DNA nanostructures prevent the formation of and convert toxic amyloid proteospecies into cytocompatible and biodegradable spherical complexes

2.1 Materials

Fmoc-protected amino acids and O-(1H-6-chlorobenzotriazole-1-yl)−1,1,3,3-tetramethyluronium hexafluorophosphate were purchased from Matrix Innovation. Piperidine was purchased from A&C American Chemicals Ltd. Tris(hydroxymethyl) aminomethane, magnesium chloride, ammonium persulfate, glacial acetic acid, and tetramethylethylenediamine were purchased from Bioshop Canada. Unlabeled DNA strands were purchased from idtDNA. Maleimide-labeled DNA strands were purchased from GeneLink. TAMg buffer was made in-house (40 mM Tris, 12.5 mM MgCl₂·6H₂O, 20 mM glacial acetic acid, pH 7.8). GelRed stain was purchased from VWR Canada. Precast 4%–15% polyacrylamide gel electrophoresis (PAGE) gels were obtained from BioRad. All solvents were of analytical grade and were purchased from Sigma-Aldrich. Deionized (DI) water was used to prepare buffer solution (pH 7.4). All solvents, reagents, and other chemicals were used as received without further purification.

2.2 Peptide synthesis, purification, and characterization

IAPP was synthesized by means of standard Fmoc solid-phase peptide synthesis and then purified using reverse-phase high-performance liquid chromatography (RP-HPLC), as previously described.^[35] Formation of the C²–C⁷ disulfide bridge was achieved by oxidation in dimethyl sulfoxide at room temperature (RT). The assembled IAPP was purified by RP-HPLC and analyzed by high-resolution mass spectrometry (HRMS) using an Agilent 1200 series HPLC coupled to an electrospray time-of-flight mass spectrometer (ESI-TOF MS) (Figure S1). Peptide fractions with a purity higher than 95% were combined and lyophilized. Subsequently, aliquots of monomerized peptide were prepared by treating the lyophilized powder in 100% hexafluoro-2-propanol (HFIP) at a concentration of 1 mM. The solution was sonicated for 30 min, filtered through a 0.22 µm hydrophilic polypropylene filter, and lyophilized. The lyophilized IAPP powder was solubilized in HFIP, sonicated for 30 min, and lyophilized a second time. Monomerized samples were kept in the frozen dried state at −80°C for a maximum period of 4 weeks.

2.3 Design and assembly of DNA nanostructures

Linear DNA nanostructures (DNA-1D) consisted of a 39-base pair (bp) complementary region (Table S1), followed by a four-thymine spacer, and a 15-base addressable overhang. The overhang permits the incorporation of overhang complements (OC, Table S1). The sequences of the pairing region and the overhang were generated using CANADA 2.0,^[36] a random DNA sequence generator. Sequences were then cross-analyzed for unwanted binding using idtDNA's Oligoanalyzer tool, based on the work of Allawi.^[37] Final sequences were ordered from idtDNA. Unmodified OC strands are used to complete the double-stranded structures bearing no ligands. For the peptide-guided complexing studies, the OC strand was conjugated to a short fragment of IAPP (20–29). Assembly of the desired structure was verified using PAGE (Figure S2, 0.002 nmol of structure per well, pre-annealed from 90 to 23.3°C over the course of 16 h in TAMg buffer; 80 V, 90 min, 4%–15% polyacrylamide). The DNA triangle (DNA-2D) is a novel design created to provide a 2D structure with three overhangs (Figure 1 and Table S1). Built around a central (Tri1) strand, three strands with regions complementary to both the Tri1 and each other were designed using CANADA2.0 to generate unique sequences. Final sequences were chosen based on cross-analyses for undesired binding. The final structure resembles a triangle with three arms, each possessing a structural binding region and an overhang region. Once the complementary sequences were selected, the structure was modeled using GIDEON,^[37] a program for DNA nanostructure design. GIDEON was used to determine if extra non-binding bases (thymines) were required at each vertex. Extra bases allow for the continuation of the DNA helix, releasing the strain between the main triangle and each arm. These bases are indicated in black in Table S1. Assembly of the desired structure was based on the predicted design and migration via PAGE. Each strand was added in a stepwise manner, to ensure that changes in mobility were as expected (Figure S2, 0.00435 nmol of structure per well, pre-annealed from 90 to 23.3°C over the course of 16 h in TAMg buffer; 80 V, 90 min, 4%–15% polyacrylamide). The DNA tetrahedron (DNA-3D) was adapted from the design of Dong et al.,^[38] with the addition of a four-thymine linker and the addressable overhang at each vertex. To verify assembly, each strand was added in a stepwise manner to ensure that changes in mobility were as expected (Figure S2, 0.00435 nmol of structure per well, pre-annealed from 90 to 23.3°C over the course of 16 h in TAMg buffer; 80 V, 90 min, 4%–15% polyacrylamide.). Each DNA strand was quantified by absorbance at 260 nm prior to use. For each structure, sufficient DNA was added to reach a final concentration of 14 µM in TAMg buffer. The overhang regions were blocked with appropriate amounts of OC, creating fully duplexed structures. The molar ratio of OC per assembly was dependent on the structure. For the linear structure, the ratio was 2:1 (OC: structure). For the triangle, the ratio was 3:1. For the tetrahedron the ratio was 4:1. Due to the prevalence of oligomer formation at high 2D and 3D DNA concentrations, the stock solutions of each structure were diluted 10× with DI water prior to assembly. The stock was mixed and then separated into polymerase chain reaction (PCR) tubes. Using a PCR machine, the tubes were heated to 90°C and then cooled to 23.3°C over the course of 16 h. Samples were recombined and lyophilized to dryness, then resuspended to the initial volume using sterile water. Assembly was verified using PAGE (80 V, 75 min).

