Allosteric DNA Research Articles

Allosteric DNAzyme-based DNA logic circuit: operations and dynamic analysis.

Recently, due to the dual roles of DNA and enzyme, DNAzyme has been widely used in the field of DNA circuit, which has a wide range of applications in bio-engineered system, information processing and biocomputing. In fact, the activity of DNAzymes was regulated by subunits assembly, pH control and metal ions triggers. However, those regulations required to change the sequences of whole DNAzyme, as separating parts and inserting extra DNA sequence. Inspired by the allosteric regulation of proteins in nature, a new allosteric strategy is proposed to regulate the activity of DNAzyme without DNA sequences changes. In this strategy, DNA strand displacement was used to regulate the DNAzyme structure, through which the activity of DNAzyme was well controlled. The strategy was applied to E6-type DNAzymes, and the operations of DNA logic circuit (YES, OR, AND, cascading and feedback) were established and simulated with the dynamic analyses. The allosteric regulation has potential to construct more complicated molecular systems, which can be applied to bio-sensing and detection.

Detecting transcription factors with allosteric DNA-Silver nanocluster switches

Sensitive and efficient detection of protein markers, such as transcription factors (TFs), is an important issue in postgenomic era. In this paper, we report a DNA nanodevice, allosteric DNA-silver nanocluster switches (AgSwitches), for TFs detection. The mechanism of this nanodevice is based on the binding-induced allostery whereby the binding between AgSwitches and TFs alters the conformation of AgSwitches. This alteration brings DNA-silver nanocluster (DNA-AgNCs) and guanine-rich enhancer sequences (GRS) into close proximity, generating fluorescent enhancement for quantifications. Our results revealed that the sequence design of AgSwitches can be rationally optimized according to stimulated free energy, and we demonstrated that this method can not only be used for detecting TFs in nuclear extracts of cells, but also be developed as a tool for screening inhibitors of TFs. Overall, this work expanded the category allosteric DNA nanodevices by first introducing DNA-AgNCs into this area, and the obtained method was efficient for TFs-related investigations.

Voltammetric determination of DNA based on regulation of DNA strand displacement using an allosteric DNA toehold

An electrochemical biosensor for determination of DNA is described that is based on the reaction of regulated DNA (reg-DNA) first with substrated DNA (subs-DNA) to form a reaction intermediate. The intermediate binds target DNA (T) by hybridization and initiates a branch migration leading to the production of complex of substrated DNA and target DNA (TC). Once TC is produced, it reacts with assisted DNA (ass-DNA) through a toehold exchange mechanism, yielding the product complex of substrated DNA and assisted DNA (CS). The target is then released back into the solution and and catalyzes the next cycle of toehold-exchange with the reaction intermediate of substrated DNA and regulated DNA (CPR). Unlike in a conventional DNA toehold that is hardwired with the branch migration domain, the allosteric DNA toehold is designed into a reg-DNA which is independent of the branch migration domain. Under the optimal experimental conditions and at a working potential as low as 0.18V, response to DNA is linear in the 1 fM to 1000 pM concentration range, and the detection limit is 0.83 fM. The assay is highly specific and can discriminate target DNA even from a single-base mismatch. It was applied to the analysis of DNA spiked plasma samples. Graphical abstract Schematic illustration of the electrochemical strategy for target DNA detection based on regulation of DNA strand displacement using an allosteric DNA toehold strategy. It can be used toanalyze DNA-spiked plasma samples and has a low detection limit of 0.83 fM.

Allosteric DNA nanoswitches for controlled release of a molecular cargo triggered by biological inputs.

Here we demonstrate the rational design of a new class of DNA-based nanoswitches which are allosterically regulated by specific biological targets, antibodies and transcription factors, and are able to load and release a molecular cargo (i.e. doxorubicin) in a controlled fashion. In our first model system we rationally designed a stem-loop DNA-nanoswitch that adopts two mutually exclusive conformations: a "Load" conformation containing a doxorubicin-intercalating domain and a "Release" conformation containing a duplex portion recognized by a specific transcription-factor (here Tata Binding Protein). The binding of the transcription factor pushes this conformational equilibrium towards the "Release" state thus leading to doxorubicin release from the nanoswitch. In our second model system we designed a similar stem-loop DNA-nanoswitch for which conformational change and subsequent doxorubicin release can be triggered by a specific antibody. Our approach augments the current tool kit of smart drug release mechanisms regulated by different biological inputs.

