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Stimuli-Responsive DNA Binding by Synthetic Systems
Abstract
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.
... Moreover, it was demonstrated that the photoisomerization of azobenzenes can effectively be used to control peptide-DNA binding. [21][22][23] In these examples, only the Z-isomers of azobenzene-peptide conjugates offer the prerequisites for the peptides to bind to DNA allowing for a photocontrol of DNA-binding properties even in living cells. [22,23] Furthermore, additional photolabile groups were introduced that turn a non-binding peptide into a DNA-binding one upon the photoinduced release of an oligodeoxynucleotide (ODN) that initially blocked the DNAbinding sequence of the peptide. ...
... [21][22][23] In these examples, only the Z-isomers of azobenzene-peptide conjugates offer the prerequisites for the peptides to bind to DNA allowing for a photocontrol of DNA-binding properties even in living cells. [22,23] Furthermore, additional photolabile groups were introduced that turn a non-binding peptide into a DNA-binding one upon the photoinduced release of an oligodeoxynucleotide (ODN) that initially blocked the DNAbinding sequence of the peptide. [22,23] Likewise, the photoinduced activation of DNA-binding ligands may also be accomplished with metal-ligand complexes. ...
... [22,23] Furthermore, additional photolabile groups were introduced that turn a non-binding peptide into a DNA-binding one upon the photoinduced release of an oligodeoxynucleotide (ODN) that initially blocked the DNAbinding sequence of the peptide. [22,23] Likewise, the photoinduced activation of DNA-binding ligands may also be accomplished with metal-ligand complexes. [24] In particular, according to the general concept of photoinduced fragmentation reactions to release bioactive fragments [25] the DNAbinding properties of sterically demanding metal complexes can be changed by photoinduced exchange or displacement of the complex ligands. ...
In the current field of photopharmacology, molecular photoswitches are applied whose interactions with DNA can be triggered or controlled by light. And although several photochromic reactions have been shown to serve this purpose well, the reversible photocycloaddition and photocycloreversion reactions have been largely neglected. This absence of research is surprising because especially the photodimerization of a DNA ligand leads to products with significant change of the size and shape which, in turn, leads to strongly diminished or even suppressed DNA association. Therefore, photocycloaddition–cycloreversion sequences have a huge potential for the photoinduced, reversible deactivation and activation of ligand–DNA interactions, as will be shown with selected examples in this Concept Article. Specifically, heterostyryl and ‐stilbene derivatives are presented whose DNA–binding properties are efficiently switched in reversible [2+2] photocycloaddition reactions. In addition, the photocontrolled DNA–binding of anthracene derivatives and their heterocyclic benzo[b]quinolizinium analogues in a [4+4] photocycloaddition, as well as the use of this reaction as part of dual–mode switches in combination with redox‐active functionalities, are highlighted. Furthermore, examples of conjugates are provided, in which the photochromic unit is bound covalently to nucleic acids or proteins, such that the photocycloaddition reaction can be used for reversible photoinduced crosslinking, ligation, or inhibition of gene expression.
... [4,[18][19][20] Another strategy to increase the selectivity of DNA-binding ligands has been realized with photo-active compounds, whose association with the target DNA can be switched on or off by light. [21][22][23][24][25][26] In these cases, a substrate, which does not bind to DNA, may be delivered close to the nucleic acid without an effect, whereas the DNA-binding ligand is formed directly upon irradiation. As this reaction is exclusively stimulated by light it can be applied with high local and temporal control and, therefore, offers a promising basis for the development of externally controllable DNA-targeting drugs. ...
... As this reaction is exclusively stimulated by light it can be applied with high local and temporal control and, therefore, offers a promising basis for the development of externally controllable DNA-targeting drugs. [27] And although this concept has been applied to regular DNA and noncanonical DNA, such as quadruplex DNA, [23,28] photo-controllable ligands for AP-DNA have not been reported, so far. To close this gap, we aimed at the design of photo-controllable AP-DNA ligands by the combination of a photoactive precursor with a sterically demanding substituent. ...
The photocyclization reaction of sterically demanding styrylpyridine derivatives was investigated and shown to depend on the type of substituent. With this method, a 2,2‐diphenyl‐1,3‐benzodioxolo‐annelated benzo[c]quinolizinium was synthesized, and its association with regular and abasic site‐containing DNA (AP‐DNA) was investigated by absorption, fluorescence, circular and linear dichroism spectroscopy. Specifically, this ligand binds preferentially to AP‐DNA relative to regular duplex DNA, and the AP‐DNA/ligand complex is formed in situ upon irradiation of the styrylpyridine substrate in the presence of the DNA.
... These regulators' specific binding to their respective promoters responds directly to metal ion levels, ensuring that the expression of genes is carefully adjusted in accordance with the concentration of metal ions present. This specificity is achieved by the recognition of metal-responsive motifs in the DNA and the subsequent alteration in the accessibility of the promoter [104,105]. ...
Heavy metal pollution from industrial activities and poor waste disposal poses significant environmental and health threats to humans and animals. This calls for sustainable approaches to the cleanup of heavy metals. This review explores metal tolerance mechanisms of bacteria such as the formation of biofilms, efflux systems, and enzymatic detoxification. These mechanisms allow bacteria communities to adapt and survive in contaminated environments. These adaptations are enhanced by mutations in the bacteria genes and by horizontal gene transfers, enabling bacteria species to survive under environmental stress while simultaneously contributing to nutrient cycling and the decomposition of organic matter. This review further explores the symbiotic interactions between bacteria, plants, and animals. These relationships enhance the metal tolerance ability of the different living organisms involved and are also very important in the bioremediation and phytoremediation of heavy metals. Plant growth-promoting rhizobacteria, Rhizobium, and Bacillus species are very important contributors to phytoremediation; they improve heavy metal uptake, improve the growth of roots, and plants resilience to stress. Moreover, this review highlights the importance of genetically engineered bacteria in closed-loop systems for optimized metal recovery. This offers environmentally friendly and sustainable options to the traditional remediation methods. Engineered Cupriavidus metallidurans CH34 and Pseudomonas putida strain 15420352 overexpressing metallothioneins have shown enhanced metal-binding capabilities, which makes them very effective in the treatment of industrial wastewaters and in biosorption applications. The use of engineered bacteria for the cleanup of heavy metals in closed-loop systems promotes the idea of a circular economy by recycling metals, thus reducing environmental waste. Multidisciplinary research that integrates synthetic biology, microbial ecology, and environmental science is very important for the advancement of metal bioremediation technologies. This review’s analysis on bacterial metal tolerance, symbiosis, and bioengineering strategies offers a pathway to effective bioremediation options, for the reclamation of heavy metal-polluted environments while promoting sustainable environmental practices.
