Peptide with Short Single-Stranded Synthetic Nucleotide Sequences

Aug 5, 2014 - purchased from Bachem Laboratories (Bubendorf, Switzerland). Synthetic nucleotide sequences were purchased from Microsynth. (Balgach ...
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Effect of the Interaction of the Amyloid β (1−42) Peptide with Short Single-Stranded Synthetic Nucleotide Sequences: Morphological Characterization of the Inhibition of Fibrils Formation and Fibrils Disassembly Jancy Nixon Abraham, Dawid Kedracki, Enora Prado, Charlotte Gourmel,† Plinio Maroni, and Corinne Nardin* Faculty of Sciences, Department of Inorganic and Analytical Chemistry, University of Geneva, Quai Ernest Ansermet 30, CH-1211, Geneva 4, Switzerland S Supporting Information *

ABSTRACT: The formation of extracellular neuritic plaques in the brain of individuals who suffered from Alzheimer’s disease (AD) is a major pathological hallmark. These plaques consist of filamentous aggregates of the amyloid beta (1−42) (Aβ42) proteins. Prevention or reduction of the formation of these fibrils is foreseen as a potential therapeutic approach. In this context, we investigated the interactions between the Aβ42 protein and polyions, in particular short single stranded synthetic nucleotide sequences. The experimental outcomes reported herein provide evidence of the inhibition of amyloid fibril genesis as well as disassembly of existing fibers through electrostatic interaction between the Aβ42 protein and the polyions. Since the polyions and the Aβ42 protein are oppositely charged, the formation of (micellar) inter polyelectrolyte complexes (IPECs) is likely to occur. Since the abnormal deposition of amyloid fibers is an archetype of AD, the outcomes of these investigations, supported by atomic force microscopy imaging in the dry and liquid states, as well as circular dichroism and Fourier transform infrared spectroscopy, are of high interest for the development of future strategies to cure a disease that concerns an ever aging population. currently assessed as more toxic than polymeric fibrils and might be the primary pathological species.7 Since the abnormal deposition of this protein is an archetype of AD, the investigation of inhibition or reduction of fibril formation thus remains one of the approaches to advance the current understanding of both AD pathophysiology and protein assembly to eventually establish potential strategies to cure a disease of global health concern. The in vitro characterization of conditions for Aβ42 synthetic polypeptide organization into oligomers and fibrils have been systematically investigated by varying time, concentration, temperature, pH and ionic strength.8 Statistical analyses by atomic force microscopy to characterize amyloid aggregation are reported as well.9 Several elicitors or inducers of fibril genesis could thus be identified. Like sodium dodecyl sulfate, which is an amyloid formation inducer,10 mutations that replace aromatic side chains promote aggregation.11 On the other hand, inhibition of fibril formation could be achieved by interaction with short amyloid fragments12a−d and cyclic peptides.12e Interaction with oligopeptides such as KLVFF, LPFFD prevents assembly of Aβ42 into fibrils and induces disassembly

1. INTRODUCTION Alzheimer’s disease (AD) is a fatal, progressive illness characterized by memory loss, cognitive deficits and behavioral changes. Structural and kinetics features evidence that the disease is associated with a protein folding disorder: the amyloid β(1−42) proteins (Aβ42) indeed organize into senile or diffuse plaques and vascular deposits according to a nucleation dependent polymerization process.1 Since amyloidosis is accompanying several diseases, the mechanism of peptide fibril formation is thus currently widely studied.2 The Aβ42 proteins exist in a variety of complex forms that differ in their number of peptide building blocks as well as in their overall conformation and biological activity.3 The Aβ42 proteins exist as monomers, which further assemble into oligomers and protofibrils that eventually aggregate into fibrils. Within hours of incubation, Aβ42 monomers assemble into a wide range of soluble oligomeric species of 5−25 nm diameter and protofibrils of length over 40 nm.4 The stable hydrophobic core is composed of the LVFF (17−20) oligopeptide and the VGSN (24−27) β turn stabilized by a salt bridge between D23 and K28.2a,5 Recent studies indicate that the residues 17−21 and 30−35, which promote Aβ42 aggregation, are involved in neurotoxicity owing to their high propensity to aggregate.6 Moreover, the prefibrillar oligomeric Aβ42 intermediates are © XXXX American Chemical Society

