Mechanism Of DNA Binding Research Articles

Antibody-Drug Conjugates with Indolinobenzodiazepine Dimer Payloads: DNA-Binding Mechanism of Indolinobenzodiazepine Dimer Catabolites in Target Cancer Cells.

DNA-targeting indolinobenzodiazepine dimer (IGN) payloads are used in several clinical-stage antibody-drug conjugates. IGN drugs alkylate DNA through the single imine moiety present in the dimer in contrast to the pyrrolobenzodiazepine dimer drugs, such as talirine and tesirine, which contain two imine moieties per dimer and cross-link DNA. This study explored the mechanism of binding of IGN to DNA in cells and to synthetic duplex and hairpin oligonucleotides. New, highly sensitive IGN-DNA binding enzyme-linked immunosorbent assay methods were developed using biotinylated IGN analogues (monoimine, diimine, and diamine IGNs) and digoxigenin-labeled duplex oligonucleotides, which allowed the measurement of drug-DNA adducts in viable cells at concentrations below IC50. Furthermore, the release of free drug from the IGN-DNA adduct upon treatment with nuclease ex vivo was tested under physiological conditions. The monoimine IGN drug formed a highly stable adduct with DNA in cells, with stability similar to that of the diimine drug analogue. Both monoimine and diimine IGN-DNA adducts released free drugs upon DNA cleavage by nuclease at 37 °C, although more free drug was released from the monoimine compared to the diimine adduct, which presumably was partly cross-linked. The strong binding of the monoimine IGN drug to duplex DNA results from both the noncovalent IGN-DNA interaction and the covalent bond formation between the 2-amino group of a guanine residue and the imine moiety in IGN.

New ruthenium polypyridyl complexes functionalized with fluorine atom or furan: Synthesis, DNA-binding, cytotoxicity and antitumor mechanism studies.

Two novel ruthenium(II) polypyridyl complexes, namely, [Ru(dmp)2(CAPIP)](ClO4)2 (Ru(II)-1) and [Ru(dmp)2(CFPIP)](ClO4)2 (Ru(II)-2), which respectively contain (E)-2-(2-(furan-2-yl)vinyl)-1H-imidazo[4,5-f][1,10]phen-anthroline (CAPIP) and (E)-2-(4-fluorostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline. (CFPIP), were first designed and characterized (dmp = 2,9-dimethyl-1,10-phenanthroline). DNA binding experiments indicated that Ru(II) complexes interact with CT DNA through intercalative mode. In addition, the complexes Ru(II)-1 and Ru(II)-2, showed remarkable cell cytotoxicity, giving the respective IC50 values of 4.1 ± 1.4 μM and 6.1 ± 1.4 μM on the A549 cancer cells. These values indicated higher activity than CAPIP, CFPIP, cisplatin (8.2 ± 1.4 μM) and other corresponding Ru(II) polypyridyl complexes. Furthermore, the Ru(II) complexes could arrive the cytoplasm through the cell membrane and accumulate in the mitochondria. Significantly, complexes Ru(II)-1 and Ru(II)-2 induced A549 cells apoptosis was mediated by increase of ROS levels and dysfunction of mitochondria, and resulted in cell cycle arrest and increased anti-migration activity on A549 cells. Overall, these results indicated that complexes Ru(II)-1 and Ru(II)-2 could be suitable candidates for further investigation as a chemotherapeutic agent in the treatment of tumors.

Large conformation shifts of Vibrio cholerae VqmA dimer in the absence of target DNA provide insight into DNA-binding mechanisms of LuxR-type receptors

Quorum sensing regulates the biofilm formation and expression of virulence factors in Vibrio cholerae, an obligate human pathogen that continues to imperil human health. Cytoplasmic transcription factor VqmA is a LuxR-type receptor ubiquitous in the Vibrio genus and one vibriophage VP882 and plays an important role in V. cholerae pathogenicity. Here we presented the X-ray crystal structure of V. cholerae VqmA–DPO complex and compared it with the previously determined VqmA–DPO–DNA complex. To our knowledge, this is the first report on the crystal structures of the same LuxR-type receptor with two conformations of binding to DNA and not binding to DNA. Based on the results of structural analysis and biochemical assays, we revealed the secondary structure of the linker region between two function domains changed significantly, and DNA binding domains were covalently linked by a disulfide bond formed by the highly conserved Cys134. Besides, the distance between two DBD monomers became longer than that in DNA-binding conformation, and two α8 helixes underwent a large conformation shift. The results of the structure-function analyses presented here improve our understanding of the complex mechanisms in the conformational changes of LuxR-type receptors caused by DNA binding.