2.4 Preparation of peptide-labeled DNA (OC-Pep) structures

Maleimide-labeled DNA strands (OC-mal) arrive as protected maleimido-2,5-dimethylfural cycloadducts. Maleimide deprotection was performed using a microwave deprotection strategy.^[39] Briefly, the cycloadduct was deprotected in 1:1 MeOH:H₂O solvent mixture with a final DNA concentration of 25 µM in 200 µL. This mix was placed in a CEM microwave reactor. A ‘dynamic’ method was prepared (T = 90°C, Power = 300 W). With this method, the temperature was maintained at T = 90°C by automatic variation of the power. The buildup of pressure kept the sample from boiling, and PowerMax was enabled to provide cooling via compressed air. The reaction was run for 90 min. Once the mixture had cooled, it was transferred to an Eppendorf tube and dried using a SpeedVac. For conjugation to the maleimide, an IAPP peptide fragment containing an additional Cys with free thiol was synthesized (CKGSGSSNNFGAILSS), purified using RP-HPLC, and analyzed by LC-HRMS (Figure S3). The conjugation reaction was then performed in TAMg buffer, with 20 µM of OC-mal and 40 µM of peptide (1:2 ratio). The reaction mixture was placed on a shaker overnight at RT. To ensure conjugation of the peptide to OC-mal, crude reaction product was evaluated by LC-MS, with a gradient of 20%–60% acetonitrile + 0.1% trifluoroacetic acid over 25 min (flow rate 1 mL/min, Agilent Zorbax 300SB-C18, 5 µm, 4.6 × 150 mm column, electrospray ionization, drying gas 12 mL/min, nebulizer pressure of 35 psig, drying gas temperature of 350°C, capillary voltage of 3000 V and fragmentor set to 70). Three peaks were identified through a combination of liquid chromatography controls and mass calculations: OC-mal eluted at 14.5 min, peptide eluted at 4.2 min, and OC-Pep eluted at 15.5 min. Analysis of peak areas revealed no leftover OC-mal, so no further purification was performed. Free peptides may have been present but are minimal in comparison to the large amounts of peptide added for subsequent experiments. Polyacrylamide gel electrophoresis (0.024 nmol DNA-Pep per lane, 90 min at 80 V) confirmed the hybridization of OC-peptide to the OC portions of the linear structure (Figure S3).

2.5 Preparation of IAPP quaternary structures

IAPP quaternary structures were prepared by dissolving the lyophilized and monomerized peptide into 20 mM Tris-HCl buffer (pH 7.4) at a concentration of 75 µM. The freshly prepared solution was used immediately as mainly monomeric species (IAPP_M), incubated under fully quiescent conditions at RT for 3 h to form oligomeric and pre-fibrillar species (IAPP_O), or incubated for 48 h to generate amyloid fibrils (IAPP_F). Solutions were then diluted to reach a final concentration of 25 µM of IAPP to which were added (or not) the DNA nanostructures.

2.6 Ultraviolet-visible spectroscopy

A Mandel UV-1280 spectrophotometer was used to record ultraviolet-visible (UV-vis) absorption spectra from 190 to 390 nm. The light path length was 10 mm, and the internal step was set to 1 nm.

