymer chains, which may interfere with access to membrane-localized c-Met.

Effect of polymer modification on c-Met inhibitory activity. To evaluate the impact of polymer conjugation on the inhibitory activity of SL1, a c-Met phosphorylation assay was performed (Fig. 4C). When cells were stimulated with 0.3 nM HGF in the presence of a 1–900 nM aptamer, SL1 showed inhibitory activity with an IC₅₀ of 16.6 nM. A decrease in inhibitory potency was observed for SL1-PEG_{40 kDa} and SL1-PMPC₂₀₀, as reflected by their increased IC₅₀ values of 107.1 and 109.9 nM, respectively. Given that their binding affinities remained comparable to that of unmodified SL1, the reduced inhibitory effect is likely due to polymer-induced steric hindrance, which may limit the accessibility of the aptamer to membrane-bound c-Met. Despite this reduction, both SL1-PEG_{40 kDa} and SL1-PMPC₂₀₀ retained the ability to inhibit c-Met phosphorylation in a dose-dependent manner, indicating that the aptamer's function remains largely intact after polymer modification. It was also confirmed that this inhibitory activity stems from the aptamer sequence and not from nonspecific effects of the oligonucleotide or polymer moiety, as inhibition was abolished in samples where the aptamers were hybridized with complementary strands (Fig. S4).

To further evaluate the biological activity, a cell proliferation assay was conducted using U87MG cells, which rely on autocrine HGF signaling and c-Met-dependent growth (Fig. 4D).³⁷ At 300 nM, both unmodified and polymer-conjugated SL1 exhibited comparable inhibitory effects, supporting that their functional activity is preserved.

In vitro function and in vivo plasma retention of the PMPC-modified RB005 aptamer

The broader applicability of PMPC₂₀₀ conjugation as an aptamer modifier was investigated using a clinically validated aptamer RB005. RB005 is a 31-nucleotide RNA aptamer (Fig. 5A)³⁸ that binds to activated coagulation factor IX (FIXa) and inhibits its enzymatic activity, thereby reducing thrombin generation and fibrin clot formation through suppression of factor X activation (Fig. 5B).^39–41 RB005 showed promising therapeutic potential and its PEGylated form, pegnivacogin, advanced to phase 2 clinical trials. However, clinical development was discontinued due to allergic reactions during the trials, likely caused by pre-existing anti-PEG antibodies in the treated patients’ plasma.⁷

Fig. 5 Activated coagulant factor IX (FIXa) aptamer RB005 and effect of PMPC modification on its anti-coagulant activity. (A) Sequence of RB005. idT: inverted dT. (B) Schematic representation of the coagulation cascade and its inhibition by RB005. (C) Activated partial thromboplastin time (aPTT) of normal human serum in the presence of RB005 and its polymer conjugates. APTT assay was performed in triplicate, and data are presented as mean ± standard deviation. (D) Time course of clotting time of plasma collected at several time points after intravenous injection of RB005 or RB005-PMPC₂₀₀. Each symbol represents the measurement from an individual mouse. Data for RB005-PEG_40kDa and RB005-PSBMA₂₀₀ are provided in Fig. S6.

Given that FIXa is a validated target for anticoagulation therapy, RB005 has potential applications under conditions such as thromboembolism associated with atrial fibrillation and deep vein thrombosis. Therefore, demonstrating that PMPC conjugation improves the pharmacokinetics of RB005 not only supports the broader utility of PMPC as an aptamer modifier but also potentially restores the clinical relevance of a discontinued therapeutic candidate.

RB005–polymer conjugates were synthesized and purified (Fig. S2B and S5), and their anticoagulant activity was evaluated using the activated partial thromboplastin time (aPTT) assay (Fig. 5C). The assay evaluates anticoagulant activity based on clotting time. RB005, RB005-PEG_{40 kDa}, and RB005-PMPC₂₀₀ all prolonged the clotting time in a dose-dependent manner, with IC₅₀ values of 30.8, 33.4, and 29.6 nM, respectively. No significant differences were observed among the RB005 and polymer conjugates, indicating that polymer modifications did not compromise the anticoagulant function of RB005.

To examine the in vivo pharmacodynamic behavior, the aPTT assay was conducted using plasma collected from mice intravenously injected (2 nmol per 20 g body weight) with RB005 or RB005-PMPC₂₀₀ (Fig. 5D).⁴² Although RB005 was originally developed to target human FIXa, previous studies have shown that it also inhibits murine FIXa activity in vivo.⁴¹ While typical pharmacokinetic studies, such as quantitative PCR, directly measure the plasma concentration, the aPTT assay evaluates the plasma retention of the aptamer in terms of its functional activity, serving as an indirect measure of its pharmacokinetic profile and reflecting the persistence of functional activity. When plasma was collected 10 min after injection, both RB005 and RB005-PMPC₂₀₀-treated groups showed prolonged clotting times, with aPTT values of 29 and 38 s, respectively, compared to plasma from PBS-injected controls (21 s). These results confirm that both aptamers remained in circulation to exhibit anticoagulant activity shortly after the injection.

