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(a) A single a-HL pore inserted in a lipid membrane allows the flow of ions in response to an applied voltage. The ionic current of the open pore is shown below. (b) A double stranded DNA containing the target CTCTC-A 2 T 2-GAGAG binding site inserted in a hairpin followed by a ssDNA overhang (see the ESI † for the full sequence). This DNA is driven to the pore by the electric field. The threading, unzipping and translocation of the DNA causes the characteristic signal shown below. (c) When the trivalent peptide is bound to the DNA there is an additional step with the DNA-protein complex on top of the pore, which causes a new high-conductance level (c1). Once the protein is detached, the reaction proceeds as with free DNA (c2-3). Below is the ionic current signal when a protein-DNA complex is analyzed.
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![Fig. 3 Top: EMSA results for Hk-gaga. Panel (a), lanes 1-5: [Hk-gaga] ¼...](publication/324405481/figure/fig2/AS:613878610280448@1523371474011/Top-EMSA-results-for-Hk-gaga-Panel-a-lanes-1-5-Hk-gaga-14-0-300-500-700-and_Q320.jpg)
Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins tha...
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... of thermodynamic and kinetic parameters in the formation of protein-DNA complexes, 17 and to our knowledge, up to now it had not been used to characterize the DNA recognition of synthetic peptide binders. 9c Briey, it works by stochastically examining DNA states in the presence of a given amount of the peptide binder, as described in Fig. 5. Typically, by analyzing up to a hundred DNA molecules, the fraction of complexes can be determined (Fig. 6, le), and the K D deduced (Fig. 6, right). When analyzing the interaction of the trivalent chimera with its target ternary binding site ðCTCTC-A 2 T 2 -GAGAG Þ we calculated a K D of 120 AE 10 nM. This result is in reasonable ...
Citations
... The single CTD of MucR/Ros can bind target DNA in vitro (27,55). Several cases of DNA binding by single zinc-finger domain proteins from eukaryotes also have been reported (56,57), while in most cases, a minimum of two zinc-fingers is required for high-affinity DNA binding and a protein harboring tandemly repeated zincfingers (as many as 37) is common in eukaryotes (54,55,58). Recently, it was found that NTD is essential for in vitro self-association of MucR homologs from Mesorhizobium loti and Brucella abortus, resulting in high-order oligomerization of MucR (59,60). ...
Bacterial adaptation is largely shaped by horizontal gene transfer, xenogeneic silencing mediated by lineage-specific DNA bridgers (H-NS, Lsr2, MvaT and Rok), and various anti-silencing mechanisms. No xenogeneic silencing DNA bridger is known for α-proteobacteria, from which mitochondria evolved. By investigating α-proteobacterium Sinorhizobium fredii, a facultative legume microsymbiont, here we report the conserved zinc-finger bearing MucR as a novel xenogeneic silencing DNA bridger. Self-association mediated by its N-terminal domain (NTD) is required for DNA-MucR-DNA bridging complex formation, maximizing MucR stability, transcriptional silencing, and efficient symbiosis in legume nodules. Essential roles of NTD, CTD (C-terminal DNA-binding domain), or full-length MucR in symbiosis can be replaced by non-homologous NTD, CTD, or full-length protein of H-NS from γ-proteobacterium Escherichia coli, while NTD rather than CTD of Lsr2 from Gram-positive Mycobacterium tuberculosis can replace the corresponding domain of MucR in symbiosis. Chromatin immunoprecipitation sequencing reveals similar recruitment profiles of H-NS, MucR and various functional chimeric xenogeneic silencers across the multipartite genome of S. fredii, i.e. preferring AT-rich genomic islands and symbiosis plasmid with key symbiosis genes as shared targets. Collectively, the convergently evolved DNA bridger MucR predisposed α-proteobacteria to integrate AT-rich foreign DNA including symbiosis genes, horizontal transfer of which is strongly selected in nature.
... Single-stranded portions are formed at desired positions of both DNA strands, and various transformations selectively occur there due to differences in reactivity and other physicochemical properties. On this strategy, site-selective scission of human genome was accomplished by using Ce(IV)/EDTA as scissors which hydrolyze only ssDNA.27 ...