Received: May 5, 2014

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temperature for 30 min. Twenty-five μL aliquots were transferred into prechilled Eppendorf tubes. HFIP was then removed under a gentle stream of nitrogen, or by leaving the tubes opened in the fume hood overnight. For overnight evaporation, the tubes were placed in a rack and covered to prevent contamination. The solution can be then stored for an extended period at −20 °C. Aβ42 fibrils were prepared by diluting 5 mM Aβ42 peptides in DMSO to 100 μM with 10 mM HCl (pH = 2), followed by 30 s vortexing and incubation at 37 °C for 24 h. The solution was further diluted with water to 10 μM for CD and imaging by AFM. Incubation with either synthetic polymers or nucleic acid strands was performed by adding 500 μM polymers or nucleotide sequences and incubation was carried out under the same conditions as for growing fibers. Incubation of nucleic acid strands with fibers was performed by adding a 10 μM solution of amyloid fibers to a 50 μM solution of oligonucleotides and incubating at room temperature for 1 h. The solution was directly used for AFM measurements. 2.2.2. Atomic Force Microscopy. AFM imaging in the alternating contact mode (AC mode) was performed using a Nanoscope IIIa scanning probe workstation equipped with a Multimode head using an A-series piezoceramic scanner (Digital Instruments, CA, U.S.). AFM probes were single-crystal silicon microcantilevers AC240TS (Olympus, Japan) of 70 kHz resonance frequency and 1.8 N m−1 spring constant with a nominal tip radius smaller than 9 nm. A total of 15 μL of the solution was spotted on freshly cleaved mica or HOPG, incubated at room temperature for 10 min, and dried under nitrogen. Liquid imaging in the AC-mode was performed with a Cypher AFM (Asylum Research, Santa Barbara, CA). AFM probes were Biolever mini BL-AC40TS (Olympus, Japan) of 30 kHz resonance frequency in liquid and 0.1 N m−1 spring constant (free oscillation amplitude (FOA) 20 nm; set point at about 70% of the FOA; scan rate 3 Hz). A total of 15 μL of the solution was spotted on freshly cleaved mica or HOPG and washed several times with the same buffer. 2.2.3. Circular Dichroism Spectroscopy. Circular dichroism (CD) spectra of the Aβ42 peptide solution (10 μM) were recorded on a JASCO J-815 CD Spectrometer (Jasco Corporation, Tokyo, Japan) at 25 °C using a bandwidth of 5.0 nm, a data pitch of 0.2 nm, a scanning speed of 100 nm min−1, and a response time of 4 s. A 0.1 cm quartz cell was used for far-UV (190−260 nm) measurements. Three scans of duplicate samples were measured and averaged. Control buffer scans were run in duplicate, averaged, and then subtracted from the samples spectra. The results of CD measurements were plotted as ellipticity (milli degree) versus wavelength (nm). 2.2.4. Fourier Transform Infrared Spectroscopy (FTIR). Spectra were recorded in the attenuated total reflectance (ATR) mode using a Fourier Transform Infrared spectrometer (Spectrum 100, PerkinElmer, U.S.A.). For each measurement, 4 μL of the sample was spread on the surface of the internal reflection element made of a diamond crystal and dried. The analysis was performed between 4000 and 450 cm−1 at a resolution of 4 cm−1 averaged over 50 scans. 2.2.5. Dynamic Light Scattering and ζ-Potential Measurements. The size of the fibers and IPECs were estimated by dynamic light scattering (DLS) at 25 °C using a Zetasizer Nanoseries from Malverin Instruments with the backscattered angle detection at 173° in optically homogeneous square polystyrene cells. The zeta potentials of samples were measured by the micro electrophoretic method using a Malvern Zetasizer Nano ZS apparatus. All the measurements were taken with samples prepared in the same molar proportion as discussed above. 2.2.6. Transmission Electron Microscopy. For transmission electron microscopy (TEM), 3 μL of solution was placed on a carbon-coated, 400-mesh copper grid. The sample was dried and was imaged with a Tecnai G2 electron microscope operating at 120 kV.