... Chemoselective reactions, which can be controlled by an external stimulus, are a powerful tool for applications in synthesis, catalysis, drug release, protein immobilization, molecular recognition, and molecular machines. [1][2][3][4][5][6][7][8] This becomes more apparent when the stimulus has to counteract an overwhelming difference in reactivities of the competing reaction centers. By virtue of such stimuli-controlled chemoselective reactions, donor/acceptor (DA) interactions within dyads can be controlled by targeting the two units individually. ...
We present the design of an anthracenyl–naphthyl (ANT‐NAPH) dyad and its application as a luminescent 4‐stage photo switch. Both segments can individually react with singlet oxygen to switch off an optical response. In their initial form the larger ANT component reacts significantly faster and thus an ANTO2‐NAPH stage is turned on, observed by optical response of the remaining NAPH. To reduce its reactivity, ANT is substituted with two pyridine rings. This concept is first investigated and quantified on ANT and NAPH as separated molecules. Upon protonation the reaction of ANT becomes significantly slower. For the three possible pyridyl isomers this effect increases along the order meta<para<ortho. With the pyridyl nitrogen in ortho position the reaction completely toggles from ANT to NAPH. Application of this concept on the dyad allows to turn on the ANT‐NAPHO2 stage with optical response of the remaining ANT. The sequence of protonation‐oxygenation‐neutralization is thereby the only possible way to isolate the unfavored form ANT‐NAPHO2. In the dyad ANT and NAPH are directly attached and their coupling constitutes a non‐oxygenated third stage, where the NAPH luminescence is quenched and ANT luminescence is enhanced. Reaction of both NAPH and ANT to ANTO2‐NAPHO2 constitutes the fourth dark stage.
... Notably, the use of light for the activation of photo-controllable DNA ligands offers several advantages because it is easy to apply, traceless, and non-invasive [19]. As a result, several photoactive compounds have been developed, whose DNAbinding properties can be efficiently switched on and off by light [14,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]. ...
The photoreactions of selected styrylpyridine derivatives to the corresponding benzo[ c ]quinolizinium ions are described. It is shown that these reactions are more efficient in aqueous solution (97–44%) than in organic solvents (78–20% in MeCN). The quinolizinium derivatives bind to DNA by intercalation with binding constants of 6–11 × 10 ⁴ M ⁻¹ , as shown by photometric and fluorimetric titrations as well as by CD- and LD-spectroscopic analyses. These ligand–DNA complexes can also be established in situ upon irradiation of the styrylpyridines and formation of the intercalator directly in the presence of DNA. In addition to the DNA-binding properties, the tested benzo[ c ]quinolizinium derivatives also operate as photosensitizers, which induce DNA damage at relative low concentrations and short irradiation times, even under anaerobic conditions. Investigations of the mechanism of the DNA damage revealed the involvement of intermediate hydroxyl radicals and C-centered radicals. Under aerobic conditions, singlet oxygen only contributes to marginal extent to the DNA damage.
... Several stimuli have been explored in the context of DNAstructure regulation and DNA hybrid architectures. 35,[47][48][49][50][51] Within these stimuli, light stands out due the traceless nature of the photon as a reactant, its spatiotemporal precise dosing and the high tunability of its energy and intensity. 51 In this way, the structure 52-58 and function 57,59-63 of DNA and ribonucleic acid (RNA) could be controlled by light. ...
Molecular recognition-driven self-assembly employing single-stranded DNA (ssDNA) as a template is a promising approach to access complex architectures from simple building blocks. Oligonucleotide-based nanotechnology and soft-materials benefit from the high information storage density, self-correction, and memory function of DNA. Here we control these beneficial properties with light in a photoresponsive biohybrid hydrogel, adding an extra level of function to the system. An ssDNA template was combined with a complementary photo-responsive unit to reversibly switch between various functional states of the supramolecular assembly using a combination of light and heat. We studied the structural response of the hydrogel at both the microscopic and macroscopic scale using a combination of UV-vis absorption and CD spectroscopy, as well as fluorescence, transmission electron, and atomic force microscopy. The hydrogels grown from these supramolecular self-assembly systems show remarkable shape-memory properties and imprinting shape-behavior while the macroscopic shape of the materials obtained can be further manipulated by irradiation.
... 6,33−35 In this context, stimuli-responsive ligands have emerged involving chemical/supramolecular triggers, although those will not be discussed in the context of this perspective, and also light. 36,37 Light offers several advantages over chemical stimuli for the regulation of G4 formation/topology in vivo as a potential therapeutic strategy or in the regulation of the G4 structure in order to generate a mechanical or spectroscopic output that can form the basis of a functional system. Unlike chemical stimuli, light can be delivered with much higher spatiotemporal precision than chemical "fuels" by controlling the wavelength, intensity, or irradiation time. ...