Peptide nucleic acid (PNA) is a DNA analog, in which the sugar-phosphate backbone in DNA is replaced by poly[N-(2-aminoethyl)glycine]. Since its discovery in the early 1990s, PNA has been widely employed in chemistry, biochemistry, medicine, nanotechnology, and many other fields. This account surveys recent developments on the design of PNA derivatives and their applications. In the first part, PNAs for sequence-specific recognition of DNA and RNA (single-strands, double-strands, G-quadruplexes, i-motifs, and others) are comprehensively covered. Modifications of nucleobases and of the main chain effectively promote both the strength of binding and the selectivity of recognition. In the second half of this account, practical applications of PNA are presented. Structural restraints, induced by complex formation of PNA with DNA and RNA substrates, lead to selective transformation of target sites to desired structures. Applications to regulation of gene expression, gene editing, construction of sophisticated nanostructures, and others are also described. Advantages and disadvantages of PNAs, compared with other sequence-recognizing molecules hitherto reported, are discussed in terms of various physicochemical and biological features.
... Among the metals of major importance for maintenance of basic activities in an organism, zinc is the second most abundant concerning the association with enzymes with known structures [1] as well as in total cellular distribution, surpassed only by iron [2]. This metal is known especially for its structural role in the regulation of gene transcription by the zinc fingers, considered as the largest group of transcription regulators [3][4][5]. In fungi, zinc binding to transcription factors and other proteins is quite similar, ranging from approximately 9% of the proteome in Aspergillus fumigatus and 7.5% in Saccharomyces cerevisiae [6]. ...
Zinc is an essential nutrient for all living organisms. However, firm regulation must be maintained since micronutrients also can be toxic in high concentrations. This notion is reinforced when we look at mechanisms deployed by our immune system, such as the use of chelators or membrane transporters that capture zinc, when threatened with pathogens, like fungi. Pathogenic fungi, on the other hand, also make use of a variety of transporters and specialized zinc captors to survive these changes. In this review, we sought to explain the mechanisms, grounded in experimental analysis and described to date, utilized by pathogenic fungi to maintain optimal zinc levels.
... Exploiting multivalent binding with polytopic ligands, Vázquez, Mascareñas, and co-workers reported the design of peptidic DNA binders that combine two modules of 'zinc finger' transcription factor (GAGA) and one peptide referred to as "AThook" (Figure 11). [59] This trimeric molecule binds DNA via "major-minor-major" groove interaction, which was shown to yield high affinity towards DNA and excellent sequenceselectivity. This 'multi-groove' binding approach is very appealing to build sophisticated DNA-templated assemblies, permitting the positioning of different units along a relatively large section of DNA. ...
... Reproduced with permission from reference. [59] Copyright 2018 The Royal Society of Chemistry. ...
DNA‐templated self‐assembly represents a rich and growing subset of supramolecular chemistry where functional self‐assemblies are programmed in a versatile manner using nucleic acids as readily‐available and readily‐tunable templates. In this review, we summarize the different DNA recognition modes and the basic supramolecular interactions at play in this context. We discuss the recent results that report the DNA‐templated self‐assembly of small molecules into complex yet precise nanoarrays, going from 1D to 3D architectures. Finally, we show their emerging functions as photonic/electronic nanowires, sensors, gene delivery vectors, and supramolecular catalysts, and their growing applications in a wide range of area from materials to biological sciences. Inspired by DNA: By making use of DNA as templates, supramolecular interactions can be exploited for generating complex yet precise nano‐arrays of small‐molecules. In this review, we summarize how this growing approach has been explored so far, present the emerging functions, and the wide applications of such self‐assemblies in materials and biological sciences.
... Beside the larger variety of pores employed (solidstate 14,26,27,36,50,65 or biological 3,48,52,62 ) and the number of different sensing strategies, there are three fundamental issues in nanopore based devices 19 : i) capture, the molecule in the bulk of the reservoir has to move in the pore region and engage the pore, ii) residence, the molecule has to reside into the pore for a time interval enough long to record a stable signal and iii) distinguishability, different molecules (or different conformation of the same molecule) have to leave different signal. ...
Nanopore based sensors constitute a promising approach to single molecule protein characterization being able, in principle, to detect sequences, structural elements and folding states of proteins and polypeptide chains. In narrow nanopores, one of the open issues concerns the coupling between unfolding and translocation. Here, we studied the Ubiquitin translocation in an α−Hemolysin nanopore, the most widely used pore for nanopore sensing, via all-atom molecular dynamics simulations. We completely characterize the co-translocational unfolding pathway finding that robust translocation intermediates are associated to rearrangement of secondary structural elements, as also confirmed by coarse grained simulations. An interesting recurrent pattern is the clogging of the α−Hemolysin constriction by a N-terminal β -hairpin. This region of Ubiquitin is the target of several post-translational modifications. We propose a strategy to detect post-translational modifications at N-terminal using α-Hemolysin nanopore based on the comparison of the co-translocational unfolding signals associated to modified and unmodified proteins.