2.7 Fluorescence spectroscopy

Fluorescence measurements were carried out at RT on a PTI Photon Technology International steady-state spectrofluorometer (QuantaMaster 40) and operated with PTI's FelixGX software with a light path length of 3 mm. For thioflavin T (ThT), used at a concentration of 40 µM, the excitation wavelength was set to 440 nm and the emission was recorded from 450 to 550 nm. For 8-anilinonaphthalene-1-sulfonic (ANS), used at a concentration of 100 µM, the excitation wavelength was set to 355 nm and the emission spectra were recorded from 385 to 585 nm. For fluorescein arsenical hairpin (FlAsH), used at a concentration of 0.5 µM and supplemented with 125 µM dithiol British anti-lewisite (BAL) and 500 µM tris(2-carboxyethyl)phosphine (TCEP), the excitation wavelength was set to 508 nm and the emission spectra were recorded from 460–540 nm. For intrinsic fluorescence spectroscopy, the tyrosine fluorescence spectra were recorded from 290–360 nm with an excitation wavelength of 274 nm. The intrinsic fluorescence of IAPP Y³⁷ in the absence of DNA was assigned as F₀, and the intrinsic fluorescence of tyrosine in the presence of DNA was assigned as F. The extent of the change in the fluorescence intensity was ascribed to (F₀ − F)/F₀.

2.8 Kinetics of IAPP fibrillization

2.9 Far-UV circular dichroism spectroscopy

Circular dichroism (CD) spectra were collected on a Jasco J-815 spectrophotometer at RT, in a 2-mm quartz cuvette. Peptide solutions were diluted in 20 mM Tris-HCl, pH 7.4 buffer to reach a concentration of 25 µM, and the CD spectra were collected from 260 to 190 nm. Curves were smoothed using a smoothing moving window of 13 data points through the Savisky-Golay algorithm.

2.10 Dynamic light scattering

Size measurements were performed using the Malvern Zetasizer Nano S90 (Malvern Panalytical) containing a 4 mW helium-neon laser with a wavelength of 633 nm and a scattering angle of 90°. Each measurement corresponded to a triplicate of 10 runs per analysis performed at 25°C. The results were presented as the number distribution (%) of particles versus hydrodynamic radii (nm) calculated from the Malvern software.

2.11 Zeta potential

Measurements were carried out using a zeta potential analyzer (ZetaPlus/Bl-PALS; Brookhaven Instrument Corp.). Each measurement corresponds to a triplicate of 10 runs per analysis.

2.12 Atomic force microscopy

IAPP solutions, with and without DNA nanostructures, were spotted on a freshly peeled mica surface. Excess liquid was removed by wicking, then the surface was rinsed twice with DI water. After being air-dried overnight, atomic force microscopy (AFM) imaging was conducted using a Bruker (MultiMode 8) microscope equipped with a Nanoscope E controller. To characterize the morphology of the resulting complexes, samples were scanned in scanasyst-air mode using a silicon nitride AFM tip (2−12 nm tip radius, k = 0.4 N/m) under ambient conditions. Gwyddion v2.59 was used to analyze images.

2.13 Attenuated total reflection-Fourier transform infrared spectroscopy

IAPP incubated for up to 72 h in 20 mM Tris-HCl, pH 7.4 with, or without 1D-, 2D-, or 3D-DNA were lyophilized and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) spectra were recorded from the solid-state sample using a Nicolet Magna 560 spectrometer equipped with a nitrogen-cooled MCT detector.

2.14 Photochemical-induced cross-linking

An IAPP_O solution (75 µM) was treated with cross-linking solution (70 µM tris-(bipyridyl)Ru(II) and 1.4 mM ammonium persulfate) and then illuminated with a 150 W incandescent bulb for 5 s. Subsequently, the reaction was quenched with 1 M dithiothreitol, and oligomers were separated by sodium dodecyl sulfate (SDS)-PAGE (10% Tris-tricine gel) and revealed using silver staining.

2.15 Dot blot analysis

IAPP_O solution (10 µL at 75 µM) was applied to a nitrocellulose membrane, allowed to air dry, and then blocked in 5% non-fat dried milk in Tris-buffered saline with Tween-20 (TBS-T) for 30 min. The membrane was incubated with an anti-oligomer rabbit polyclonal primary antibody (A11) for 2 h and then washed three times in TBS-T. The membrane was incubated for 1 h with a Horseradish peroxidase-conjugated secondary antibody, diluted at 1:10,000 in 5% non-fat dried milk in TBS-T. Finally, after three washes of the blot in TBS-T, the signal from the secondary antibody was detected by enhanced chemiluminescence.

2.16 Proteolytic digestion by proteinase K

Stability against proteolysis was assessed by subjecting the IAPP fibrils and IAPP_F/DNA-1D complexes to 120 U/mL of proteinase K for 1 h and digestion was evaluated by matrix-assisted laser desorption ionization-TOF (MALDI-TOF) MS.