G-quadruplex (G4) oligonucleotide secondary structures have recently attracted significant attention as therapeutic targets owing to their occurrence in human oncogene promoter sequences and the genome of pathogenic organisms. G4s also demonstrate interesting catalytic activities in their own right, as well as the ability to act as scaffolds for the development of DNA-based materials and nanodevices. Owing to this diverse range of opportunities to exploit G4 in a variety of applications, several strategies to control G4 structure and function have emerged. Interrogating the role of G4s in biology requires the delivery of small-molecule ligands that promote its formation under physiological conditions, while exploiting G4 in the development of responsive nanodevices is normally achieved by the addition and sequestration of the metal ions required for the stabilization of the folded structure. Although these strategies prove successful, neither allows the system in question to be controlled externally. Meanwhile, light has proven to be an attractive means for the control of DNA-based systems as it is noninvasive, can be delivered with high spatiotemporal precision, and is orthogonal to many chemical and biological processes. A plethora of photoresponsive DNA systems have been reported to date; however, the vast majority deploy photoreactive moieties to control the stability and assembly of duplex DNA hybrids. Despite the unique opportunities afforded by the regulation of G-quadruplex formation in biology, catalysis, and nanotechnology, comparatively little attention has been devoted to the design of photoresponsive G4-based systems. In this Perspective, we consider the potential of photoresponsive G4 assemblies and examine the strategies that may be used to engineer these systems toward a variety of applications. Through an overview of the main developments in the field to date, we highlight recent progress made toward this exciting goal and the emerging opportunities that remain ripe for further exploration in the coming years.
... A promising strategy is the development of new stimuli-responsive nucleic acid binders in which a given stimulus promotes or hinders the nucleic acid binding to the molecule and, therefore, modulates the biological activity associated with the nucleic acid metabolism. Mascareñas, Vazquez, and co-workers have finely designed several stimuli-responsive systems based mainly on synthetic polypeptide helices that bind to DNA upon light irradiation and metal coordination and are compiled in a recent review [8]. Among the triggering stimuli, metal coordination is one of the most challenging strategies due to the abundance of metal ions in cells that can occupy the coordination sites of the synthetic systems and hamper the envisaged DNA interaction process. ...
Nucleic acids are essential biomolecules in living systems and represent one of the main targets of chemists, biophysics, biologists, and nanotechnologists. New small molecules are continuously developed to target the duplex (ds) structure of DNA and, most recently, RNA to be used as therapeutics and/or biological tools. Stimuli-triggered systems can promote and hamper the interaction to biomolecules through external stimuli such as light and metal coordination. In this work, we report on the interaction with ds-DNA and ds-RNA of two aza-macrocycles able to coordinate Zn²⁺ metal ions and form binuclear complexes. The interaction of the aza-macrocycles and the Zn²⁺ metal complexes with duplex DNA and RNA was studied using UV thermal and fluorescence indicator displacement assays in combination with theoretical studies. Both ligands show a high affinity for ds-DNA/RNA and selectivity for ds-RNA. The ability to interact with these duplexes is blocked upon Zn²⁺ coordination, which was confirmed by the low variation in the melting temperature and poor displacement of the fluorescent dye from the ds-DNA/RNA. Cell viability assays show a decrease in the cytotoxicity of the metal complexes in comparison with the free ligands, which can be associated with the observed binding to the nucleic acids.
Current anticancer therapies suffer from issues such as off‐target side effects and the emergence of drug resistance; therefore, the discovery of alternative therapeutic approaches is vital. These can include the development of drugs with different modes of action, and the exploration of new biomolecular targets. For the former, there has been increasing interest in drugs that are activated by an external stimulus (e. g. light irradiation) to generate cytotoxic chemicals such as reactive oxygen species (ROS). For the latter, significant efforts are being directed to explore non‐canonical DNA and RNA structures (e. g. guanine‐quadruplexes), as alternative biomolecular targets. Herein we report the synthesis of a library of 21 new platinum(II)‐Salphen complexes (square planar platinum(II) complexes coordinated to tetradentate O,N,N,O‐Schiff base ligands), and the investigation, for all complexes, of their photophysical and photochemical properties, their interactions with duplex and quadruplex DNA, and their cytotoxicity against HeLa cancer cells both in the dark and upon light irradiation. Thanks to the intrinsic phosphorescence of the platinum(II) complexes, confocal microscopy was used for six of the complexes to determine their cellular permeability and localisation in two cancer cell lines (HeLa and U2OS). Altogether, these studies have allowed us to identify two lead platinum(II) complexes with high guanine‐quadruplex DNA affinity and selectivity, good cell permeability and nuclear localisation, and high cytotoxicity against HeLa cancer cells upon irradiation with no detected cytotoxicity in the dark.
We present the design of an anthracenyl⎯naphthyl (ANT⎯NAPH) dyad and its application as a luminescent 4‐stage photo switch. Both segments can individually react with singlet oxygen to switch off an optical response. In their initial form the larger ANT component reacts significantly faster and thus an ANTO2⎯NAPH stage is turned on, observed by optical response of the remaining NAPH. To reduce its reactivity, ANT is substituted with two pyridine rings. This concept is first investigated and quantified on ANT and NAPH as separated molecules. Upon protonation the reaction of ANT becomes significantly slower. For the three possible pyridyl isomers this effect increases along the order meta<para<ortho. With the pyridyl nitrogen in ortho position, the reaction completely toggles from ANT to NAPH. Application of this concept on the dyad allows to turn on the ANT⎯NAPHO2 stage with optical response of the remaining ANT. The sequence of protonation‐oxygenation‐neutralization is thereby the only possible way to isolate the unfavored form ANT⎯NAPHO2. In the dyad ANT and NAPH are directly attached and their coupling constitutes a non‐oxygenated third stage, where the NAPH luminescence is quenched and ANT luminescence is enhanced. Reaction of both NAPH and ANT to ANTO2⎯NAPHO2 constitutes the fourth dark stage.
Peptides work as both functional molecules to modulate various biological phenomena and self-assembling artificial materials. The introduction of photoresponsive units to peptides allows the spatiotemporal remote control of their structure and function upon light irradiation. This article overviews the photoresponsive peptide design, interaction with biomolecules, and applications in self-assembling materials over the last 30 years. Peptides modified with photochromic (photoisomerizable) molecules, such as azobenzene and spiropyran, reversibly photo-controlled the binding to biomolecules and nanostructure formation through self-assembly. Photocleavable molecular units irreversibly control the functions of peptides through cleavage of the main chain and deprotection by light. Photocrosslinking between peptides or between peptides and other biomolecules enhances the structural stability of peptide assemblies and complexes. These photoresponsive peptides spatiotemporally controlled the formation and dissociation of peptide assemblies, gene expressions, protein–drug interactions, protein–protein interactions, liposome deformation and motility, cytoskeleton structure and stability, and cell functions by appropriate light irradiation. These molecular systems can be applied to photo-control biological functions, molecular robots, artificial cells, and next-generation smart drug delivery materials.