of already formed fibers. The extent of the interactions depends on the binding affinity between the oligopeptide and the protein, in particular the hydrophobic interactions between the short peptide segments and the full length Aβ42 protein. Moreover these short peptides, which are charged (lysine (+ ve) and aspartic acid (-ve)) also engage in electrostatic interactions. Proteins were shown to reduce amyloid load through bioencapsulation by oral delivery,13a as well as delay Aβ42 fibril formation,13b or prevent fibrillation and associated toxicity.13c The interactions between amyloid fibrils and polyanions such as DNA, ATP and heparin has been reported.14 Since both oligomers and fibers are currently assigned as toxic, the present approach is focused on identifying how to inhibit both fibril formation and disassemble fibrils. Although coupling of nucleotide and peptide sequences to synthetic polymers to induce their self-assembly in aqueous solution has been investigated recently,15 the formation of (micellar) interpolyelectrolyte complexes (IPECs) between a nucleotide and a peptide sequence is scarcely reported. Structure formation of IPECs occurs upon mixing of polyelectrolytes of opposite charge in aqueous solution.16 When this coassembly occurs between copolymers, the resultant structures are generically named as complex coarcevate core micelles or micellar IPECs. One of the polyions engaged in the structure formation might be a biopolymer such as a peptide or a nucleotide sequence. Investigations with the latter have been particularly carried out for potential applications in gene therapy.17 We describe herein the inhibition of amyloid fibril genesis as well as disassembly of existing fibers through electrostatic interaction between the Aβ42 protein and polyions, in particular single stranded short synthetic nucleic acid strands. The experimental outcomes supported by atomic force microscopy imaging and spectroscopy enables assuming the formation of interpolyelectrolyte complexes, in line with recent reports on charge dependent retardation of Aβ42 fibril genesis through interaction with hydrophilic proteins18a as well as complex formation with charged conjugated polymers.18b

2. EXPERIMENTAL SECTION 2.1. Materials. Synthetic full-length Aβ(1−42) peptides were purchased from Bachem Laboratories (Bubendorf, Switzerland). Synthetic nucleotide sequences were purchased from Microsynth (Balgach, Switzerland). Nucleotide sequences used in this study were 5′-CTA GTC GAG TAG-3′ (MW 3685 Da), 5′-AAA GAG AGA GAG-3′ (MW 3776 Da) and 5′-TCT GAG-3′ (MW 1807 Da) respectively. DMSO and HCl were purchased from AppliChem (Darmstadt, Germany). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from Fluka (Switzerland). Poly(acrylic acid), (MW 5000) was purchased from Acros Organics (Geel, Belgium) and poly(ethylene imine) (MW 2500) was purchased from Polysciences (Warrington, Pennsylvania). Poly[2-(3-butenyl)-2-oxazoline] (PBOX) was kindly provided by Prof. Schlaad (University of Potsdam, Germany).19 The isolated PBOX chains had an average length of 43 repeat units, corresponding to a number-average molar mass (Mn) of 5380 g mol−1, by 1H NMR end group analysis, and a dispersity index (ratio of weight- over number-average molar mass, Mw/Mn) of 1.05, by size exclusion chromatography (SEC). Sodium chloride was purchased from Sigma-Aldrich (Steinheim, Denmark). 2.2. Methods. 2.2.1. Controlled Aggregation of Aβ42 Fibrils. The lyophilized peptide was first pretreated with HFIP. All the work with HFIP was done in fume hood with adequate protection. The peptide (1 mg) was dissolved in HFIP (450 μL) in ice cold conditions, to obtain a final concentration of 0.5 mM. The HFIP solution was sonicated for 5 min, vortexed gently and incubated at room