2.17 Cell viability assays

Rat pancreatic β-cell line (INS-1E) were cultured in black wall clear bottom 96-well plates treated for tissue culture at a density of 25 k cells/well in RPMI-1640 media containing 10% of fetal bovine serum (FBS), 100 UI/mL penicillin, 100 mg/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 50 mM mercaptoethanol, and 2 mM L-glutamine. After 48 h incubation in a humidified atmosphere of 5% CO₂ and 95% air at 37°C, cells were treated by the direct addition of IAPP_O (25 µM), with or without the co-addition of 1.6 µM of 1D, 2D, or 3D DNA nanostructures, and then incubated for 5 h. For the evaluation of the cytotoxicity of the IAPP-DNA complexes, obtained by preincubation of IAPP (75 µM) with DNAs (4.8 µM) in 20 mM Tris-HCl buffer (pH 7.4) for 72 h, cells were treated with 25 µM of IAPP/DNA complexes and then incubated for 24 h. Cellular viability was evaluated using the resazurin metabolic assay and determined in % from the fluorescence ratio of peptide-treated sample to vehicle-treated control (20 mM Tris-Buffer, pH 7.4).^[40] Data of a least three independent assays performed in triplicate were averaged and expressed as the mean ± standard error of mean (S.E.M.). For Live/Dead assays, INS-1E cells were plated in 12-well plates at a density of 150,000 cells/well for 48 h before treatment. Fluorescent microscopy analyses were performed after the incubation with 4 µM of ethidium homodimer-1 and 2 µM of calcein-AM for 45 min incubation.

2.18 Lactate dehydrogenase release assay

3.1 DNA nanostructures modulate IAPP self-assembly

Owing to their programmability and addressability, DNA molecules have emerged as versatile nanoscale materials for numerous biomedical applications including vectors for targeted drug delivery as well as sensors for diagnostics and theranostics.^[41] Short ssDNA has been shown to be a potent modulator of amyloid formation of the Aβ peptide,^[18] however ssDNA is highly flexible and less thermodynamically stable compared to dsDNA, especially when assembled into 2D and 3D architectures.^[42] In this context, the present study explored a new paradigm to modulate amyloid formation and its associated toxicity via a DNA-mediated scavenging mechanism. Through the rational design of DNA sequences, almost any desired shape can be constructed with nanometer-scale precision.^[43] Small and ordered 1D, 2D, and 3D dsDNA nanostructures were assembled from individual DNA strands (Table S1 and Figure 1). The linear dsDNA strands used in this study were longer relative to the ssDNA previously used,^[18] to ensure that all regions remained stably hybridized during the increased temperatures required for in vitro cell studies, especially the overhang regions. The DNA tetrahedron is based on a common motif that has been used in multiple in vitro and in vivo studies,^[44] The 2D structure is an original design, created to have approximately the same number of bps (and anionic phosphate groups) as the tetrahedron. DNA stability over 1-week under experimental conditions was confirmed by UV-vis spectroscopy (Figure S4).