A disulfide-functionalized photoactive DNA ligand is presented that enables the control of its DNA-binding properties by a combination of a photocycloaddition reaction and the redox reactivity of the sulfide/disulfide functionalities. In particular, the initially applied ligand binds to DNA by a combination of intercalation and groove-binding of separate benzo[b]quinolizinium units. The association to DNA is interrupted by an intramolecular [4 + 4] photocycloaddition to the non-binding head-to-head cyclomers. In turn, the subsequent cleavage of these cyclomers with dithiothreitol (DTT) regains temporarily a DNA-intercalating benzoquinolizinium ligand that is eventually converted into a non-binding benzothiophene. As a special feature, this sequence of controlled deactivation, recovery and internal shut-off of DNA-binding properties can be performed directly in the presence of DNA.
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.
A disulfide-functionalized bis-benzo[b]quinolizinium is presented that is transformed quantitatively into its cyclomers in a fast intramolecular [4 + 4] photocycloaddition. Both the bis-quinolizinium and the photocyclomers react with glutathione (GSH) or dithiothreitol (DTT) to give 9-(sulfanylmethyl)benzo[b]quinolizinium as the only product. As all components of this reaction sequence have different DNA-binding properties, it enables the external control and switching of DNA association. Hence, the bis-benzo[b]quinolizinium binds strongly to DNA and is deactivated upon photocycloaddition to the non-binding cyclomers. In turn, the subsequent cleavage of the cyclomers with DTT regains a DNA-intercalating benzoquinolizinium ligand. Notably, this sequence of controlled deactivation and recovery of DNA-binding properties can be performed directly in the presence of DNA.
The understanding of sequence-specific DNA minor groove interactions has recently made major steps forward and as a result, the goal of development of compounds that target the minor groove is an active research area. In an effort to develop biologically active minor groove agents, we are preparing and exploring the DNA interactions of diverse diamidine derivatives with a 5'-GAATTC-3' binding site using a powerful array of methods including, biosensor-SPR methods, and X-ray crystallography. The benzimidazole-thiophene module provides an excellent minor groove recognition component. A central thiophene in a benzimidazole-thiophene-phenyl aromatic system provides essentially optimum curvature for matching the shape of the minor groove. Comparison of that structure to one with the benzimidazole replaced with an indole shows that the two structures are very similar, but have some interesting and important differences in electrostatic potential maps, the DNA minor groove binding structure based on x-ray crystallographic analysis, and inhibition of the major groove binding PU.1 transcription factor complex. The binding KD for both compounds is under 10 nM and both form amidine H-bonds to DNA bases. They both have bifurcated H-bonds from the benzimidazole or indole groups to bases at the center of the -AATT- binding site. Analysis of the comparative results provides an excellent understanding of how thiophene compounds recognize the minor groove and can act as transcription factor inhibitors.
Herein, we describe an approach for the on-demand disassembly of dimeric peptides using a palladium-mediated cleavage of a designed self-immolative linker. The utility of the strategy is demonstrated for the case of dimeric basic regions of bZIP transcription factors. While the dimer binds designed DNA sequences with good affinities, the peptide-DNA complex can be readily dismounted by addition of palladium reagents that trigger the cleavage of the spacer, and the release of unfunctional monomeric peptides.
RNA is an emerging drug target that opens new perspectives in the treatment of viral and bacterial infections, cancer and a range of so far incurable genetic diseases. Among the various strategies towards the design and development of selective and efficient ligands for targeting and detection of therapeutically relevant RNA, photoswitchable RNA binders represent a very promising approach due to the possibility to control the ligand-RNA and protein-RNA interactions by light with high spatiotemporal resolution. However, the field of photoswitchable RNA binders still remains underexplored due to challenging design of lead structures that should combine high RNA binding selectivity with efficient photochemical performance. The aim of this highlight article is to describe the development of photoswitchable noncovalent RNA binders and to outline the current situation and perspectives of this emerging interdisciplinary field.
Short, complementary DNA single strands with mismatched base pairs cannot undergo spontaneous formation of duplex DNA (dsDNA). Mismatch binding ligands (MBLs) can compensate this effect, inducing the formation of the double helix and thereby acting as a molecular glue. Here, we present the rational design of photoswitchable MBLs that allow for reversible dsDNA assembly by light. Careful choice of the azobenzene core structure results in excellent band separation of the E and Z isomers of the involved chromophores. This effect allows for efficient use of light as an external control element for duplex DNA formation and for an in-depth study of the DNA-ligand interaction by UV-Vis, SPR, and CD spectroscopy, revealing a tight mutual interaction and complementarity between the photoswitchable ligand and the mismatched DNA. We also show that the configuration of the switch reversibly dictates the conformation of the DNA strands, while the dsDNA serves as a chiral clamp and translates its chiral information onto the ligand inducing a preference in helical chirality of the Z isomer of the MBLs.
DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In this Review, we first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. We then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, we consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, we discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases. G-quadruplexes (G4s) are structures formed in guanine-rich DNA or RNA, which are linked to transcription, translation, chromatin biology, genome instability and RNA modifications. Recent studies connect G4 formation with cancer-cell lethality and indicate that G4s could be therapeutic targets.
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.
The generation of catalytically active metalloproteins inside living mammalian cells is a major research challenge at the interface between catalysis and cell biology. Herein we demonstrate that basic domains of bZIP transcription factors, mutated to include two histidine residues at i and i+4 positions, react with palladium(II) sources to generate catalytically active, stapled pallado‐miniproteins. The resulting constrained peptides are efficiently internalized into living mammalian cells, where they perform palladium‐promoted depropargylation reactions without cellular fixation. Control experiments confirm the requirement of the peptide scaffolding and the palladium staple for attaining the intracellular reactivity.