3. RESULTS To ensure structural homogeneity, amyloid fibrils were assembled under controlled aggregation conditions following the procedure reported by Stine et al.8 Within 24 h of incubation, long fibers of several micrometers length and diameter of less than 5 nm developed. For AFM imaging, the B

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solution was diluted 10 times so that the final concentration of fibers is 10 μM. To rule out any surface effect, Aβ42 fibrils were observed by AFM on both freshly cleaved hydrophobic highly ordered pyrolytic graphite (HOPG; Figure 1A) and hydrophilic

Figure 1D, incubation of Aβ42 with this synthetic not watersoluble polymer does not prevent fibril formation. Upon incubation of the Aβ42 amyloid with nucleic acid strands, the negative charges of the phosphate group along the nucleotide sequence might interact with the positively charged amino acid residues of the amyloid protein. Incubating the amyloid with a 12 nucleotide-long sequence, 5′-CTAGTCGACTAG-3′ (G stands for guanine, C for cytosine, T for thymine, and A for adenine) at a molar ratio of 1:5 caused inhibition of fibril formation (Figure 2A, height profile in Figure S2B). This effect is independent of the length or composition of the nucleic acid strands. No fibrils were observed upon incubation of the peptide with either the 5′-TCTGAG-3′ (6mer; Figure 2C) or the 5′-AAAGAGAGAGAG-3′ (12-mer; Figure 2D). The corresponding height profiles are shown in Figure S2. The height profile suggests that the complexes formed have a height of 2−5 nm. It also has to be noticed that the nucleic acid sequences dissolve as single molecules in solution (Figure S4). The hydrophobic interactions between the bases and sugars of the DNA and the hydrophobic core of the amyloid may also contribute to the inhibition of fibril genesis, but the experimental results reported above with PAA, PEI, and PBOX strongly support the crucial role of electrostatic interactions. Aβ42 fibril formation is described by a nucleation polymerization process.1 Recently, Jeong et al. evidenced the existence of secondary nucleation sites on the surface of the Aβ42 fibrils.9a They further emphasized the particular role of the Aβ42 monomer concentration in determining the structural properties and homogeneity of the fibrils formed, as evidenced as well by Stine et al.8 To ensure that the inhibition of fibril formation observed upon incubation of the Aβ42 monomers with the nucleic acid strands is not an effect of concentration upon drying on the surface, we performed comparable AFM analyses in the liquid state (Figure 3). No fibrils were formed upon incubation of the peptide and nucleotide sequences. Ill-defined structures were observed instead, when the Aβ42 peptide was incubated with the 5′-AAAGAGAGAGAG-3′ (12-mer; Figure 3B), the 5′-CTAGTCGACTAG-3′ (12-mer; Figure 3C), and the 5′-TCTGAG-3′ (6-mer; Figure 3D). The corresponding height profiles obtained by AFM performed in the liquid state are displayed in Figure S5. Figure S6 reveals by AFM in the liquid state that the polyions are molecularly dissolved. The effect of interaction of nucleic acid strands on the disassembly of fibers was further studied. Since the previous experiments evidence that inhibition of fibril growth is irrespective of the length or composition of the nucleic acid strand, experiments were conducted with 5′-TCTGAG-3′ as a representative example. As can be observed by AFM, subsequent to incubation with the nucleotide sequences, fibrils

Figure 1. AFM imaging on HOPG of (A) Aβ42 fibers (10 μM) assembled under controlled aggregation conditions; Fibril formation was inhibited upon incubation of Aβ42 with (B) poly(acrylic acid) (50 μM), (C) poly(ethylene imine) (50 μM), and (D) poly(oxazoline) (50 μM) does not affect fibrils formation.