Monomerized IAPP (IAPP_M) was incubated in the presence or absence of DNA nanostructures in Tris-buffer, pH 7.4 at RT under fully quiescent conditions. The kinetics of IAPP fibrillization were evaluated over time by means of fluorescence spectroscopy in a microplate reader. Although ThT fluorescence is usually used to probe amyloid formation with good selectivity,^[45] ThT exhibits significant emission in the presence of DNA nanostructures^[46] that overlaps with amyloid binding (Figure S5), precluding the use of this method. Thus, an alternative strategy based on the fluorogenic FlAsH dye was used to probe the kinetics of IAPP aggregation in the presence of DNA nanostructures. FlAsH is a derivative of fluorescein whose fluorescence quantum yield is dramatically amplified upon its binding to a tetracysteine motif. Accordingly, under reducing conditions that do not affect IAPP fibrillization, a non-contiguous tetracysteine FlAsH-binding site is formed upon the convergence of N-terminal domains that each contain a Cys²-Cys⁷ motif (Figure 1A), leading to a fluorescent signal.^[47] Thus, this method is well-suited to investigate the kinetics of IAPP aggregation by monitoring the formation of quaternary complexes in the presence of dsDNA. As shown in Figure 2A, a prototypical sigmoidal growth curve was obtained for IAPP in the absence of DNA by monitoring FlAsH fluorescence. The extracted lag-time of 3.3 ± 0.1 h and half-time (t_1/2) of 5.0 ± 0.2 h were within the range of the kinetics data obtained using ThT fluorescence under these conditions (Figure 2A). In sharp contrast, incubation of 25 µM IAPP in the presence of 1.6 µM of linear (1D), triangular (2D), or tetrahedral (3D) dsDNA nanostructures, induced a sharp increase of FlAsH fluorescence with the absence of any observable lag phase (Figure 2B,C). This observation indicates that the dsDNA nanostructures accelerate the formation of the split-tetracysteine motif through the convergence of the N-terminal domain of IAPP molecules. Different concentrations of dsDNA were evaluated to define the minimal concentration of dsDNA that modulates the kinetics of aggregation. At concentrations of 0.4 µM and above, the accelerating effect of dsDNA is observed with the quasi-absence of a lag phase. In contrast, at a concentration of 0.2 µM dsDNA, a prototypical sigmoidal growth curve of IAPP alone was observed, suggestive of the minimal effect of dsDNA on the kinetics of IAPP aggregation (Figure S6). This observation might be partially explained by the charge ratio between IAPP and DNA. IAPP is a positively charged peptide displaying a net charge of +3 at physiological pH (N-terminus, Lys¹, Arg¹¹; Figure 1A), whereas dsDNA is polyanionic with a charge of −2 per bp. The linear structure contains 58 bps and therefore an overall charge of −116. Thus, the opposing charges likely promote their mutual binding through electrostatic interactions and DNA nanostructures likely potentiate IAPP self-assembly by an electrostatic-type mechanism.^{[48, 49]} More specifically, it is likely that the negatively charged DNA interacts with the positively charged N-terminal domain of IAPP, increasing the latter's local concentration, and promoting intermolecular FlAsH binding. Varying the DNA concentration changes the overall negative/positive (N/P) charge ratio. At low DNA concentrations (0.2 µM dsDNA), the N/P ratio was only 0.3, resulting in a loss of templating effects that are expected to facilitate IAPP assembly. In contrast, higher concentrations of DNA increased the ratio of N/P to 0.62, 1.24, and 2.48 for DNA concentrations of 0.4, 0.8, and 1.6 µM, respectively, suggesting a threshold charge ratio for DNA-promoted IAPP assembly exists at a N/P ratio between ∼0.3 and 0.6. No significant differences were observed between the kinetics of IAPP assembly in the presence of the DNA 2D and 3D nanostructures, both of which had relatively high N/P ratios of ∼7.6. Nevertheless, a trend in the maximum intensity of FlAsH was observed as 3D < 2D < 1D, suggesting the influence of the DNA structure on the morphology of the assemblies formed in the presence of IAPP, with greater convergence of the N-termini of IAPP in the presence of 1D and 2D DNA nanostructures.

The ability of DNA nanostructures to modulate IAPP self-assembly was further evaluated by performing biophysical analyses and AFM imaging after prolonged incubation of IAPP with DNA. CD spectroscopy revealed that IAPP alone, that is, in the absence of DNA, exhibited a random coil secondary structure with a negative band at 202 nm immediately after its solubilization (0 h), which shifted towards a β-sheet-rich conformation after 72 h of incubation under fully quiescent conditions (Figure S7A). This secondary structure conversion is associated with a sharp increase in ThT fluorescence (Figure S7B), indicative of the formation of the cross-β-sheet quaternary organization.^[50] Similarly, FlAsH and ANS fluorescence, respectively, revealed the gathering of adjacent N-terminal regions and the formation of hydrophobic clusters upon 72 h incubation (Figure 3A,B and Figure S7C,D). A modest change to FlAsH fluorescence intensity over incubation with DNA nanostructures was observed, despite changes observed by other techniques (Figure 3A). For IAPP, the formation of amyloid fibrils was confirmed by AFM, with images showing a network of typical fibrils with two major distinctive morphologies: flat ribbons and twisted filaments (Figure 3C and Figure S7E). This morphological heterogeneity is typical for IAPP fibrils assembled in vitro.^[51] Because of the strong background signal, CD spectroscopy and ThT fluorescence could not be performed in the presence of DNA nanostructures. Remarkably, incubation of 1D, 2D, or 3D dsDNA nanostructures with monomerized IAPP for 72 h led to the formation of spherical assemblies with the complete absence of fibrillar aggregates (Figure 3C), indicating that these entities inhibit IAPP fibril formation by modulating the mechanism of self-assembly. These spherical aggregates were characterized by the presence of solvent-accessible hydrophobic patches, as observed with the sharp increase in the intensity of ANS fluorescence (Figure 3B). Dynamic light scattering (DLS) analyses revealed that the DNA/peptide complexes had hydrodynamic radii in the 300–400 nm range, independent of the identity of the DNA nanostructure (Figure 3D). These spherical complexes were not observable for the dsDNA nanostructures incubated in the absence of peptides (Figure S8). The formation of spherical assemblies was dependent on DNA concentration, with (predominantly) typical filaments observed at a DNA concentration of 0.2 µM and spherical aggregates at concentrations above 0.4 µM (Figure S9). This observation correlates with the kinetics of aggregation measured at different IAPP:DNA ratios (Figure S6).