Molecular motors that operate with high efficiency using visible light are attractive for numerous applications. Here the synthesis and characterisation of three novel visible light switchable 2nd generation molecular motors is presented. Two of them are based on push-pull systems with the third one possessing an extended π-system. With a maximum effective excitation wavelength of 530 nm we designed the most red-shifted artificial rotary motor known to date. All three motors benefit from efficient switching to the metastable isomer, high quantum yields and excellent photostability setting them apart from visible light switchable motors reported previously. The activation barriers of the rate-limiting thermal helix inversion could be accurately predicted using DFT calculations and differences between the motors can be explained by distinct transition state structures. Enantiomers of push-pull motors were successfully separated and their helical twisting power in E7 liquid crystals was determined.
In this Minireview, we highlight recent advances in the design of transition metal complexes for photodynamic therapy (PDT) and photoactivated chemotherapy (PACT), and discuss the challenges and opportunities for the translation of such agents into clinical use. New designs for light‐activated transition metal complexes offer photoactivatable prodrugs with novel targeted mechanisms of action. Light irradiation can provide spatial and temporal control of drug activation, increasing selectivity and reducing side‐effects. The photophysical and photochemical properties of transition metal complexes can be controlled by the appropriate choice of the metal, its oxidation state, the number and types of ligands, and the coordination geometry.
G‐quadruplexes (G4) are four‐stranded structures formed from guanine‐rich oligonucleotides. Their defined 3D structures and polymorphic nature set them apart from classical nucleic acid morphology and suggest a range of potential applications in the development of functional materials. Meanwhile, the occurrence of G4 across the genomes of animals, plants and pathogens suggests roles for these structures in biology that may be exploited for therapeutic effect. Hundreds of G4 ligands are reported to bind these sequences with high specificity and affinity, but such ligands can also be engineered to do more than simply associate with G4 in a straightforward host‐guest fashion. Ligands have been developed that can switch G4 topology, direct the selective covalent modification of nucleic acid structures, or respond to external stimuli to permit spatiotemporal control of their activity. Herein we survey the main themes of such “value‐added” G4 ligands and consider the opportunities and challenges of their potential applications.
DNA–peptide interactions are involved in key life processes, including DNA recognition, replication, transcription, repair, organization, and modification. Development of tools that can influence DNA–peptide binding non‐invasively with high spatiotemporal precision could aid in determining its role in cells and tissues. Here, the design, synthesis, and study of photocontrolled tools for sequence‐specific small peptide‐DNA major and minor groove interactions are reported, shedding light on DNA binding by transcriptionally active peptides. In particular, photoswitchable moieties were implemented in the peptide backbone or turn region. In each case, DNA binding was affected by photochemical isomerization, as determined in fluorescent displacement assays on model DNA strands, which provides promising tools for DNA modulation.
The polymorphic nature of G‐quadruplex (G4) DNA structures points to a range of potential applications in nanodevices and an opportunity to control G4 in biological settings. Light is an attractive means for the regulation of oligonucleotide structure as it can be delivered with high spatiotemporal precision. However, surprisingly little attention has been devoted towards the development of ligands for G4 that allow photoregulation of G4 folding. We report a novel G4‐binding chemotype derived from stiff‐stilbene. Surprisingly however, whilst the ligand induces high stabilization in the potassium form of human telomeric DNA, it causes the unfolding of the same G4 sequence in sodium buffer. This effect can be reversed on demand by irradiation with 400 nm light through deactivation of the ligand by photo‐oxidation. By fuelling the system with the photolabile ligand, the conformation of G4 DNA was switched five times.
PROTEIN-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix–turn–helix1 or the zinc-finger2. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element—the leucine-zipper—and an adjoining basic region which mediates DNA binding3. Here we describe circular dichroism and 1NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN44,5. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent α-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide6. The GCN4-p dimer is ∼70% helical at 25 °C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the α-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial α-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high α-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 °C and that its folded, α-helical conformation is stabilized by binding to DNA.
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 that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction.
A fragment of the DNA basic region (br) of the GCN4 bZIP transcription factor has been modified to include two His residues at designed i, and i+4 positions of its N-terminus. The resulting monomeric peptide (brHis2) does not bind to its consensus target DNA site (5’-GTCAT-3’). However, addition of cis-Pd(en)Cl2 promotes a high-affinity interaction with exquisite selectivity for this sequence. The peptide-DNA complex is disassembled by addition of a slight excess of a palladium chelator, and the interaction can be reversibly switched multiple times by playing with controlled amounts of either the metal complex or the chelator. Importantly, while the peptide brHis2 fails to translocate across cell membranes on its own, addition of the palladium reagent induces an efficient cell internalization of this peptide. In short, we report 1) a designed, short peptide that displays highly selective, major groove DNA binding, 2) a reversible, metal-dependent DNA interaction, 3) a metal-promoted cell internalization of this basic peptide.
Guanine quadruplexes (GQs) are compact four-stranded DNA structures that play a key role in the control of a variety of biological processes, including gene transcription. Bulky ruthenium complexes featuring a bipyridine, a terpyridine, and one exchangeable ligand ([Ru(terpy)(bpy)X](n+) ) are able to metalate exposed guanines present in the GQ of the c-MYC promoter region that are not involved in quadruplex base pairing. qRT-PCR and western-blot experiments indicated that the complexes promote a remarkable increase in the expression of this oncogene. We also show that exchangeable thioether ligands (X=RSR', Met) allow regulation of the metalating activity of the complex with visible light.
Given the prevalent role of α-helical motifs on protein surfaces in mediating protein-protein and protein-DNA interactions, there have been significant efforts to develop strategies to induce α-helicity in short, unstructured peptides to interrogate such interactions. Toward this goal, we have recently introduced hybrid metal coordination motifs (HCMs). HCMs combine a natural metal-binding amino acid side chain and a synthetic chelating group that are appropriately positioned in a peptide sequence to stabilize an α-helical conformation upon metal coordination. Here, we present a series of short peptides modified with HCMs consisting of a His and a phenanthroline group at i and i + 7 positions that can induce α-helicity in a metal-tunable fashion as well as direct the formation of discrete dimeric architectures for recognition of biological targets. We show that the induction of α-helicity can be further modulated by secondary sphere interactions between amino acids at the i + 4 position and the HCM. A frequently cited drawback of the use of peptides as therapeutics is their propensity to be quickly digested by proteases; here, we observe an enhancement of up to ∼100-fold in the half-lives of the metal-bound HCM-peptides in the presence of trypsin. Finally, we show that an HCM-bearing peptide sequence, which contains the DNA-recognition domain of a bZIP protein but is devoid of the obligate dimerization domain, can dimerize with the proper geometry and in an α-helical conformation to bind a cognate DNA sequence with high affinities (Kd ≥ 65 nM), again in a metal-tunable manner.