freshly cleaved mica (Figure S1). The corresponding representative height profile is shown in Figure S2B, which evidences the formation of fibrils of infinite length and diameter of about 5−10 nm. To reveal at first the role of electrostatic interaction in fibrils formation, incubation of the amyloid sequence with sodium chloride (NaCl), poly(acrylic acid) (PAA), and poly(ethylene imine) (PEI) at the same molar ratio than amyloid to nucleic acid strand, that is, 1:5, was performed and structure formation induced in the same conditions as in which the fibrils were grown. As can be observed in Figure S3, NaCl affects the formation of the amyloid fibrils. Likewise, incubation with PAA affects the fibril formation in a similar way as PEI does (Figure 2A and B, respectively). These experimental outcomes clearly point toward the role of electrostatic interactions between the oppositely charged amyloid proteins and synthetic polymers in inhibiting the fibril growth. This observation is further supported upon incubation with the hydrophobic poly[2-(3-butenyl)-2-oxazoline] (PBOX). Owing to a chain configuration close to that of a polypeptide, poly(2-oxazoline)s are in general considered for usage in biomedical applications.20 As can be observed in

Figure 2. AFM imaging on HOPG of Aβ42 peptide incubated with (A) 5′-CTA GTC GAC TAG-3′, (B) 5′-AAA GAG AGA GAG-3′, (C) 5′-TCT GAG-3′ (50 μM). C

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Figure 5. TEM images showing (A) complexes formed when Aβ42 (10 μM) was coincubated with 5′-TCTGAG-3′ (50 μM; inhibition of fibril genesis) and (B) complexes formed by disassembly of Aβ42 fibers (10 μM) upon incubation with 5′-TCTGAG-3′ (50 μM).

Figure 3. Liquid state AFM imaging on HOPG of (A) Aβ42 fibers (10 μM) assembled under controlled aggregation conditions; Fibril formation was inhibited upon incubation of Aβ42 with (B) 5′-CTA GTC GAC TAG-3′, (C) 5′-AAA GAG AGA GAG-3′, (D) 5′-TCT GAG-3′ (50 μM).

of already formed fibers on the β-sheet fingerprint. CD spectroscopy upon incubation of the amyloid protein with nucleotide sequences as well as with PEI and PAA are shown in Figure 6. All the data were treated with a baseline correction

are no longer visible whereas small spherical structures are observed (Figures 4 and S7). The dissolution of fibers is clearly

Figure 4. AFM imaging on HOPG of Aβ42 fibers (10 μM) incubated with nucleic acid strand (5′-TCT GAG-3′ (50 μM), fibers are disrupted): (A) dry state; (B) liquid imaging.

visible by AFM in both dry and liquid states (the appearance of elongated structures upon imaging in the dry state might be a concentration effect). These results are further supported by DLS and preliminary SAXS measurements (Figure S8). DLS evidence, upon addition of the nucleic acid strands to the fibrils, the disappearance of a slow diffusion time, and appearance of a faster diffusion coefficient, which corresponds to spherical structures of about 200 nm diameter, a size comparable to that of the structures obtained upon incubation of the amyloid peptide with the nucleotide sequences. The 30 mV ζ-potential of the fibrils decreases to −21 mV upon incubation of the fibers with the nucleic acid strand. Upon incubation of Aβ42 peptides with nucleotide sequences, a negative value of −13 mV was measured. To further assess the morphology of the structures, transmission electron microscopy (TEM) imaging was performed (Figure 5). These experiments revealed the formation of structures of size of 152 nm upon incubation of the amyloid peptide with the nucleic acid strands (mean of 20 random structures), whereas upon incubation of the fibrils with the nucleotide sequences, the size is in average 328 nm. The characterization of the size and the kinetics of formation of soluble equilibrium structures are currently in progress by scattering investigations. We eventually resorted to circular dichroism and FTIR spectroscopy in order to assess the effect of the interaction with polyions over the course of fibril genesis as well as disassembly