Next, this effect, most likely mediated through electrostatic interactions, was tested in solutions of physiologically relevant ionic strength. Thus, monomeric IAPP was incubated in the presence of DNA nanostructures in 20 mM Tris, pH 7.4, supplemented with 150 mM NaCl. Results revealed the formation of similar spherical complexes with a total absence of amyloid fibrils after 3 days of incubation (Figure S10), indicating that IAPP-DNA interactions can occur in the presence of physiologically relevant salt concentrations. Finally, the effect of functionalizing the DNA nanostructures on this inhibitory effect of amyloid formation was tested by appending ligands that could be used for future in vivo targeting strategies. To do so, the nucleating core region of IAPP (segment 20–29 (I₁₀)) was covalently attached to the flanking region of the 1D dsDNA nanostructure by Michael addition and the resulting peptide-labeled DNA structure was incubated in the presence of IAPP. Interestingly, the same effect on IAPP amyloid formation was observed in the presence of 1D-DNA functionalized with the I₁₀ peptide (Figure S11), indicating that dsDNA can be functionalized while maintaining its inhibitory effect on amyloid self-assembly. Overall, these results revealed that linear, triangular, and tetrahedral dsDNA promoted the self-assembly of IAPP_M into spherical structures, preventing the formation of amyloid fibrils.

3.2 DNA nanostructures convert cytotoxic oligomers into spherical complexes

Going beyond previous works and considering that transient prefibrillar assemblies are the most toxic species of the amyloid pathway, the effects of these DNA nanostructures on pre-assembled oligomers were tested. First, the time-resolved toxicity of IAPP proteospecies was evaluated by monitoring the viability of INS-1E pancreatic cells after incubation with proteospecies pre-assembled for different times.^[52] This allowed the identification of the mixture of IAPP assemblies having the highest cytotoxicity, and that can subsequently be used for evaluating the effect of DNA nanostructures. As shown in Figure 4A, IAPP cytotoxicity correlated with self-assembly time with the pre-fibrillar proteospecies obtained after 3 h incubation at RT being the most toxic (15% viability of control cells). Accordingly, IAPP oligomers (IAPP_O) were prepared by incubating the monomerized peptide for 3 h under fully quiescent conditions (20 mM Tris-HCl, pH 7.4, concentration of 75 µM, RT) before being exposed to DNA nanostructures. The formation of oligomers/pre-fibrillar species under these conditions was confirmed by ThT and ANS fluorescence, CD spectroscopy, AFM, dot blot analysis, and photochemical cross-linking (PICUP). A predominantly random coil conformation was observed in the CD spectra (i.e., sharp band at 202 nm and weak signal at 222 nm) alongside weak ThT and ANS fluorescence (Figure S7A,B). Dot blot analysis conducted with the oligomer-specific A11 antibody and SDS analysis upon PICUP confirmed the formation of oligomeric assemblies (Figure 4B,C).

As expected, when this heterogeneous mixture of prefibrillar aggregates was left to incubate for an additional 72 h in the absence of DNA, a dense network of ANS- and FlAsH-positive fibrils was obtained (Figure 4D,E). Strikingly, incubation of pre-assembled IAPP_O with 1D-, 2D-, or 3D-DNA nanostructures led to the formation of spherical aggregates with a complete absence of fibrils (Figure 4F). These assemblies closely resembled those obtained by the incubation of IAPP_M with the DNA nanostructures (Figure 3D). Moreover, immediately after mixing the DNA nanostructures with IAPP_O solutions, that is, experimental dead time of around 4 min, a strong increase of ANS fluorescence was observed, which likely correlates with the quasi-instantaneous binding of the oligomeric species to DNA thereby generating a high density of hydrophobic patches (Figure 4E). Over 72 h of incubation, these DNA-peptide clusters likely rearrange into core-shell type interpolyelectrolyte complexes in which the hydrophobic IAPP becomes more buried inside the core while the polyanionic DNA sequester to the solvent-exposed shell, as inferred by the decrease of ANS fluorescence. Similarly, FlAsH fluorescence, which reports on the convergence of N-terminal domains of adjacent IAPP molecules, also increased immediately upon the addition of the DNA nanostructures to the IAPPo solutions, before subsequently decreasing over time (Figure 4E). Again, this suggests a rapid clustering of IAPP prefibrillar species onto the DNA nanostructures, leading to the formation of complexes. The height of these clusters observed by AFM imaging and the hydrodynamic radii by DLS were within the range of those observed for the IAPP_M-DNA complexes. Overall, these results indicate that dsDNA nanostructures avidly interact with highly cytotoxic IAPP oligomeric species, shifting the amyloid fibril formation pathway towards the formation of spherical complexes.