We describe the synthesis of designed peptidic modules that self-assemble in specific DNA sequences of 12 base pairs in the presence of Ni(II) salts. The modules consist of modified fragments of transcription factors that have been appropriately engineered to include metal-chelating His and bipyridine ligands.
We report the rational design of a DNA-binding peptide construct composed of the DNA-contacting regions of two transcription factors (GCN4 and GAGA) linked through an AT-hook DNA anchor. The resulting chimera, which represents a new, non-natural DNA binding motif, binds with high affinity and selectivity to a long composite sequence of 13 base pairs (TCAT-AATT-GAGAG).
We have synthesized oligoarginine conjugates of selected DNA-binding agents (a bisbenzamidine, acridine, and thiazole orange), and demonstrated that their DNA binding and cell internalization can be inhibited by appending a negatively charged oligoglutamic tail through a photolabile linker. Irradiation with UV light releases the parent octaarginine conjugates, thus restoring their cell internalization and biological activity. Preliminary assays using zebrafish embryos demonstrates the potential of this prodrug strategy for controlling in vivo cytotoxicity.
We report the development of chimeric DNA binding peptides comprised of the DNA binding fragment of natural transcription factors (basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif. The resulting peptide conjugates display high DNA affinity and excellent sequence selectivity. Furthermore, the the AT-Hook motif also favors the cell internalization of the conjugates.
The unprecedented use of anthracene photodimerization within a protein or peptide system is explored through its incorporation into a DNA-binding peptide, derived from the GCN4 transcription factor. This study demonstrates an effective and dynamic interplay between a photoreaction and a peptide-DNA assembly, with each process able to exert control over the other.
The basic DNA recognition region of the GCN4 protein comprising 23 amino acids has been modified to contain two optimally positioned cysteines which have been linked and stapled using cross-linkers of suitable lengths. This results in stapled peptides with a stabilized α-helical conformation which allows for DNA binding and concurrent enhancement of cellular uptake.
We report a light-sensitive histidine building block for Fmoc/tBu solid-phase peptide synthesis in which the imidazole side chain is coordinated to a ruthenium complex. We have applied this building block for the synthesis of caged-histidine peptides that can be readily deprotected by irradiation with visible light, and demonstrated the application of this approach for the photocontrol of the activity of Ni(II)-dependent peptide nucleases.
We report a photolabile biselectrophilic Ru(ii) complex that can be used for homo- or heterodimerization of cysteine-containing peptides. The resulting dimers can be efficiently disassembled by long-wavelength light. As proof-of-concept, we describe the preparation of homo- and heterodimeric bZIP peptides whose DNA-binding properties can be turned off using visible light.
We report the selective modification of cysteine residues engineered in peptides that have two additional cysteine residues as part of a Cys2His2 zinc finger motif. The chemoselective modification is achieved, thanks to the protecting effect exerted by the zinc cation upon coordination with the native cysteines and histidines of the zinc-finger fold. The strategy allows a straightforward synthesis of DNA binding zinc finger constructs.
Introduction:
DNA G-quadruplexes are four-stranded DNA structures and are widely distributed in functional regions of the human genome, such as telomeres and gene promoter regions. G-quadruplex structures can play important roles, including in immunoglobulin gene rearrangements, DNA replication, gene transcription, and are viewed as valid therapeutic targets in human cancer diseases. Design of G-quadruplex binders that target these structures and regulate related gene functions through stabilization of these structures are emerging as an exciting new class of potential anticancer agents. Besides as drug candidate, DNA G-quadruplex binders can also serve as excellent probes, helping the further exploration of biological functions of G-quadruplex and early diagnosis of G-quadruplex-related disease.
Areas covered:
This review provides an overview of current knowledge on patents of DNA G-quadruplex binders from 2008 to 2013. Information is collected from an extensive search, covering Derwent Innovations Index, Espacenet, SciFinder, and Google patent search.
Expert opinion:
With the accumulating evidence of G-quadruplex as an effective drug target, an increasing number of DNA G-quadruplex binders with diverse structural features were developed. These binders are either used as drug candidates targeting G-quadruplex, or as probes for diagnostic purpose in genomic study. This review would mainly focus on the patents published after 2008 (including 2008). In order to cover all the diverse structural types of DNA G-quadruplex binders, some patents published before 2008 would be mentioned as well.
The use of a caged G-quadruplex ligand allows for transcriptional control of quadruplex-containing genes using UV light as an external trigger. An important oncogene, SRC, involved in the initiation and proliferation of epithelial tumours is shown to be significantly downregulated in cells treated by the caged ligand in synergy with UV light treatment.
One of the strategies used by nature to regulate gene expression relies on the stimuli-controlled combination of DNA-binding proteins. This in turn determines the target-binding site within the genome, and thereby whether a particular gene is activated or repressed. Here we demonstrate how a designed basic region leucine zipper-based peptide can be directed towards two different DNA sequences depending on its dimerization arrangement. While the monomeric peptide is non-functional, a C-terminal metallo-dimer recognizes the natural ATF/CREB-binding site (5'-ATGA cg TCAT-3'), and a N-terminal disulphide dimer binds preferentially to the swapped sequence (5'-TCAT cg ATGA-3'). As the dimerization mode can be efficiently controlled by appropriate external reagents, it is possible to reversibly drive the peptide to either DNA site in response to such specific inputs. This represents the first example of a designed molecule that can bind to more than one specific DNA sequence depending on changes in its environment.
Caging of the amidinium moieties of two representative bisbenzamidine DNA binders with suitable photoresponsive protecting groups suppresses their DNA-binding, which can then be fully recovered upon irradiation with UV light that removes the caging groups, regenerating the parent compounds. Moreover, a caged aza-pentamidine (©1a) is inert against Candida albicans, but displays a potent growth inhibitory activity after irradiation with UV light.