Molecular architecture of the SYCP3 fibre and its interaction with DNA.

The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic recombination. A hallmark of the SC is the presence of its constituent protein SYCP3 on the chromosome axis. During SC assembly, SYCP3 is deposited on both axes of the homologue pair, forming axial elements that fuse into the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Our findings suggest that SYCP3 deposition on the chromosome axis might take place by polymerization into a fibre that is fastened to the chromosome surface via DNA binding.

The DNA-binding mechanism of the TCS response regulator ArlR from Staphylococcus aureus

ArlRS is an essential two-component system in Staphylococcus aureus that regulates the transcription of virulence factors and participate in numerous pathogenic and symbiotic processes. In this work, we identified different DNA binding properties and oligomerization states among the DNA-binding domain of ArlR (ArlRDBD) and the phosphorylated and unphosphorylated full-length ArlR. Based on a 2.5-Å resolution crystal structure of ArlRDBD and subsequent mutagenesis experiments, we confirmed the DNA-binding site of ArlR and the preferred binding sequences in the agr promoter that enables the DNA recognition process. Finally, we propose a putative transcription regulation mechanism for ArlR. This work will facilitate our understanding of the DNA binding affinity regulatory mechanism between the phosphorylated and unphosphorylated response regulator in the two-component system.

Design, synthesis and DNA interaction studies of new fluorescent platinum complex containing anti-HIV drug didanosine

Forming coordination complexes with nucleoside analogues may be helpful in studying anti-tumour activity of them. Therefore, to improve the clinical efficacy of nucleoside analogue and design new ones, a new fluorescent platinum (Pt) complex with anti-human immunodeficiency virus drug didanosine (ddI); K[PtCl(OCH3)2(ddI)]; was synthesized and characterized. The ultraviolet–visible (UV-vis) spectroscopy, infrared, thermogravimetric analysis, mass assignments and elemental analysis confirmed the preparation of the complex. The molecular ion peaks seen at the positive mass spectrum of Pt complex confirm coordination of the drug to metal centre. The interaction of this complex with calf thymus DNA (ct-DNA) was studied using several spectroscopic techniques such as UV absorption, fluorescence spectroscopy and dynamic viscosity measurements. Hyperchromism of the band in the UV-vis spectra and the intrinsic binding constant (0.56 ± 0.25) × 104 M−1, decreasing in Hoechst-DNA fluorescence by adding Pt complex concentration and also relatively small changes in DNA viscosity indicated that this complex could interact as a groove-binder. According to the UV spectra and the fluorescence quenching of the complex in our case seems to be primarily caused by complex formation between the Pt complex and DNA. The thermodynamic parameters showed that hydrogen bond and van der Waals interactions play main roles in the binding of Pt complex to ct-DNA. The free energy values are negative, showing the spontaneity of the Pt complex–DNA binding. The docking simulation was performed and the results confirm a preference of groove site of synthesized complex on DNA helix. The knowledge gained from this study will be helpful to further understand the DNA binding mechanism and can also provide much fruitful information for designing a new type of anti-cancer drugs.Communicated by Ramaswamy H. Sarma

Population balance modeling of assembly formation; accounting for the H-NS protein oligomerization and DNA binding mechanisms

The results obtained in this study demonstrate H-NS oligomerization in solution is not sufficient in forming large structures of H-NS-DNA assemblies indicating oligomerization along bacterial DNA is crucial. We present a comprehensive and novel multi-scale modeling scheme to study protein-protein and protein-DNA interactions, with the focus on population balance modeling of assemblies formed by the H-NS protein and bacterial DNA. The model helps understanding how the concentration of H-NS influences the formation of H-NS-DNA assemblies. The presented model covers the two underlying molecular mechanisms involved in assembly formation, H-NS oligomerization in solution and H-NS-DNA binding. Within protein oligomerization interactions, protein molecules are entitled to dimerize, propagate, recombine and deform (break). In addition, DNA binding and unbinding interactions are included to account for formation of filaments along DNA. All mechanisms have been modeled with their associated forward (formation) and backward (deformation) interactions. The results obtained agree well with former experimental studies.

Recent Development in Indole Derivatives as Anticancer Agents for Breast Cancer.