Regulation of DNA Strand Displacement Using an Allosteric DNA Toehold.

Toehold-mediated DNA strand displacement is the fundamental basis for the construction and operation of diverse DNA devices, including circuits, machines, sensors, and reconfigurable structures. Controllable activation and regulation of toeholds are critical to construct devices with multistep, autonomous, and complex behaviors. A handful of unique toehold activation mechanisms, including toehold-exchange, associative toehold, and remote toehold, have been developed and are often combined to achieve desired strand displacement behaviors and functions. Here we report an allosteric DNA toehold (A-toehold) design that allows the flexible regulation of DNA strand displacement by splitting an input strand into an A-toehold and branch migration domain. Because of its simplicity, the A-toehold mechanism can be a useful addition to the current toolbox of DNA strand displacement techniques. We demonstrated that A-toehold enabled a number of interesting functions that were previously shown using more sophisticated DNA strand displacement systems, including (1) continuously tuning the rate of strand displacement, (2) dynamic control of strand displacement reactions, and (3) selective activation of multiple strand displacement reactions. Moreover, by combining A-toehold and toehold-exchange mechanisms, we have successfully constructed a noncovalent DNA catalysis network that resembles an allosteric enzyme.

Dynamic Modulation of DNA Hybridization Using Allosteric DNA Tetrahedral Nanostructures.

The fixed dynamic range of traditional biosensors limits their utility in several real applications. For example, viral load monitoring requires the dynamic range spans several orders of magnitude; whereas, monitoring of drugs requires extremely narrow dynamic range. To overcome this limitation, here, we devised tunable biosensing interface using allosteric DNA tetrahedral bioprobes to tune the dynamic range of DNA biosensors. Our strategy takes the advantage of the readily and flexible structure design and predictable geometric reconfiguration of DNA nanotechnology. We reconfigured the DNA tetrahedral bioprobes by inserting the effector sequence into the DNA tetrahedron, through which, the binding affinity of DNA tetrahedral bioprobes can be tuned. As a result, the detection limit of DNA biosensors can be programmably regulated. The dynamic range of DNA biosensors can be tuned (narrowed or extended) for up to 100-fold. Using the regulation of binding affinity, we realized the capture and release of biomolecules by tuning the binding behavior of DNA tetrahedral bioprobes.

Label-free technology for the amplified detection of microRNA based on the allosteric hairpin DNA switch and hybridization chain reaction

By using the allosteric hairpin DNA switch, a novel assay for the detection of microRNA (miRNA) let-7a via a hybridization chain reaction (HCR) was introduced. Briefly, the hairpin DNA switch probe is a single-stranded DNA consisting of a streptavidin (SA) aptamer sequence, a target binding sequence and a certain sequence that acts as a trigger of the HCR. In the presence of target let-7a, the hairpin DNA switch would open and expose the stem region sequences, where a part of this sequence acts as initiator sequence strands for the HCR and triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers, another part is the SA aptamer sequence which activates its binding affinity to SA on SA-coated magnetic particles. The hybridization event could be sensitively detected via an instantaneous derivatization reaction between a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotides within the target, the hairpin DNA switch probe, and HCR helices to form an unstable CL intermediate for the generation of light. Our results show that the coupling of the hairpin DNA switch probe and the HCR for the amplified detection of let-7a achieves a better performance (e.g. wide linear response range: 0.1-1000 fmol, low detection limit: 0.1 fmol, and high specificity). Furthermore, this approach could be easily applied to the detection of let-7a in human lung cells, and extended to detect other types of miRNA and proteins such as PDGF based on aptamers. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment.