Guanine-rich sequences of DNA can assemble into tetrastranded structures known as G-quadruplexes. It has been suggested that these secondary DNA structures could be involved in the regulation of several key biological processes. In the human genome, guanine-rich sequences with the potential to form G-quadruplexes exist in the telomere as well as in promoter regions of certain oncogenes. The identification of these sequences as novel targets for the development of anticancer drugs has sparked great interest in the design of molecules that can interact with quadruplex DNA. While most reported quadruplex DNA binders are based on purely organic templates, numerous metal complexes have more recently been shown to interact effectively with this DNA secondary structure. This Review provides an overview of the important roles that metal complexes can play as quadruplex DNA binding molecules, highlighting the unique properties metals can confer to these molecules.
Here, we report the application of surface-enhanced Raman scattering (SERS) spectroscopy as a rapid and practical tool for assessing the formation of coordinative adducts between nucleic acid guanines and ruthenium polypyridyl reagents. The technology provides a practical approach for the wash-free and quick identification of nucleic acid structures exhibiting sterically accessible guanines. This is demonstrated for the detection of a quadruplex-forming sequence present in the promoter region of the c-myc oncogene, which exhibits a non-paired, reactive guanine at a flanking position of the G-quartets.
Due to their important biological functions, G-quadruplex (G4) DNA structures have been identified as potential drug targets. Consequently, the interactions of a large number of compounds with G4s and their biological effects have been investigated. While the majority of G4 binders reported to date are based on purely organic molecules, over the past two decades metal complexes have received increasing attention. This chapter provides an overview of metal complexes as G4 binders including a discussion of their properties, in vitro characterization of their affinity and selectivity to G4 structures and cellular studies.
We report a high-affinity photoswitchable DNA binder, which displays different nucleosome-binding capacity upon visible-light irradiation. Both photochemical and DNA-recognition properties were examined by UV-Vis, HPLC, CD spectroscopy, NMR, FID assays,...
Inspired by natural transcription factors (TFs), researchers have explored the potential of artificial peptides for the recognition of specific DNA sequences, developing increasingly sophisticated systems that not only display excellent DNA binding properties, but also are endowed with new properties not found in their natural counterparts. Here we review some of the developments in the field of artificial peptide-based DNA binders, focusing on the supramolecular and molecular design aspects of such systems.
There has been increasing interest in the development of small molecules that can selectively bind to G-quadruplex DNA structures. The latter have been associated to a number of key biological processes and therefore are proposed to be potential targets for drug development. In this paper we report the first example of a reduction-activated G-quadruplex DNA binder. We show that a new octahedral platinum(IV)-salphen complex does not interact with DNA in aqueous media at pH 7.4; however, upon addition of bio-reductants such as ascorbic acid or glutathione, the compound readily reduces to the corresponding square planar platinum(II) complex. In contrast to the parent platinum(IV) complex, the in situ generated platinum(II) complex binds to HTelo and c-Myc G-quadruplex DNA with affinity constants up to 106 M-1.
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
Gripping compounds! A homodimer is formed by linking two monomers of b-ZIP protein basic regions through a photoresponsive azobenzene unit. By simple irradiation at appropriate wavelenghths, this dimer can be switched between isomers which have moderate (trans) and high (cis) sequence-specific affinity for gripping the major groove of DNA (see scheme). BR = basic region, ds = double-stranded.
At specific DNA sites, nickel(II) salts promote the assembly of designed components, namely a bis(histidine)-modified peptide that is derived from a bZIP transcription factor and a bis(benzamidine) unit that is equipped with a bipyridine. This programmed supramolecular system with emergent properties reproduces some key characteristics of naturally occurring DNA-binding proteins, such as bivalence, selectivity, responsiveness to external agents, and reversibility.
We report the construction of conjugates between three variants of the helix 3 region of a Q50K engrailed homeodomain and bisbenzamidine minor-groove DNA binders. The hybrid featuring the sequence of the native protein failed to bind to DNA; however, modifications that increased the α-helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interaction. Groovy attraction: Conjugates of the helix 3 region of a Q50K engrailed homeodomain with bisbenzamidine minor-groove DNA binders failed to bind DNA. However, modifications that increase the α-helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interaction.
The design of artificial peptide dimers containing polypyridine switching domains, for which metal-ion coordination is shown to regulate DNA binding, is reported. Short peptides, based on the basic domain of the GCN4 transcription factor (GCN4bd), dimerised with either 2,2'-bipyridine (bipy(GCN4bd)2 ) or 2,2':6',2''-terpyridine (terpy(GCN4bd)2 ) linker units, undergo a conformational rearrangement on Cu(II) and Zn(II) coordination. Depending on the linker substitution pattern, this is proposed to alter the relative alignment of the two peptide moieties, and in turn regulate DNA binding. Circular dichroism and UV-visible spectroscopy reveal that Cu(II) and Zn(II) coordination promotes binding to DNA containing the CRE target site, but to a differing and opposite degree for the two linkers, and that the metal-ion affinity for terpy(GCN4bd)2 is enhanced in the presence of CRE DNA. Binding to DNA containing the shorter AP1 target site, which lacks a single nucleobase pair compared to CRE, as well as half-CRE, which contains only half of the CRE target site, was also investigated. Cu(II) and Zn(II) coordination to terpy(GCN4bd)2 promotes binding to AP1 DNA, and to a lesser extent half-CRE DNA. Whereas, bipy(GCN4bd)2 , for which interpeptide distances are largely independent of metal-ion coordination and less suitable for binding to these shorter sites, displays allosteric ineffective behaviour in these cases. These findings for the first time demonstrate that biomolecular recognition, and specifically sequence-selective DNA binding, can be controlled by metal-ion coordination to designed switching units, non-native regulation sites, in artificial biomolecules. We believe that in the future these could find a wide range of applications in biotechnology.