Breast Cancer (BC) is the second most common cause of cancer related deaths in women. Due to severe side effects and multidrug resistance, current therapies like hormonal therapy, surgery, radiotherapy and chemotherapy become ineffective. Also, the existing drugs for BC treatment are associated with several drawbacks such as poor oral bioavailability, non-selectivity and poor pharmacodynamics properties. Therefore, there is an urgent need for the development of more effective and safer anti BC agents. This article explored in detail the possibilities of indole-based heterocyclic compounds as anticancer agents with breast cancer as their major target. Recent literature related to indole derivatives endowed with encouraging anti BC potential is reviewed. With special focus on BC, this review offers a detailed account of multiple mechanisms of action of various indole derivatives: aromatase inhibitor, tubulin inhibitor, microtubule inhibitor, targeting estrogen receptor, DNA-binding mechanism, induction of apoptosis, inhibition of PI3K/AkT/NFkB/mTOR, and HDAC inhibitors, by which these derivatives have shown promising anticancer potential. Exhaustive literature survey indicated that indole derivatives are associated with properties of inducing apoptosis and disturbing tubulin assembly. Indoles are also associated with the inhibition of NFkB/mTOR/PI3K/AkT and regulation of estrogen-mediated activity. Furthermore, indole derivatives have been found to modulate critical targets such as topoisomerase and HDAC. These derivatives have shown significant activity against breast cancer cells. In BC, indole derivatives seem to be quite competent and act through various mechanisms that are well established in case of BC. This review has shown that indole derivatives can further be explored for the betterment of BC chemotherapy. A lot of potential is still hidden which demands to be discovered for upgrading BC chemotherapy.

A Decade Decoded: Spies and Hackers in the History of TAL Effectors Research.

Transcription activator-like effectors (TALEs) from the genus Xanthomonas are proteins with the remarkable ability to directly bind the promoters of genes in the plant host to induce their expression, which often helps bacterial colonization. Metaphorically, TALEs act as spies that infiltrate the plant disguised as high-ranking civilians (transcription factors) to trick the plant into activating weak points that allow an invasion. Current knowledge of how TALEs operate allows researchers to predict their activity (counterespionage) and exploit their function, engineering them to do our bidding (a Manchurian agent). This has been possible thanks particularly to the discovery of their DNA binding mechanism, which obeys specific amino acid-DNA correspondences (the TALE code). Here, we review the history of how researchers discovered the way these proteins work and what has changed in the ten years since the discovery of the code. Recommended music for reading this review can be found in the Supplemental Material.

Crystal Structures and Nuclear Magnetic Resonance Studies of the Apo Form of the c-MYC:MAX bHLHZip Complex Reveal a Helical Basic Region in the Absence of DNA.

The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix–loop–helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate helical conformations. We have also assigned the NMR spectra of the c-MYC basic helix–loop–helix Zip motif in the absence of MAX and showed that the basic region has an intrinsic helical propensity even in the absence of its dimerization partner. The presence of helical structure in the basic regions in the absence of DNA suggests that the molecular recognition occurs via a conformational selection rather than an induced fit. Our work provides both insight into the mechanism of DNA binding and structural information to aid in the development of MYC inhibitors.

Abstract 230: Antibody-drug conjugates (ADCs) with indolinobenzodiazepine dimer (IGN) payloads: DNA-binding mechanism of IGN catabolites in target cancer cells