However, an hour after injection, plasma from RB005-injected mice showed clotting times of 22 s, similar to the PBS control, indicating rapid clearance of RB005 from the circulation. In contrast, plasma from the RB005-PMPC₂₀₀ group maintained a prolonged aPTT of 36–39 s even 1–4 h after injection, which is nearly identical to the value observed at 10 min post-injection. These results show that PMPC conjugation prolongs the circulation time of RB005, maintaining its presence in circulation for at least 4 hours post-injection. Furthermore, the results for the RB005-PEG_{40 kDa} group were nearly identical to those of the RB005-PMPC₂₀₀ group (Fig. S6). Interestingly, however, RB005-PSBMA₂₀₀ exhibited a significant reduction in the duration of activity.

Together, these results demonstrate an additional example that PMPC conjugation enhances the pharmacokinetics of aptamers, supporting its potential as a PEG alternative for therapeutic aptamers.

Conclusions

Building on previous findings that PMPC conjugation improves the pharmacokinetics of aptamers and exhibits reduced reactivity to anti-PEG antibodies,¹⁵ this study evaluated its suitability as a polymeric modifier by investigating its effects on the aptamer structure, biological function, and circulation behavior. Compared to PSBMA, a zwitterionic polymer that shares the same backbone structure, PMPC increased the molecular size of the aptamer more effectively, indicating that the superior size-increasing effect of PMPC is attributable to its unique hydration and side-chain structure,¹⁹ rather than to zwitterionic character alone.

Biophysical and binding analysis showed that PMPC conjugation preserved the secondary structure and target binding affinity of the DNA aptamer SL1, with minor differences detected in thermodynamic parameters involved in aptamer folding, consistent with trends observed for polymers under molecular crowding conditions. In a cell-based assay, polymer-conjugated SL1 showed slightly reduced binding levels and decreased inhibitory activity, likely due to steric hindrance that occurs when targeting membrane-bound c-Met in the crowded environment of the cell surface. For SL1, the observed increase in IC₅₀ reflects a trade-off, where some in vitro potency is sacrificed to achieve superior in vivo pharmacokinetics provided by PMPC conjugation. By contrast, the anticoagulant RNA aptamer RB005, which targets FIXa, a soluble protein, retained its activity even after PMPC conjugation, with no increase in the IC₅₀ value observed. This result is consistent with our previous finding for an IFN-gamma aptamer, which also targets a soluble protein and fully maintains its inhibitory activity against IFN-gamma-induced signaling even after PMPC conjugation.¹⁵ This finding highlights the target-dependent nature of polymer conjugation effects and suggests that polymer modification may be particularly effective for aptamers directed against soluble targets, where steric hindrance is less pronounced compared to crowded membrane-associated environments.⁴³

Importantly, from a therapeutic perspective, PMPC conjugation enabled RB005 to maintain functionally active plasma levels for at least 4 hours in vivo, and notably, its anticoagulant activity at this time point exceeded that of unmodified RB005 at 10 min post-injection, demonstrating a significant pharmacokinetic enhancement. By evading recognition by pre-existing anti-PEG antibodies (Fig. S7) and enabling prolonged functional activity of conjugated aptamers in circulation, PMPC satisfies key criteria for aptamer modification and offers a viable alternative to PEG. Taken together, these findings establish PMPC as a promising aptamer modifier to enhance pharmacokinetics while preserving key structural features and function.

Author contributions

S. S. conceived the project; S. C. and S. S. designed the experiments; S. C. and M. T. carried out experiments and analyzed data, with support from J. M., Y. S., Y. N., A. S., K. Y., D. M., and S. S.; S. S. provided resources and supervision; S. C. and S. S. wrote the manuscript, and all authors commented on and approved the manuscript.

Conflicts of interest

The authors declare the following competing financial interests: the authors (S. C., Y. S. and S. S.) have filed a patent application (PCT/JP2022/022601). All other authors declare that they have no competing interests.

Ethical approval

All animal experiments were performed with the approval of the Animal Care and Use Committee of the University of Tokyo, in accordance with the guidelines for the care and use of laboratory animals as stated by this institution.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5bm01078k.

Acknowledgements

This research was partially supported by JSPS KAKENHI [Grant Number JP24KJ0742 (to S. C.)] and [Grant Number 22H05049 and 25H01283 (to K. Y.)]. We thank Prof. Takai and Dr Masuda for their support in measuring DLS.

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