Novel polymer linkers for single molecule AFM force spectroscopy

Tong, Zenghan; Mikheikin, Andrey; Krasnoslobodtsev, Alexey V.; Lv, Zhengjian; Lyubchenko, Yuri L.

doi:10.1016/j.ymeth.2013.02.019

Cited by 30 publications

(46 citation statements)

References 18 publications

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“…A plausible explanation for this finding is that the density of the peptide coverage is very low, so non-apically located peptides are not able to interact efficiently with the counterpart on the substrate. This conclusion is confirmed by our very recent results obtained with DNA pulling experiments in which fully homogeneous PA tether was used 24 . The experiments produced the contour length distribution as narrow as 3 nm.…”

Section: Discussionsupporting

confidence: 81%

Nanoprobing of misfolding and interactions of amyloid β 42 protein

Kim

Lyubchenko

2014

Nanomedicine: Nanotechnology, Biology and Medicine

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The assembly of amyloid β (Aβ) proteins into nanostructures is currently considered a major pathway of Alzheimer's disease development, but the molecular mechanisms of this self-assembly process remains unclear. Recently, we showed that single-molecule AFM force spectroscopy (SMFS) is capable of probing the dynamics and interaction between Aβ40 peptides, and these studies allowed us to shed new light on transiently existing Aβ40 misfolding states. In this study, we applied the same SMFS approach to characterize the misfolding of Aβ42 peptide, the most toxic Aβ alloform. The quantitative analysis of SMFS data demonstrated that Aβ interaction leads to the formation of dimers with a lifetime in the range of a second. Interaction via C-terminal segments prevailed at pH 7, but interaction within the peptide center prevailed at acidic pH levels. The difference in the misfolding properties for Aβ40 and Aβ42 peptides and the mechanisms of amyloid nanoassembly are discussed. KeywordsAmyloid beta; misfolding; single-molecule force spectroscopy; contour length; dimerization pathways Aberrant folding, misfolding, and aggregation of amyloid proteins are pathological hallmarks of a large class of neurodegenerative diseases. 1, 2 The assembly of amyloid β (Aβ) proteins in nanoaggeragtes is a ubiquitous phenomenon for a number of neurodegenerative disorders, including Alzheimer's disease (AD) (reviewed in 3, 4 ). Aβ proteins containing 42 and 40 residues (Aβ42 and Aβ40, respectively) are the major species, and Aβ42, which differs from Aβ40 by two extra C terminal residues (I41 and A42), has an elevated aggregation propensity and neurotoxicity compared to Aβ40. 5,6 Traditional © 2013 Elsevier Inc. All rights reserved. * Corresponding author: Yuri L. Lyubchenko, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, Phone: 402-559-1971; Fax: 402-559-9543, ylyubchenko@unmc. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. structural techniques, such as solid-state NMR, AFM, and electron microscopy, are widely used for their characterization of large aggregates, such as amyloid fibrils. However, these powerful techniques are not amenable to studies of oligomers that exist transiently and convert from one species to another, so novel approaches are needed for oligomers studies. The photocrosslinking method, developed for the stabilization of oligomers in Teplow lab 7 was instrumental in the spectroscopic characterization of Aβ42 oligomers 8 . It was shown that the transition from the monomer to the dimer is rapid and then proceeds gradually for h...

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Section: Discussionsupporting

confidence: 81%

Nanoprobing of misfolding and interactions of amyloid β 42 protein

Kim

Lyubchenko

2014

Nanomedicine: Nanotechnology, Biology and Medicine

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“…All measurements were done in HEPES buffer (10 mM HEPES, 100 mM NaCl, PH 7.0). The data analysis was performed as described 20, 35, 37, 38 . Briefly, DFS data was obtained for pulling experiments performed at different velocities (100-3000 nm/sec).…”

Section: Methodsmentioning

confidence: 99%

Probing of miniPEGγ-PNA–DNA Hybrid Duplex Stability with AFM Force Spectroscopy

Dutta

Armitage

Lyubchenko³

2016

Biochemistry

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Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplex. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. (2011) Journal of Organic Chemistry 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γMPPNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that γMPPNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ∼ 0.030 ± 0.01 sec-1 for γMPPNA-DNA hybrid duplex vs. 0.375 ± 0.18 sec-1 for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γMPPNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γMPPNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