Metallo-regulation of the bimolecular triplex formation of a peptide nucleic acid

Peptide nucleic acid (PNA) conjugates incorporating a bipyridine unit were prepared. The bipyridine was built into the loop moiety of PNAs that were designed to specifically form a hairpin and a PNA/DNA bimolecular triplex. While the thermal stability of the hairpin structure was only minimally affected by Cu(2+) addition, the PNA/DNA bimolecular triplex structure was significantly destabilized by complexation with Cu(2+). The melting temperature of the bimolecular triplex decreased by 17.4 °C in the presence of Cu(2+). This corresponds to more than a 1000 fold decrease in the binding constant for bimolecular triplex formation. Upon complexation, the bipyridine unit underwent a drastic conformational change which accounts for the observed differences in the thermal stabilities of the triplex upon binding. The bipyridine-PNA conjugate may be useful as an allosteric DNA carrier that releases the DNA in response to a certain metal ion concentration.

Lifetime Heterogeneity of DNA-Bound dppz Complexes Originates from Distinct Intercalation Geometries Determined by Complex–Complex Interactions

Despite the extensive interest in structurally explaining the photophysics of DNA-bound [Ru(phen)(2)dppz](2+) and [Ru(bpy)(2)dppz](2+), the origin of the two distinct emission lifetimes of the pure enantiomers when intercalated into DNA has remained elusive. In this report, we have combined a photophysical characterization with a detailed isothermal titration calorimetry study to investigate the binding of the pure Δ and Λ enantiomers of both complexes with [poly(dAdT)](2). We find that a binding model with two different binding geometries, proposed to be symmetric and canted intercalation from the minor groove, as recently reported in high-resolution X-ray structures, is required to appropriately explain the data. By assigning the long emission lifetime to the canted binding geometry, we can simultaneously fit both calorimetric data and the binding-density-dependent changes in the relative abundance of the two emission lifetimes using the same binding model. We find that all complex-complex interactions are slightly unfavorable for Δ-[Ru(bpy)(2)dppz](2+), whereas interactions involving a complex canted away from a neighbor are favorable for the other three complexes. We also conclude that Δ-[Ru(bpy)(2)dppz](2+) preferably binds isolated, Δ-[Ru(phen)(2)dppz](2+) preferably binds as duplets of canted complexes, and that all complexes are reluctant to form longer consecutive sequences than triplets. We propose that this is due to an interplay of repulsive complex-complex and attractive complex-DNA interactions modulated by allosteric DNA conformation changes that are largely affected by the nature of the ancillary ligands.

176 N3-Substituted temozolomide analogs overcome methylguanine DNA methyltransferase and mismatch repair

DNA aptamers and DNA enzymes (DNAzymes or deoxyribozymes) are single-stranded DNA molecules with ligand-binding and catalytic capabilities, respectively. Allosteric DNA enzymes (aptazymes) are deoxyribozymes whose activity can be regulated by the binding state of an appended aptamer domain and have many potential uses in the fields of drug discovery and diagnostics. In this report, we describe a simple, yet potentially general, DNA aptazyme rational design strategy that requires no structural characterization of the constituent deoxyribozymes and aptamers. It is based on the concept originally developed in our laboratory for the design of structure-switching signaling aptamers that change structural states from a DNA–DNA duplex to a DNA–target complex upon target binding. In our new strategy, an antisense oligonucleotide is used to regulate the enzymatic activity of a linked aptamer–deoxyribozyme by annealing with a stretch of nucleotides on each side of the aptamer–DNAzyme junction. Structural reorganization of the aptamer domain upon target binding relieves the suppressive effect of this regulatory oligonucleotide on the attached DNA enzyme. Consequently, the target-binding event triggers the catalytic action of the aptazyme. We have demonstrated this concept using two RNA-cleaving deoxyribozymes, each adjoined to a DNA aptamer that binds ATP. These allosteric DNA enzymes exhibit the same ligand-binding specificity as the parental DNA aptamer and show up to 30-fold rate enhancement in the presence of ATP. The described methodology provides a convenient approach for rationally designing catalytic DNA-based biosensors.