Abstract A DNA-interacting indolinobenzodiazepine dimer (IGN) payload was designed with a single reactive imine group towards the goal of eliminating DNA cross-linking and avoiding related toxicities, while conferring a strong binding of the IGN scaffold to duplex DNA. Several IGN ADCs, wherein the payload is linked via a peptide or hindered disulfide, are currently being evaluated in the clinic. In contrast to our lead IGNs, DNA-interacting pyrrolobenzodiazepine (PBD)-based ADC payloads, such as talirine and tesirine, contain two reactive imine groups that can cross-link DNA. Here, we investigated the mechanism of binding of IGN catabolites with DNA in target cancer cells, and with model duplex DNA or hairpin oligonucleotides. Hairpin and duplex oligonucleotides, designed for high melting temperatures (around 50-60 °C), were custom synthesized with labels. Model IGN catabolites bearing a single imine (mono-imine) or two imine groups (di-imine) were synthesized with a biotin label. Sensitive assays were developed to measure IGN-DNA binding in cells at sub-cytotoxic concentrations (lower than IC50) to allow studies of DNA adduct stability and repair. The mono-imine IGN molecules bind readily to oligonucleotides, generating stable adducts as determined by gel filtration and reversed phase HPLC analysis. To investigate the binding of unconjugated IGNs with cellular DNA, cancer cells were incubated with mono- and di-imine IGNs for a short-term, followed by wash and further incubation in fresh media. Both mono-and di-imine IGN molecules remained bound to genomic DNA even at 2 days, suggesting a potent interaction with cellular DNA. The time course of binding of IGN to DNA in cells was slower than that observed with model oligonucleotides, as expected because the tightly coiled cellular DNA presumably binds IGN only after the unwinding of DNA during cell cycle or transcription. Upon DNA cleavage by an added nuclease, free IGN was released from IGN adducts of model oligonucleotides and from genomic DNA of cells that had been treated with unconjugated IGN or IGN ADC. This dissociation of IGN from IGN-DNA adducts only upon cleavage with nuclease suggests that a strong non-covalent interaction between IGN and duplex DNA stabilizes the adduct. The amount of free IGN released from cellular DNA adduct upon nuclease treatment was about 2-fold greater for mono-imine IGN than di-imine IGN, presumably because di-imine IGN was partly cross-linked to cellular DNA. Mono-imine IGN-DNA adducts could potentially be repaired by cellular endonucleases via a DNA cleavage mechanism. In conclusion, the mono-imine IGN payload molecules form highly stable adducts with DNA, which dissociate upon DNA cleavage at physiological temperature. Citation Format: Rajeeva Singh, Luke Harris, Paulin Salomon, Emily E. Reid, Michael L. Miller, Ravi V. Chari, Thomas A. Keating. Antibody-drug conjugates (ADCs) with indolinobenzodiazepine dimer (IGN) payloads: DNA-binding mechanism of IGN catabolites in target cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 230.

Abstract 2758: Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor

Abstract The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers, including hepatocellular carcinoma, pancreatic cancer, oral cancer, breast cancer and non-small cell lung cancer. However, the functional mechanisms of human HDGF had been puzzling because of a lack of essential knowledge of its intact structure in complex with DNA. We present the first crystal structures of the human HDGF PWWP domain (residues 1 - 100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions. Note: This abstract was not presented at the meeting. Citation Format: Chun-Jung Chen, Li-Jin Hsu. Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2758.

Native mass spectrometry identifies an alternative DNA-binding pathway for BirA from Staphylococcus aureus

An adequate supply of biotin is vital for the survival and pathogenesis of Staphylococcus aureus. The key protein responsible for maintaining biotin homeostasis in bacteria is the biotin retention protein A (BirA, also known as biotin protein ligase). BirA is a bi-functional protein that serves both as a ligase to catalyse the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates biotin biosynthesis, biotin transport and fatty acid elongation. The mechanism of BirA regulated transcription has been extensively characterized in Escherichia coli, but less so in other bacteria. Biotin-induced homodimerization of E. coli BirA (EcBirA) is a necessary prerequisite for stable DNA binding and transcriptional repression. Here, we employ a combination of native mass spectrometry, in vivo gene expression assays, site-directed mutagenesis and electrophoretic mobility shift assays to elucidate the DNA binding pathway for S. aureus BirA (SaBirA). We identify a mechanism that differs from that of EcBirA, wherein SaBirA is competent to bind DNA as a monomer both in the presence and absence of biotin and/or MgATP, allowing homodimerization on the DNA. Bioinformatic analysis demonstrated the SaBirA sequence used here is highly conserved amongst other S. aureus strains, implying this DNA-binding mechanism is widely employed.

Characterization of the Novel DNA Binding Activity of the BRG1 At-Hook-Bromodomain