3.3 dsDNA nanostructures disassemble stable amyloid fibrils into spherical complexes

Amyloid fibrils are highly stable structures that are resistant to thermal denaturation, chemical treatment with mild denaturants, and proteolytic degradation.^[53] This stability results in the life-long and massive accumulation of amyloid deposits in vivo, which ultimately cause tissue dysfunction despite the low cytotoxicity of individually organized fibrils.^[54] Considering that it has been astonishingly reported that ssDNA could disassemble Aβ fibrils,^[18] and in light of the results above, a comparable test of the effect of dsDNA nanostructures on mature IAPP fibrils (IAPP_F) was warranted. Fibrils were prepared by incubating the monomerized peptide for 48 h in 20 mM Tris-HCl pH 7.4 under quiescent conditions at a concentration of 75 µM, a procedure known to reach the saturation phase.^[55] The formation of amyloid fibrils was confirmed by ThT fluorescence, CD spectroscopy, and AFM imaging (Figure S7). The resulting fibrils were characterized by intense FlAsH and ANS fluorescence signals, respectively resulting from the convergence of N-terminal domains and the presence of hydrophobic domains (Figure 5A,B). These amyloid fibrils also resisted thermal denaturation and proteolytic degradation by proteinase K, as confirmed by AFM imaging, CD spectroscopy, and MALDI-TOF MS (Figures S12 and S13). As expected, incubation of these pre-assembled fibrils in the absence of DNA for an additional 72 h did not significantly alter FlAsH or ANS fluorescence, nor the morphology of the fibrils (Figure 5). In sharp contrast, the presence of 1.6 µM of either 1D-, 2D-, or 3D- DNA nanostructures in the IAPP_F mixture led to the total disappearance of the fibrillar aggregates and the formation of spherical nanostructures (Figure 5C,D), with similar shapes and sizes to those reported for IAPP_M (Figure 3) and IAPP_O (Figure 4). This mesoscopic morphological conversion was accompanied by a slight decrease in FlAsH and ANS fluorescence (Figure 5A,B). The mixtures obtained after incubation with DNA were subsequently exposed to proteinase K to evaluate whether IAPP could be digested within these spherical complexes. As observed by MALDI-TOF MS, after incubation with the protease, IAPP originating from stable amyloid fibrils within the DNA-peptide complexes were digested to an extent similar to IAPP_M, in contrast to IAPP_F that were fully resistant to enzymatic hydrolysis (Figure S13). These observations indicate that DNA nanostructures not only have the capacity to interact with monomeric and oligomeric IAPP proteospecies but can also disassemble thermodynamically stable and proteolytically resistant amyloid fibrils into spherical aggregates, in which the polypeptide can be degraded by proteolysis.

3.4 DNA binding promotes the random coil conformation of IAPP

Results obtained thus far revealed that the interaction of IAPP with DNA nanostructures induced the formation of spherical complexes, regardless of the peptide's initial secondary or quaternary structure (i.e., randomly coiled monomers, heterogeneous oligomers/prefibrillar species, or organized β-sheet-rich amyloid fibrils). In fact, for all IAPP assembly states, the resulting spherical structures obtained upon incubation with DNA nanostructures showed similar characteristics in terms of morphology, hydrophobicity, size, and peptide clustering. IAPP-dsDNA interactions were further investigated by monitoring changes to the spectroscopic properties of IAPP and DNA molecules. As described above, monomers, oligomers, and fibrils were obtained by dispersing the lyophilized and monomerized IAPP in 20 mM Tris-HCl buffer (pH 7.4) at 75 µM. Each solution was then diluted to reach a final concentration of 25 µM of IAPP in the absence or in the presence of 1.6 µM of DNA nanostructure. As observed in Figure 6, in the absence of peptide, the UV-Vis absorption spectrum of dsDNA showed a broad band in the UV region with maximum absorption at 260 nm that does not overlap with the absorption maximum of IAPP species (Figure S14). To assess the direct interaction between DNA and the proteospecies, the UV-Vis absorption spectra of IAPP/DNA mixtures were scrutinized for shifts in the position of the maximum bands.^[56] The spectra of the 1D-, 2D-, and 3D-DNA nanostructures in the presence of IAPP_M, IAPP_O, and IAPP_F species were characterized by hyperchromism and slight bathochromism (redshift) (Figure 6 and Figure S15). Combined with zeta potential measurements (Figure S16), these observations indicate direct binding of dsDNA to the different peptide proteospecies.

