7,325 research outputs found
Programmable Control of Nucleation for Algorithmic Self-Assembly
Algorithmic self-assembly, a generalization of crystal growth processes, has
been proposed as a mechanism for autonomous DNA computation and for bottom-up
fabrication of complex nanostructures. A `program' for growing a desired
structure consists of a set of molecular `tiles' designed to have specific
binding interactions. A key challenge to making algorithmic self-assembly
practical is designing tile set programs that make assembly robust to errors
that occur during initiation and growth. One method for the controlled
initiation of assembly, often seen in biology, is the use of a seed or catalyst
molecule that reduces an otherwise large kinetic barrier to nucleation. Here we
show how to program algorithmic self-assembly similarly, such that seeded
assembly proceeds quickly but there is an arbitrarily large kinetic barrier to
unseeded growth. We demonstrate this technique by introducing a family of tile
sets for which we rigorously prove that, under the right physical conditions,
linearly increasing the size of the tile set exponentially reduces the rate of
spurious nucleation. Simulations of these `zig-zag' tile sets suggest that
under plausible experimental conditions, it is possible to grow large seeded
crystals in just a few hours such that less than 1 percent of crystals are
spuriously nucleated. Simulation results also suggest that zig-zag tile sets
could be used for detection of single DNA strands. Together with prior work
showing that tile sets can be made robust to errors during properly initiated
growth, this work demonstrates that growth of objects via algorithmic
self-assembly can proceed both efficiently and with an arbitrarily low error
rate, even in a model where local growth rules are probabilistic.Comment: 37 pages, 14 figure
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Structure-based inhibitors of amyloid beta core suggest a common interface with tau.
Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline
Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches
Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.
Production and analysis of synthetic Cascade variants
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR assoziiert) ist ein
adaptives Immunsystem in Archaeen und Bakterien, das fremdes genetisches Material mit Hilfe von
Ribonukleoprotein-Komplexen erkennt und zerstört. Diese Komplexe bestehen aus einer CRISPR RNA
(crRNA) und Cas Proteinen. CRISPR-Cas Systeme sind in zwei Hauptklassen und mehrere Typen
unterteilt, abhängig von den beteiligten Cas Proteinen. In Typ I Systemen sucht ein Komplex namens
Cascade (CRISPR associated complex for antiviral defence) nach eingedrungener viraler DNA während
einer Folgeinfektion und bindet die zu der eingebauten crRNA komplementäre Sequenz. Anschließend
wird die Nuklease/Helikase Cas3 rekrutiert, welche die virale DNA degradiert (Interferenz).
Das Typ I System wird in mehrere Subtypen unterteilt, die Unterschiede im Aufbau von Cascade
vorweisen. Im Fokus dieser Arbeit steht eine minimale Cascade-Variante aus Shewanella putrefaciens
CN-32. Im Vergleich zur gut untersuchten Typ I-E Cascade aus Escherichia coli fehlen in diesem Komplex
zwei Untereinheiten, die gewöhnlicher Weise für die Zielerkennung benötigt werden. Dennoch ist der
Komplex aktiv. Rekombinante I-Fv Cascade wurde bereits aus E. coli aufgereinigt und es war möglich,
den Komplex zu modifizieren, indem das Rückgrat entweder verlängert oder verkürzt wurde. Dadurch
wurden synthetische Varianten mit veränderter Protein-Stöchiometrie erzeugt.
In der vorliegenden Arbeit wurde I-Fv Cascade weiter mit in vitro Methoden untersucht. So wurde die
Bindung von Ziel-DNA beobachtet und die 3D Struktur zeigt, dass strukturelle Veränderungen im
Komplex die fehlenden Untereinheiten ersetzen, möglicherweise um viralen Anti-CRISPR Proteinen zu
entgehen. Die Nuklease/Helikase dieses Systems, Cas2/3fv, ist eine Fusion des Cas3 Proteins mit dem
Interferenz-unabhängigen Protein Cas2. Ein unabhängiges Cas3fv ohne Cas2 Untereinheit wurde
aufgereinigt und in vitro Assays zeigten, dass dieses Protein sowohl freie ssDNA als auch Cascadegebundene Substrate degradiert. Das komplette Cas2/3fv Protein bildet einen Komplex mit dem Protein
Cas1 und zeigt eine reduzierte Aktivität gegenüber freier ssDNA, möglicherweise als
Regulationsmechanismus zur Vermeidung von unspezifischer Aktivität.