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“…We hypothesized that adding fl exibility into the DNA system using a hexaethylene glycol phosphate group (Glen Research Corporation, 'Spacer-18', SP18) would widen the annealing window. Although SP18 contains a charged phosphate moiety, its persistence length in buffers with high ionic strength (e.g., 0.5 M NaCl used here) was found to be the same as polyethylene glycol [ 26 ] ( L p = 2.8 Å) which is substantially less than single [ 27 ] ( L p = 7.5 Å) or double [ 28 ] ( L p = 500 Å) stranded DNA. Ligand fl exibility was explored by adding various subunits of SP18 to the DNA at the fl exible spacer region (Figure 1 After annealing (A), the supernatant is separated from the aggregates to obtain a melting curve determined by UV-Vis spectroscopy, where each point is determined by a distinct sample annealed at a different temperature (B) (the curve is a best-fi t sigmoidal), and the aggregates are examined by SAXS (C).…”

Section: Communicationmentioning

confidence: 96%

Oligonucleotide Flexibility Dictates Crystal Quality in DNA‐Programmable Nanoparticle Superlattices

et al. 2014

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this sense, the particles act as "programmable atom equivalents" (PAEs), where each particle behaves as an "atom" with bonding behavior that can be tuned via the DNA interconnects. The synthetic tailorability afforded by DNA allows independent control over the superlattice connectivity and nanoparticle core, thereby enabling the design and synthesis of colloidal crystals with widely varying symmetry, scale, and composition. [14][15][16][17][18][19] While DNAmediated assembly is a useful approach for spatially arranging noble metal nanoparticles, this method has not succeeded in forming crystals with short rigid DNA strands, especially for larger diameter particles. [ 14 ] This inability to crystallize large particles with small separations is likely a result of kinetic jamming behavior, which is often seen in colloidal systems [ 20 ] and may be related to the rigidity of the double-stranded DNA ligands.Initial evidence for the importance of fl exibility came from the observation that certain PAE systems which form amorphous aggregates when linked with fully duplexed DNA will crystallize if a single unpaired base is added to the DNA strands that link the particles. [ 12 ] Given that single-stranded DNA has a shorter persistence length than double-stranded DNA, this can be rationalized by considering that adding some degree of fl exibility may be required to allow the packing of the inherently polydisperse particles into a regular lattice. Further, it is well known that the phase behavior of colloids depends on their range of interaction; long-range interactions result in a "softer" pair-potential that may allow for crystallization, while short-range interactions often result in jamming and gel formation. [ 21 ] Based on these observations, we hypothesize that adding fl exibility to the DNA ligand could allow each strand to explore a wider space, thereby increasing the interaction range and decreasing jamming during PAE crystallization.Herein, we explore the relationship between crystallization and linker fl exibility by systematically adding various lengths of a fl exible oligomer to the DNA ligand ( Figure 1 ). Through annealing at different temperatures, we fi nd that the highest quality crystals (defi ned here as crystals with the largest grain sizes and lowest microstrain as measured by X-ray diffraction) are formed at temperatures close to the melting temperature. Signifi cantly, increasing ligand fl exibility increases the temperature range under which crystallization occurs while simultaneously minimizing microstrain and maximizing domain size.In a typical assembly experiment, PAE superlattices were observed by correlated synchrotron small-angle X-ray scattering (SAXS) and UV-vis spectroscopy ( Figure 2 ), a set of measurements that allowed for the simultaneous determination of structural and thermodynamic information. Specifi cally, gold nanoparticles (AuNPs) were coated with a dense, oriented Superlattices composed of noble metal nanoparticles display considerable promise for the development of optical m...

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Novel polymer linkers for single molecule AFM force spectroscopy

Cited by 30 publications

References 18 publications

Nanoprobing of misfolding and interactions of amyloid β 42 protein

Nanoprobing of misfolding and interactions of amyloid β 42 protein

Probing of miniPEGγ-PNA–DNA Hybrid Duplex Stability with AFM Force Spectroscopy

Oligonucleotide Flexibility Dictates Crystal Quality in DNA‐Programmable Nanoparticle Superlattices

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