Since CD spectroscopy could not be used due to the strong signal of DNA, ATR-FTIR spectroscopy was employed to gain insight into the secondary structure of IAPP species and to monitor conformational changes associated with the interaction of the peptide with DNA nanostructures. The amide III region of the spectrum (∼1175–1310 cm^–1) is mainly associated with C-N stretching and NH bending vibrations of the amide group and is highly sensitive to the secondary structure of the polypeptide chain (i.e., α-helix, β-sheet, β-turn, and random coil).^{[57, 58]} In particular, this region does not overlap with the spectrum of DNA, contrary to the amide I region, nor does it overlap with the absorption of water.^[58-60] Figure 7 shows the FTIR spectra of IAPP species obtained during the different phases of the fibrillization process. The spectrum of IAPP_M was characterized by a random-coil secondary structure with a band at 1209 cm⁻¹ and a shoulder at ∼1186 cm⁻¹. The FTIR spectrum of IAPP_O was characterized by the same band at 1209 cm⁻¹ with a shoulder at ∼1186 cm⁻¹ as well as a new band at 1226 cm⁻¹ corresponding to the oligomeric transition state from random-coil to β-sheet structure. In contrast, the FTIR spectrum of IAPP_F displayed a clear signature of a β-sheet secondary conformation with a characteristic band at 1226 cm⁻¹ and a shoulder at ∼1181 cm⁻¹. In the presence of DNA nanostructures, no band was observed at 1226 cm⁻¹ for any of the IAPP-DNA mixtures, and all FTIR spectra were indicative of a random coil peptide secondary structure (Figure 7 and Figure S17). Next, we harnessed the intrinsic fluorescence of IAPP to further investigate the interaction of DNA nanostructures with IAPP species. As shown in Figure S18, the fluorescence intensity associated with the emission of Tyr³⁷ was significantly quenched when DNA nanostructures were added to the solutions, further confirming the binding of IAPP to DNA. Overall, these results suggest a templating-based mechanism, in which the negatively charged phosphate backbone of DNA avidly interacts with the positive charges of IAPP, forming nanoscale IPECs in which the peptide is locked in a disordered non-amyloidogenic conformation.

3.5 DNA nanostructures prevent IAPP cytotoxicity by generating non-toxic complexes

The cytocompatibility of the spherical IAPP-DNA complexes was evaluated using INS-1E pancreatic cells, a model commonly used to study IAPP-associated toxicity. While the incubation of INS-1E cells with 25 µM IAPP_M for 24 h reduced their viability by 60%, the pre-formed IAPP-DNA complexes (i.e., 25 µM IAPP and 1.6 µM DNA pre-incubated for 72 h), did not affect cellular viability, nor did the DNA nanostructures alone affect the viability (Figure 8A). For instance, exposure to pre-formed spherical complexes assembled from IAPP_M and DNA-1D maintained over 95% viability of the treated control cells. Next, the activity of the cytoplasmic enzyme LDH, a common indicator of plasma membrane permeabilization, was measured in the presence of DNA-1D, IAPP-DNA-1D complexes, and IAPP alone. The LDH activity measured in the cell media after 6 h treatment indicated that both IAPP_M and IAPP_O avidly disrupted the plasma membrane (Figure 8B). In sharp contrast, DNA alone as well as pre-assembled IAPP-DNA complexes induced no significant increase of LDH activity in the cell media, indicating a lack of perturbation of the plasma membrane. It is known that pre-fibrillar oligomers are the most toxic IAPP species and contribute to pancreatic β-cell death in T2D.^{[61, 62]} Accordingly, it would be interesting to evaluate whether the DNA nanostructures could trap pre-assembled toxic oligomers and convert them into cytocompatible complexes. As described above, IAPP was pre-incubated at 75 µM in 20 mM Tris-HCl for 3 h to obtain IAPP_O, and these proteospecies reduced cellular viability by almost 90% after 5 h of incubation with the cells (Figure 8C). Strikingly, the co-addition of 1.6 µM DNAs in the cell culture, that is, without pre-incubation, fully abrogated the cytotoxicity of 25 µM IAPP_O. This result was confirmed by fluorescence microscopy analysis using the LIVE/DEAD assay, with the IAPP_O/DNA-1D showing quasi-absence of red-labelled dead cells (Figure 8D). These observations indicated that DNA nanostructures promptly interact with toxic oligomers in complex biological media containing high concentrations of a diversity of biomolecules, including those from FBS, and convert them into non-toxic proteospecies.