Weiterhin wurde ein Prozess namens „RNA wrapping“ etabliert. Synthetische Cascade-Komplexe
wurden erzeugt, in denen die grundlegende RNA-Bindung des charakteristischen Cas7fv RückgratProteins auf eine ausgewählte RNA gelenkt wird. Diese spezifische Komplexbildung kann in vivo durch
eine Repeat-Sequenz der crRNA stromaufwärts der Zielsequenz und durch Bindung des Cas5fv Proteins
initiiert werden. Die erzeugten Komplexe beinhalten die ersten 100 nt der markierten RNA, die
anschlieĂźend isoliert werden kann. Innerhalb der Komplexe ist die RNA stabilisiert und geschĂĽtzt vor
Degradation durch RNasen. Komplexbildung kann außerdem genutzt werden, um ReportergenTranskripte stillzulegen. Zusätzlich wurden erste Hinweise geliefert, dass das Rückgrat der synthetischen
Komplexe durch Fusion mit weiteren Reporterproteinen modifiziert werden kann.CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) is an
adaptive immune system of Archaea and Bacteria. It is able to target and destroy foreign genetic
material with ribonucleoprotein complexes consisting of CRISPR RNAs (crRNAs) and certain Cas proteins.
CRISPR-Cas systems are classified in two major classes and multiple types, according to the involved Cas
proteins. In type I systems, a ribonucleoprotein complex called Cascade (CRISPR associated complex for
antiviral defence) scans for invading viral DNA during a recurring infection and binds the sequence
complementary to the incorporated crRNA. After target recognition, the nuclease/helicase Cas3 is
recruited and subsequently destroys the viral DNA in a step termed interfere nce.
Multiple subtypes of type I exist that show differences in the Cascade composition. This work focuses on
a minimal Cascade variant found in Shewanella putrefaciens CN-32. In comparison to the well-studied
type I-E Cascade from Escherichia coli, this complex is missing two proteins usually required for target
recognition, yet it is still able to provide immunity. Recombinant I-Fv Cascade was previously purified
from E. coli and it was possible to modulate the complex by extending or shortening the backbone,
resulting in synthetic variants with altered protein stoichiometry.
In the present study, I-Fv Cascade was further analyzed by in vitro methods. Target binding was
observed and the 3D structure revealed structural variations that replace the missing subunits,
potentially to evade viral anti-CRISPR proteins. The nuclease/helicase of this system, Cas2/3fv, is a fusion
of the Cas3 protein with the interference-unrelated protein Cas2. A standalone Cas3fv was purified
without the Cas2 domain and in vitro cleavage assays showed that Cas3fv degrades both free ssDNA as
well as Cascade-bound substrates. The complete Cas2/3fv protein forms a complex with the protein
Cas1 and was shown to reduce cleave of free ssDNA, potentially as a regulatory mechanism against
unspecific cleavage.
Furthermore, we established a process termed “RNA wrapping”. Synthetic Cascade assemblies can be
created by directing the general RNA-binding ability of the characteristic Cas7fv backbone protein on an
RNA of choice such as reporter gene transcripts. Specific complex formation can be initiated in vivo by
including a repeat sequence from the crRNA upstream a given target sequence and binding of the
Cas5fv protein. The created complexes contain the initial 100 nt of the tagged RNA which can be
isolated afterwards. While incorporated in complexes, RNA is stabilized and protected from degradation
by RNases. Complex formation can be used to silence reporter gene transcripts. Furthermore, we
provided initial indications that the backbone of synthetic complexes can be modified by addition of
reporter proteins
The relationship between amyloid structure and cytotoxicity
Self-assembly of proteins and peptides into amyloid structures has been the subject of intense and focused research due to their association with neurodegenerative, age-related human diseases and transmissible prion diseases in humans and mammals. Of the disease associated amyloid assemblies, a diverse array of species, ranging from small oligomeric assembly intermediates to fibrillar structures, have been shown to have toxic potential. Equally, a range of species formed by the same disease associated amyloid sequences have been found to be relatively benign under comparable monomer equivalent concentrations and conditions. In recent years, an increasing number of functional amyloid systems have also been found. These developments show that not all amyloid structures are generically toxic to cells. Given these observations, it is important to understand why amyloid structures may encode such varied toxic potential despite sharing a common core molecular architecture. Here, we discuss possible links between different aspects of amyloidogenic structures and assembly mechanisms with their varied functional effects. We propose testable hypotheses for the relationship between amyloid structure and its toxic potential in the context of recent reports on amyloid sequence, structure, and toxicity relationships
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