Molecular dynamics simulations reveal disruptive self-assembly in dynamic peptide libraries

There is a significant interest in the use of unmodified self-assembling peptides as building blocks for functional, supramolecular biomaterials. Recently, dynamic peptide libraries (DPLs) have been proposed to select self-assembling materials from dynamically exchanging mixture of dipeptide inputs in the presence of a nonspecific protease enzyme, where peptide sequences are selected and amplified based on their self-assembling tendencies. It was shown that the results of DPL of mixed sequences (e.g. starting from a mixture of dileucine, L2 and diphenylalanine, F2) did not give the same outcome as the separate L2 and F2 libraries (which give rise to formation of F6 and L6), implying that interaction between these sequences could disrupt the self-assembly. In this study, coarse grained molecular dynamic (CG-MD) simulations are used to understand the DPL results for F2, L2 and mixed libraries. CG-MD simulations demonstrate that interactions between precursors can cause the low formation yield of hexapeptides in mixtures of dipeptides and show that this ability to disrupt is influenced by the concentration of the different species in the DPL. The disrupting self-assembly effect between the species in DPL is an important effect to take into account in dynamic combinatorial chemistry as it affects the possible discovery of new materials. The work shows that combined computational and experimental screening can be used complementary and in combination provide a powerful means to discover new supramolecular peptide nanostructures

CHARMM force field parameterization protocol for self-assembling peptide amphiphiles : the Fmoc moiety

Aromatic peptide amphiphiles are known to self-assemble into nanostructures but the molecular level structure and the mechanism of formation of these nanostructures is not yet understood in detail. Molecular dynamic simulations using the CHARMM force field have been applied to a wide variety of peptide-based systems to obtain molecular level details of processes that are inaccessible with experimental techniques. However, this force field does not include parameters for the aromatic moieties which dictate the self-assembly of these systems. The standard CHARMM force field parameterization protocol uses hydrophilic interactions for the non-bonding parameters evaluation. However, to effectively reproduce the self-assembling behaviour of these molecules, the balance between the hydrophilic and hydrophobic nature of the molecule is essential. In this work, a modified parameterization protocol for the CHARMM force field for these aromatic moieties is presented. This protocol is applied for the specific case of the Fmoc moiety. The resulting set of parameters satisfies the conformational and interactions analysis and is able to reproduce experimental results such as the Fmoc-S-OMe water/octanol partition free energy and the self-assembly of Fmoc-S-OH and Fmoc-Y-OH into spherical micelles and fibres, respectively, while also providing detailed information on the mechanism of these processes. The effectiveness of the parameters for the Fmoc moiety validates the protocol as a robust approach to paramterise this class of compounds

Metastable hydrogels from aromatic dipeptides

We demonstrate that the well-known self-assembling dipeptide diphenylalanine (FF) and its amidated derivative (FF-NH2) can form metastable hydrogels upon sonication of the dipeptide solutions. The hydrogels show instantaneous syneresis upon mechanical contact resulting in rapid expulsion of water and collapse into a semi-solid gel

Elucidation of the bonding of a near infrared dye to hollow gold nanospheres : a chalcogen tripod

Infrared surface enhanced Raman scattering (SERS) is an attractive technique for the in situ detection of nanoprobes in biological samples due to the greater depth of penetration and reduced interference compared to SERS in the visible region. A key challenge is to understand the surface layer formed in suspension when a specific label is added to the SERS substrate in aqueous suspension. SERS taken at different wavelengths, theoretical calculations, and surface-selective sum frequency generation vibrational spectroscopy (SFG-VS) were used to define the surface orientation and manner of attachment of a new class of infrared SERS label with a thiopyrylium core and four pendant 2-selenophenyl rings. Hollow gold nanospheres (HGNs) were used as the enhancing substrate and two distinct types of SERS spectra were obtained. With excitation close to resonance with both the near infrared electronic transition in the label (max 826 nm) and the plasmon resonance maximum (690 nm), surface enhanced resonance Raman scattering (SERRS) was obtained. SERRS indicates that the major axis of the core is near to perpendicular to the surface plane and SFG-VS obtained from a dried gold film gave a similar orientation with the major axis at an angle 64°-85° from the surface plane. Longer excitation wavelengths give SERS with little or no molecular resonance contribution and new vibrations appeared with significant displacements between the thiopyrylium core and the pendant selenophene rings. Analysis using calculated spectra with one or two rings rotated indicates that two rings on one end are rotated towards the metal surface to give an arrangement of two selenium and one sulphur atoms directly facing the gold structure. The spectra, together with a space filled model, indicate that the molecule is strongly adsorbed to the surface through the selenium and sulphur atoms in an arrangement which will facilitate layer formation

Supplementary Materials for CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology

This PDF file includes: Figs. S1 to S10 Legends for tables S1 to S6Other Supplementary Material includes the following: Tables S1 to S6Peer reviewe

CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA “bait” oligonucleotides restore poly-GR–associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.This work was supported by the U.S. National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Aging (NIA) grant R01NS104219 (E.K.); NIH/NINDS grant R21NS107761 (E.K.); AFM-Telethon French Muscular Dystrophy Association Trampoline Grant #23648 (J.A.O.); AFM-Telethon postdoctoral fellowship (J.A.O.); Ramon y Cajal fellowships RYC2019-026980-I (J.A.O.) and RYC2021-033294-I (I.R.S.); Gipuzkoa Foru Aldundia 2019-FELL-000017-01 (I.R.S.); Maria de Maeztu Units of Excellence CEX2021-001159-M (J.A.O.) and MDM-2017-0720 (I.R.S.); NINDS grants R01NS097850 and R01NS131409 (J.K.I.); Department of Defense grants PR211919 and W81XWH2110131 (J.K.I.); John Douglas French Alzheimer’s Foundation (J.K.I.); Center for Regenerative Nanomedicine at the Simpson Querrey Institute (S.I.S. and T.D.C.); Intramural Research Program, NIH, National Cancer Institute (NCI), Center for Cancer Research (M.B. and S.L.W.); Les Turner ALS Foundation (E.K.); and New York Stem Cell Foundation (J.K.I. and E.K.).With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2021-001159-M (J.A.O.)).Peer reviewe

Enhanced neuron growth and electrical activity by a supramolecular netrin-1 mimetic nanofiber

Neurotrophic factors are essential not only for guiding the organization of the developing nervous system but also for supporting the survival and growth of neurons after traumatic injury. In the central nervous system (CNS), inhibitory factors and the formation of a glial scar after injury hinder the functional recovery of neurons, requiring exogenous therapies to promote regeneration. Netrin-1, a neurotrophic factor, can initiate axon guidance, outgrowth, and branching, as well as synaptogenesis, through activation of deleted in colorectal cancer (DCC) receptors. We report here the development of a nanofiber-shaped supramolecular mimetic of netrin-1 with monomers that incorporate a cyclic peptide sequence as the bioactive component. The mimetic structure was found to activate the DCC receptor in primary cortical neurons using low molar ratios of the bioactive comonomer. The supramolecular nanofibers enhanced neurite outgrowth and upregulated maturation as well as pre- and postsynaptic markers over time, resulting in differences in electrical activity similar to neurons treated with the recombinant netrin-1 protein. The results suggest the possibility of using the supramolecular structure as a therapeutic to promote regenerative bioactivity in CNS injuries.We are grateful to the following funding sources: the NIH National Institute of Biomedical Imaging and Bioengineering (NIBIB) 5R01EB003806 (S.I.S.), the Center for Regenerative Nanomedicine (CRN) at the Simpson Querrey Institute for BioNanotechnology (S.I.S.), Grant PID2021-124839OA-I00 funded by the Ministry of Science and Innovation of Spain (MCIN/AEI/10.13039/501100011033) (Z.A.), US National Institutes of Health (NIH) National Institute on Neurological Disorders and Stroke (NINDS) and NIH National Institute on Aging (NIA) R01NS104219 (E.K), and the New York Stem Cell Foundation (E.K.). E.K. is a Les Turner ALS Investigator and a New York Stem Cell Foundation–Robertson Investigator. C.S.S. was supported by an NSF graduate research fellowship program grant (DGE-1842165). Z.A., I.R.S., and J.A.O. were supported by Ramon y Cajal fellowships RYC2020-028732-I, RYC2021–033294-I, and RYC2019–026980-I, respectively, from the Spanish Ministry of Science. T.D.C. acknowledges funding support from an American Australian Association Fellowship. M.V.-P was supported by an FPU mobility grant from the Spanish Ministry of Universities.Peer reviewe

Artificial extracellular matrix scaffolds of mobile molecules enhance maturation of human stem cell-derived neurons

Human induced pluripotent stem cell (hiPSC) technologies offer a unique resource for modeling neurological diseases. However, iPSC models are fraught with technical limitations including abnormal aggregation and inefficient maturation of differentiated neurons. These problems are in part due to the absence of synergistic cues of the native extracellular matrix (ECM). We report on the use of three artificial ECMs based on peptide amphiphile (PA) supramolecular nanofibers. All nanofibers display the laminin-derived IKVAV signal on their surface but differ in the nature of their non-bioactive domains. We find that nanofibers with greater intensity of internal supramolecular motion have enhanced bioactivity toward hiPSC-derived motor and cortical neurons. Proteomic, biochemical, and functional assays reveal that highly mobile PA scaffolds caused enhanced β1-integrin pathway activation, reduced aggregation, increased arborization, and matured electrophysiological activity of neurons. Our work highlights the importance of designing biomimetic ECMs to study the development, function, and dysfunction of human neurons

Regenerative Medicine: Advances from Developmental to Degenerative Diseases

Chronic tissue and organ failure caused by an injury, disease, ageing or congenital defects represents some of the most complex therapeutic challenges and poses a significant financial healthcare burden. Regenerative medicine strategies aim to fulfil the unmet clinical need by restoring the normal tissue function either through stimulating the endogenous tissue repair or by using transplantation strategies to replace the missing or defective cells. Stem cells represent an essential pillar of regenerative medicine efforts as they provide a source of progenitors or differentiated cells for use in cell replacement therapies. Whilst significant leaps have been made in controlling the stem cell fates and differentiating them to cell types of interest, transitioning bespoke cellular products from an academic environment to off-the-shelf clinical treatments brings about a whole new set of challenges which encompass manufacturing, regulatory and funding issues. Notwithstanding the need to resolve such issues before cell replacement therapies can benefit global healthcare, mounting progress in the field has highlighted regenerative medicine as a realistic prospect for treating some of the previously incurable conditions