Determination of Allosteric Effects and Interstrand Bidentate Interactions in DNA‐Peptide Molecular Recognition

AbstractTo study DNA allostery, quantitative DNase I footprinting studies were carried out on a newly designed peptide His‐Hyp‐Lys‐Lys‐(Py)4‐Lys‐Lys‐NH2 (HypKK‐10) containing the XHypKK (Hyp = hydroxyproline) and polyamide motifs. The interconnection of DNA footprints of peptides HypKK‐10 and the parent peptide PyPro‐12 supports the proposal that interaction network cooperativity is preferred in DNA‐peptide interactions between multiple recognition sites. A simple method of determining interstrand bidentate interactions between the peptide moieties and DNA bases is introduced. It is envisaged that interstrand bidentate interactions also participate in the relay of conformational changes to recognition sites on the complementary strands. Circular dichroism studies of the titration of peptide HypKK‐10 with an oligonucleotide duplex indicate that this peptide binds in a dimeric fashion to DNA in the minor groove. This work may prompt the design of new DNA binding ligands for the study of DNA‐peptide allosteric interactions and DNA interaction network.

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with μM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.

P.1.b.007 Developmental expression and activity of carboxylesterase 1 and carboxylesterase 2 in mouse and human

Sensitive and efficient detection of protein markers, such as transcription factors (TFs), is an important issue in postgenomic era. In this paper, we report a DNA nanodevice, allosteric DNA-silver nanocluster switches (AgSwitches), for TFs detection. The mechanism of this nanodevice is based on the binding-induced allostery whereby the binding between AgSwitches and TFs alters the conformation of AgSwitches. This alteration brings DNA-silver nanocluster (DNA-AgNCs) and guanine-rich enhancer sequences (GRS) into close proximity, generating fluorescent enhancement for quantifications. Our results revealed that the sequence design of AgSwitches can be rationally optimized according to stimulated free energy, and we demonstrated that this method can not only be used for detecting TFs in nuclear extracts of cells, but also be developed as a tool for screening inhibitors of TFs. Overall, this work expanded the category allosteric DNA nanodevices by first introducing DNA-AgNCs into this area, and the obtained method was efficient for TFs-related investigations.

Dynamic Allosteric Control of Noncovalent DNA Catalysis Reactions

Allosteric modulation of catalysis kinetics is prevalent in proteins and has been rationally designed for ribozymes. Here, we present an allosteric DNA molecule that, in its active configuration, catalyzes a noncovalent DNA reaction. The catalytic activity is designed to be modulated by the relative concentrations of two DNA regulator molecules, one an inhibitor and the other an activator. Dynamic control of the catalysis rate is experimentally demonstrated via three cycles of up and down regulation by a factor of over 10. Unlike previous works, both the allosteric receptor and catalytic core are designed, rather than evolved. This allows flexibility in the sequence design and modularity in synthetic network construction.

Allostery: DNA Does It, Too

Allostery is a central concept for understanding protein function and regulation. It is less well appreciated that DNA is allosteric, too, and that DNA conformational changes can by coupled to protein binding interactions on the DNA lattice. Allosteric DNA interactions are emerging as important features in the assembly of the molecular machines that regulate transcription.

Targeted chemical wedges reveal the role of allosteric DNA modulation in protein-DNA assembly.

The cooperative assembly of multiprotein complexes results from allosteric modulations of DNA structure as well as direct intermolecular contacts between proteins. Such cooperative binding plays a critical role in imparting exquisite sequence specificity on the homeobox transcription factor (Hox) family of developmental transcription factors. A well-characterized example includes the interaction of Hox proteins with extradenticle (Exd), a highly conserved DNA binding transcription factor. Although direct interactions are important, the contribution of indirect interactions toward cooperative assembly of Hox and Exd remains unresolved. Here we use minor groove binding polyamides as structural wedges to induce perturbations at specific base steps within the Exd binding site. We find that allosteric modulation of DNA structure contributes nearly 1.5 kcal/mol to the binding of Exd to DNA, even in the absence of direct Hox contacts. In contrast to previous studies, the sequence-targeted chemical wedges reveal the role of DNA geometry in cooperative assembly of Hox-Exd complexes. Programmable polyamides may well serve as general probes to investigate the role of DNA modulation in the cooperative and highly specific assembly of other protein-DNA complexes.