... This is because stability of the folded structures needs to be maintained by fewer interactions, and also active sites are to be designed within a small structural fold. However, miniaturized proteins are of substantial importance in therapeutics and biotechnology [6][7][8][9] . ...
Nature has primarily exploited helical proteins, over β-sheets, for heme/multi-heme coordination. Understating of heme–protein structures has motivated the design of heme proteins utilizing coiled-coil helical structure. By contrast, de novo designed β-sheet proteins are less successful. However, designing proteins with discretely folded β-sheet structures encoding specific functions would have great potential for the development of new synthetic molecules e.g. enzymes, inhibitors. Here we report the design and characterization of multi-heme binding four-, six-, eight-, and twelve-stranded β-sheet mini-proteins (<40 amino acids) and proteins. Atomic-resolution structures demonstrate an expected β-sheet structural topology. The designed β-sheet mini-proteins pack or latch multiple hemes with high affnity in versatile orientations either by stacking or sideways, mimicking naturally occuring multi-heme protein conduits. The designed multi-stranded β-sheet heme proteins could serve as a platform for the generation of novel synthetic β-sheet protein mimics.
We report the modelling of the DNA complex of an artificial miniprotein composed of two zinc finger modules and an AT-hook linking peptide. The computational study provides for the first time a structural view of these types of complexes, dissecting interactions that are key to modulate their stability. The relevance of these interactions was validated experimentally. These results confirm the potential of this type of computational approach for studying peptide-DNA complexes and suggest that they could be very useful for the rational design of non-natural, DNA binding miniproteins.
ConspectusDNA is the molecule responsible for the storage and transmission of the genetic information in living organisms. The expression of this information is highly regulated. In eukaryotes, it is achieved mainly at the transcription level thanks to specialized proteins called transcription factors (TFs) that recognize specific DNA sequences, thereby promoting or inhibiting the transcription of particular genes. In many cases, TFs are present in the cell in an inactive form but become active in response to an external signal, which might modify their localization and DNA binding properties or modulate their interactions with the rest of the transcriptional machinery. As a result of the crucial role of TFs, the design of synthetic peptides or miniproteins that can emulate their DNA binding properties and eventually respond to external stimuli is of obvious interest. On the other hand, although the B-form double helix is the most common DNA secondary structure, it is not the only one with an essential biological function. Guanine quadruplexes (GQs) have received considerable attention due to their critical role in the regulation of gene expression, which is usually associated with a change in the GQ conformation. Thus, the development of GQ probes whose properties can be controlled using external signals is also of significant relevance.In this Account, we present a summary of the recent efforts toward the development of stimuli-responsive synthetic DNA binders with a particular emphasis on our own contributions. We first introduce the structure of B and GQ DNAs, and some of the main factors underlying their selective recognition. We then discuss some of the different approaches used for the design of stimulus-mediated DNA binders. We have organized our discussion according to whether the interaction takes place with duplex or guanine quadruplex DNAs, and each section is divided according to the nature of the stimulus (i.e., physical or chemical). Regarding physical stimuli, light (through the incorporation of photolabile protecting groups or photoisomerizable agents) is the most common input for the activation/deactivation of DNA binding events. With respect to chemical signals, the use of metals (through the incorporation of metal-coordinating groups in the DNA binding agent) has allowed the development of a wide range of stimuli-responsive DNA binders. More recently, redox-based systems have also been used to control DNA interactions.This Account ends with a "Conclusions and Outlook" section highlighting some of the general lessons that have been learned and future directions toward further advancing the field.
The nickel(II)‐mediated self‐assembly of a multimeric DNA binder is described. The binder is composed of two metal‐chelating peptides derived from a bZIP transcription factor (brHis2) and one short AT‐hook domain equipped with two bipyridine ligands (HkBpy2). These peptides reversibly assemble in the presence of NiII ions at selected DNA sequences of 13 base pairs.
Glass capillary-based nanopore is exploited for single-molecule conformational sensing of multi-arm DNA concatemers during translocation. Both translocation frequency and orientation preference were found related with the number of arms of the DNA concatemers.