Figure 6. Circular dichroism of Aβ42 peptide fibrils (10 μM) showing (A) β-sheet structures when assembled under controlled aggregation conditions; in the presence of (B) 5′-CTAGTCGACTAG-3′; (C) 5′AAAGAGAGAGAG-3′; (D) 5′-TCTGAG-3′ (50 μM), (E) incubation of amyloid fibers (10 μM) and TCTGAG (50 μM), (F) incubation of Aβ42 and poly(acrylic acid) (50 μM), (G) poly(ethylene imine) and (H) poly(oxazoline) (50 μM), the 218 nm negative band vanishes.

and the signal of molecularly dissolved polyions subtracted. The peak at 218 nm assigned to β-sheet structures is a characteristic of the Aβ42 fibrils. This peak is not visible upon incubation with either the nucleic acid strands (Figure 6B−D) or the polyions (Figure 6F,G). Upon incubation of fibers with the nucleic acid strand (Figure 6E), a small shoulder is visible at 217 nm solely. A weak signal at 216 nm is also visible upon incubation of Aβ42 with PBOX, which does not prevent fibers formation (Figure 6H). Since both synthetic polymers and nucleotide sequences do not reveal the negative band at 218 nm (Figure S9), CD spectroscopy indicates that incubation of the Aβ42 peptides or fibrils with short single stranded nucleotide sequences disabled the organization of the peptide into β-sheets. The results of these investigations further confirm that there is no specific effect of the nucleotide sequence composition or length. However, deconvolution of the CD spectra did not enable a clear identification of organization within the structures. D

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noncovalent interactions in particular hydrogen bonding between the base pairs, π−π stacking between the aromatic rings of the bases and electrostatic interactions owing to the presence of the negative charges of the phosphate groups along the nucleotide sequence backbone. These interactions between negatively charged nucleic acid sequences and positively charged polymers were shown to cause a conformational transition from a B to a C DNA form prior to the formation of structures on surfaces.25 Based on our experimental results, we assume the formation of an IPEC between the negatively charged nucleotides (or PAA) and the positively charged amino acid residues of amyloid Aβ42 (Scheme 1).

FTIR is an efficient tool for distinguishing amyloid oligomer from fibrils based on their spectral features in the amide I region (1700−1600 cm−1).21 The frequencies of the amide I band can be correlated closely to the secondary structure of the proteins.22 As displayed in Figure 7, the ATR-FTIR spectrum of Aβ42 shows a broad band centered at 1661 cm−1, assigned to either

Scheme 1. Putative Mechanism of Aβ42 Peptide Fibril Formation Inhibition by the Formation of an Interpolyelectrolyte Complex between the Peptide and the Nucleotide Sequences

Figure 7. ATR-FTIR spectra in the amide I region of (A) 5′TCTGAG-3′ (50 μM), (B) Aβ42 peptide before fibril genesis (10 μM), (C) Aβ42 fibrils assembled under controlled aggregation conditions (10 μM), (D) incubation of Aβ42 and 5′-TCTGAG-3′ (10 and 50 μM), (E) incubation of amyloid fibers and 5′-TCTGAG-3′ (10 and 50 μM).

random coils or helical structures.22 In the absence of nucleic acid strands, the FTIR spectrum of Aβ42 fibrils (Figure 7, spectrum C) reveals a typical band at 1630 cm −1 , corresponding to the hydrogen-bonded β-sheet structures. The relatively narrow width of this peak is further indicative of stable and long β-strands stabilized by strong hydrogen bonds,23 as expected for stable structures such as amyloid fibrils.24 Upon incubation of the fibers with nucleic acid strands, the intensity of this peak decreases drastically and a new peak emerged at 1674 cm−1 (Figure 7E), which is characteristic of the β turn of Aβ42 oligomers. This is an indication of the disruption of hydrogen-bonded β-sheet structures upon interaction with the nucleic acid strands. Moreover, the ATRFTIR spectrum recorded with Aβ42 monomers incubated with the nucleic acid strands (Figure 7, spectrum (A)) reveals a broad band at 1661 cm−1 corresponding to the sum of Aβ42 monomers and nucleic acid strands spectra (Figure 7, spectrum D). Thus, the inhibition of the fibrillation process in the presence of nucleic acid strands is further evidenced by FTIR spectroscopy. These results provide strong experimental evidence in support of the AFM and CD measurements.