Organometallic ruthenium(II) complexes [(η(6)-arene)Ru(en)Cl](+) (arene = e.g., biphenyl (1), dihydrophenanthrene, tetrahydroanthracene) show promising anticancer activity both in vitro and in vivo and are cytotoxic to cisplatin-resistant cancer cells, implying that these monofunctional complexes have a different mechanism of action from that of bifunctional cisplatin. We demonstrate here that complex 1 binds selectively to the guanine base in the 15-mer single-stranded oligodeoxynucleotides (ODNs) 5'-CTCTCTX7G8Y9CTTCTC-3' [X = Y = T; X = C, Y = A; X = A, Y = T; X = T, Y = A] to form thermodynamically stable adducts, but thymine bases (T7/T11 or T6/T11) compete kinetically with guanine for binding to 1. The T-bound monoruthenated species eventually convert to diruthenated products via a second step of binding at G or/and to G-bound monoruthenated species through dissociation of the diruthenated adducts. Complex 1 was further shown to bind preferentially to the middle T in a sequence rather than to a T near the terminus and favor coordination to a 5'-T compared to a 3'-T. Interestingly, the T bases in the human telomeric G-quadruplex sequence (5'-AGGGTTAGGGTTAGGGTTAGGG-3') were found to be more competitive both kinetically and thermodynamically with G bases for binding to 1. These results suggest that thymine bases play a unique role in the pathways of ruthenation of DNA by organoruthenium anticancer complexes and illustrate that kinetic studies can provide new insight into the mechanism of action of metallodrugs in addition to study of the structures and functions of the thermodynamically stable end products.
Transcription factors (TFs) are specialized proteins that play a key role in the regulation of genetic expression. Their mechanism of action involves the interaction with specific DNA sequences, which usually takes place through specialized domains of the protein. However, achieving an efficient binding usually requires the presence of the full protein. This is the case for bZIP and zinc finger TF families, which cannot interact with their target sites when the DNA binding fragments are presented as isolated monomers. Herein it is demonstrated that the DNA binding of these monomeric peptides can be restored when conjugated to aza-bisbenzamidines, which are readily accessible molecules that interact with A/T-rich sites by insertion into their minor groove. Importantly, the fluorogenic properties of the aza-benzamidine unit provide details of the DNA interaction that are eluded in electrophoresis mobility shift assays (EMSA). The hybrids based on the GCN4 bZIP protein preferentially bind to composite sequences containing tandem bisbenzamidine-GCN4 binding sites (TCAT⋅AAATT). Fluorescence reverse titrations show an interesting multiphasic profile consistent with the formation of competitive nonspecific complexes at low DNA/peptide ratios. On the other hand, the conjugate with the DNA binding domain of the zinc finger protein GAGA binds with high affinity (KD ≈12 nM) and specificity to a composite AATTT⋅GAGA sequence containing both the bisbenzamidine and the TF consensus binding sites.
Appending negatively charged Glu(8) tails to a peptide dimer derived from the GCN4 transcription factor leads to an effective suppression of its DNA binding. The specific DNA recognition can be restored by irradiation with UV light by using a photolabile linker between the acidic tail and the DNA binding peptide.
Durch gleichzeitige Wechselwirkung mit der großen und der kleinen Furche der DNA kann eine bifunktionelle Modellverbindung an doppelsträngige DNA binden (siehe Bild); diese Modellverbindung wird durch Verknüpfung der basischen Region eines b-ZIP-Proteins (GCN4) an ein mit dem Antibiotikum Distamycin verwandtes Tripyrrol gebildet.
Photoactivated telomerase inhibition: The biological activities of caged compounds are masked by photolabile protecting groups, and can be unmasked with spatiotemporal specificity by light. We describe the design and synthesis of a caged G-quadruplex (G4) ligand. On in situ activation by UV light irradiation the compound showed significant G4-stabilizing activity and telomerase-inhibitory activity.
The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein-DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non-natural agents that can reproduce the DNA-recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double-stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability.
G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochemical properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-molecule ligands.
Bend and stretch… bend and stretch… An azobenzene derivative was used to induce reversible stretching and folding of G-quadruplex DNA upon photoirradiation (see picture). The G quadruplex formed in the presence of the trans isomer was dissociated by irradiation with UV light, and the resulting open oligomer was refolded into a G quadruplex under visible light. This nanodevice thus converts light directly into mechanical work.
Light switching of the activity of a coiledcoil protein, the AP-1 transcription factor, in living cells was made possible by the introduction of a designed azobenzenecross-linked dominant negative peptide, XAFosW (red and yellow in the picture). In the dark, XAFosW showed decreased helical content and decreased affinity for target Jun proteins (green); irradiation at 365 nm enhanced helicity and target affinity. Chemical Equation Presentation
Hand in Hand: Zwei Paare von Iminodiessigsäure(Ida)-Einheiten wurden in das Leucin-Zippersegment des GCN4-bZIP-Proteins so eingebaut, dass sich die Ida-Einheiten eines jeden Paars an den Positionen i und i+2 befanden. Komplexbildung zwischen den Ida-Gruppen und CoII bewirkte eine Destabilisierung der helicalen Struktur, was ein reversibles Schalten der Bindung des Proteins an die Zielposition AP-1 ermöglichte (siehe Bild).
A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region
that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization. A peptide model for the basic region
of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide
bond. The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4 degrees C. DNA binding
is sequence-specific as judged by deoxyribonuclease I footprinting. Circular dichroism spectroscopy suggests that the peptide
adopts a helical structure when bound to DNA. These results demonstrate directly that the GCN4 basic region is sufficient
for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein
dimerization. Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.
The X-ray structure of the GCN4-bZIP protein bound to DNA containing the ATF/CREB recognition sequence has been refined at 2.2 A. The water-mediated interactions between the basic domain and DNA are revealed, and combined with a more accurate description of the direct contacts, further clarify how binding specificity is achieved. Water molecules extend the interactions of both invariant basic domain residues, asparagine 235 and arginine 243, beyond their direct base contacts. The slight bending of the basic domain alpha-helix around the DNA facilitates the linking of arginine 241, 243 and 245 to main-chain carbonyl oxygen atoms via water molecules, apparently stabilizing interactions with the DNA.