Use of Binding Enthalpy To Drive an Allosteric Transition

The Escherichia coli biotin repressor is an allosteric DNA binding protein and is activated by the small molecule bio-5'-AMP. Binding of this small molecule promotes transcription repression complex assembly between the repressor and the biotin operator of the biotin biosynthetic operon. The ability of the adenylate to activate the assembly process reflects its effect on biotin repressor dimerization. Thus concomitant with small molecule binding the free energy of repressor dimerization becomes more favorable by approximately -4 kcal/mol. The structural, dynamic, and energetic changes in the repressor monomer that accompany allosteric activation are not known. In this work the thermodynamics of binding of four allosteric activators to the repressor have been characterized by isothermal titration calorimetry. While binding of two of the effectors results in relatively modest activation of the dimerization process, binding of the other two small molecules, including the physiological effector, leads to large changes in repressor dimerization energetics. Results of the calorimetric measurements indicate that strong effector binding is accompanied by an enthalpically costly transition in the protein. This transition is "paid for" by the enthalpy that would have otherwise been realized from the formation of noncovalent bonds between the ligand and repressor monomer.

Structural Basis for Allosteric Control of the Transcription Regulator CcpA by the Phosphoprotein HPr-Ser46-P

Carbon catabolite repression (CCR) is one of the most fundamental environmental-sensing mechanisms in bacteria and imparts competitive advantage by establishing priorities in carbon metabolism. In gram-positive bacteria, the master transcription regulator of CCR is CcpA. CcpA is a LacI-GalR family member that employs, as an allosteric corepressor, the phosphoprotein HPr-Ser46-P, which is formed in glucose-replete conditions. Here we report structures of the Bacillus megaterium apoCcpA and a CcpA-(HPr-Ser46-P)-DNA complex. These structures reveal that HPr-Ser46-P mediates a novel two-component allosteric DNA binding activation mechanism that involves both rotation of the CcpA subdomains and relocation of pivot-point residue Thr61, which leads to juxtaposition of the DNA binding regions permitting “hinge” helix formation in the presence of cognate DNA. The structure of the CcpA-(HPr-Ser46-P)- cre complex also reveals the elegant mechanism by which CcpA family-specific interactions with HPr-Ser46-P residues Ser46-P and His15 partition the high-energy CCR and low-energy PTS pathways, the latter requiring HPr-His15-P.

Structure-switching allosteric deoxyribozymes

DNA aptamers and DNA enzymes (DNAzymes or deoxyribozymes) are single-stranded DNA molecules with ligand-binding and catalytic capabilities, respectively. Allosteric DNA enzymes (aptazymes) are deoxyribozymes whose activity can be regulated by the binding state of an appended aptamer domain and have many potential uses in the fields of drug discovery and diagnostics. In this report, we describe a simple, yet potentially general, DNA aptazyme rational design strategy that requires no structural characterization of the constituent deoxyribozymes and aptamers. It is based on the concept originally developed in our laboratory for the design of structure-switching signaling aptamers that change structural states from a DNA–DNA duplex to a DNA–target complex upon target binding. In our new strategy, an antisense oligonucleotide is used to regulate the enzymatic activity of a linked aptamer–deoxyribozyme by annealing with a stretch of nucleotides on each side of the aptamer–DNAzyme junction. Structural reorganization of the aptamer domain upon target binding relieves the suppressive effect of this regulatory oligonucleotide on the attached DNA enzyme. Consequently, the target-binding event triggers the catalytic action of the aptazyme. We have demonstrated this concept using two RNA-cleaving deoxyribozymes, each adjoined to a DNA aptamer that binds ATP. These allosteric DNA enzymes exhibit the same ligand-binding specificity as the parental DNA aptamer and show up to 30-fold rate enhancement in the presence of ATP. The described methodology provides a convenient approach for rationally designing catalytic DNA-based biosensors.

Allosteric DNA and ultrafast electron transfer of drug with DNA

Allosteric DNA and ultrafast electron transfer of drug with DNA