In the case of interactions between PEI and Aβ42, IPECs are formed by electrostatic interaction between positively charged PEI and negatively charged residues along the amyloid. As the amyloid protein is constituted of 48% of hydrophobic amino acids, the role of hydrophobic interactions between the nucleic acid strands and the amyloid protein and fibers cannot be ruled out. However, the results of the reported experiments, in particular, the effect of PEI and PAA and absence of effect of the hydrophobic PBOX polymer, strongly support the crucial role of electrostatic interactions in inducing the formation of a micellar interpolyelectrolyte complex composed of a hydrophobic peptide core stabilized by electrostatic interactions between the oppositely charged Aβ42 amino acids and polyions at the outer rim. The putative mechanism is further supported by the negative value of the ζ-potential in both cases, that is, upon inhibition of fibrils genesis and disassembly of fibers, which are characterized by a positive ζ-potential. Charge reversal is in agreement with the hypothesis of the formation of an interpolyelectrolyte complex composed of a hydrophobic peptide core stabilized by electrostatic interactions between the oppositely charged Aβ42 amino acids and polyions at the outer rim.

4. DISCUSSION The nucleic acid strands are model nucleotide sequences that are linear and do not self-hybridize, characterized by a melting temperature above the one used to induce fibril formation under controlled aggregation. The amyloid sequence (H2N-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-OH) is composed of the positively charged amino acid lysine (K), arginine (R), and histidine (H) residues, along with negatively charged residues such as glutamic acid and aspartic acid, depending on pH. Nucleic acid strands are known to undergo several modes of

5. CONCLUSION In this work, we have demonstrated the inhibition of amyloid fibril genesis as well as disassembly of fibers on incubation with polyions. Of particular interest is the formation of structures of about 80 nm radius upon interaction with short single-stranded synthetic nucleic acid strands. Irrespective of the length and composition of the nucleotide sequences, the incubation with Aβ42 resulted in inhibition of fibril growth. Instead, small spherical structures with an average size of 150 nm were formed. The experimental results from incubation of the Aβ42 peptide with sodium chloride as well as with either a synthetic E

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polyanion or a polycation also showed disturbance of fibril formation. The major role of hydrophobic interactions could be ruled out by incubation with a non-water-soluble polymer. IPECs are probably formed between the peptide and either synthetic polyions or nucleic acid strands through interaction between monomers or nucleic acids and amino acids of opposite charges. Incubation of the amyloid fibers with synthetic oligonucleotide sequences induces their disassembly. These experimental results are supported by AFM and TEM imaging, scattering experiments as well as CD and FTIR spectroscopy. The identification of the thermodynamics and kinetics of the process is under progress. If the resulting complexes are less toxic than the prefibrillar oligomers or fibers will be investigated in the future, since these experimental outcomes would be of particular relevance for future AD drug developments.

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ASSOCIATED CONTENT

S Supporting Information *

Control AFM experiments, CD controls, DLS, and preliminary SAXS results are presented. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address †

School of Medicine, Institute of Inflammation and Repair, University of Manchester, Oxford Road, Manchester, M13 9PT, U.K. (C.G.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The University of Geneva and the SNSF (PPOOP2−128380) are greatly acknowledged for financial support. We especially thank Kenneth Adea for his experimental support. Dr. Naomi Sakai is deeply acknowledged for her help with CD experiments. Dr. Nidhi Gour, Dr. Kien X. Ngo, and Ilyès Safir are thanked for useful discussions.

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dx.doi.org/10.1021/bm501004q | Biomacromolecules XXXX, XXX, XXX−XXX