The packaging of eukaryotic DNA with histones into chromatin acts to both compact it into the nucleus as well as provide a mechanism for regulation of the genome. Spatial and temporal remodeling of chromatin structure is critical for all DNA-templated processes and dysregulation of these pathways is associated with a number of diseases. The BRG1/BRM associated factor (BAF) complex, is an ATP-dependent remodeling complex that plays an important role in gene regulation, several subunits of which are mutated in human cancers. The Brahma related gene 1 (BRG1) subunit of BAF, which provides the ATPase activity, contains a bromodomain (BD) at its C-terminus. BDs are well-characterized readers of acetylated lysines on histones, and the BRG1-BD has been shown to bind H3K14ac. However, we have recently discovered that in addition to binding acetyl-lysine, the BRG1-BD associates with DNA, a novel function for BDs. In addition, we have demonstrated that an adjacent AT-hook contributes to a multivalent mechanism of association with DNA, increasing affinity and specificity (Morrison et al., Nature Communications, 2017). Notably, the newly identified DNA binding interface harbors several cancer-associated mutations. Here we present our continued studies on this newly recognized composite DNA binding domain. We use systematic evolution of ligands by exponential enrichment (SELEX-seq) to generate a biophysical model of sequence specificity for the domain that we are exploring structurally using NMR spectroscopy. To determine the kinetic and thermodynamic basis of association we use biolayer interferometry (BLI). In addition, we are investigating the molecular mechanism of DNA binding and how it is altered in the context of the nucleosome. Together, our results are revealing the molecular details of how this novel DNA binding domain functions to navigate chromatin, and how dysregulation of the ATBD may impact BAF function.

Quantifying Anticancer Drug Doxorubicin Binding to DNA using Optical Tweezers

Doxorubicin is a successful anticancer drug approved for use in the 1970s and is considered to be one of the most effective cancer treatment methods today. Although Doxorubicin has positive survival statistics it has very negative side effects in many cases. Bleeding from the soles of the palms and feet, along with excruciating pain is often exhibited through the administration of this drug. Based on the preliminary findings utilizing optical tweezers we anticipate that this study will provide critical information about the drug binding mechanism. Single molecule biophysics techniques have provided useful insight into the DNA-binding mechanisms of small molecules. For this reason, in this study we quantify the binding kinetics and affinity of Doxorubicin to DNA using optical tweezers. The optical tweezers allow us to trap a single DNA molecule and manipulate it in the presence of various concentrations of Doxorubicin. Preliminary results suggest that DNA melting facilitates the intercalation process in the nanomolar range, implying a higher binding strength than the previously reported micromolar range.

Influence of DNA-binding compounds with cancer preventive activity on the mechanisms of gene expression regulation

The presented review is devoted to the analysis of molecular mechanisms of action for different natural DNA-tropic compounds with established tumor preventive activity. Here we present their cancer preventive effects observed in vivo, mechanisms of DNA binding, influence on epigenetic regulation and “housekeeping” protein function. Additionally, the influence of these compounds on DNA helix parameters is discussed that should impact on epigenetic regulation of gene expression and formation of topologically associated domains.

DNA-binding mechanism of spiropyran photoswitches: the role of electrostatics.

The binding mechanism of the protonated open form of three spiropyran derivatives into a 12-mer (poly-dAT)2 has been unveiled by means of computational methods. It is found that the electrostatic term in the probe:DNA binding energy, modulates the binding mode, providing new guidelines for the design of spiropyran photoswitches with specific binding modes to DNA.

Structural Analysis of the Active Site and DNA Binding of Human Cytidine Deaminase APOBEC3B.

APOBEC3 (A3) proteins, a family of human cytidine deaminases, protect the host from endogenous retro-elements and exogenous viral infections by introducing hypermutations. However, overexpressed A3s can modify genomic DNA to promote tumorigenesis, especially A3B. Despite their overall similarity, A3 proteins have distinct deamination activity. Recently determined A3 structures have revealed the molecular determinants of nucleotide specificity and DNA binding. However, for A3B, the structural basis for regulation of deamination activity and the role of active site loops in coordinating DNA had remained unknown. Using advanced molecular modeling followed by experimental mutational analysis and dynamics simulations, we investigated the molecular mechanism of DNA binding by A3B-CTD. We modeled fully native A3B-DNA structure, and we identified Arg211 in loop 1 as the gatekeeper coordinating DNA and critical residue for nucleotide specificity. We also identified a unique autoinhibited conformation in A3B-CTD that restricts access and binding of DNA to the active site. Our results reveal the structural basis for DNA binding and relatively lower catalytic activity of A3B and provide opportunities for rational design of specific inhibitors to benefit cancer therapeutics.

Structural and Biochemical Analysis of the Citrate-Responsive Mechanism of the Regulatory Domain of Catabolite Control Protein E from Staphylococcus aureus.

Catabolite control protein E (CcpE) is a LysR-type transcriptional regulator that positively regulates the transcription of the first two enzymes of the TCA cycle, namely, citZ and citB, by sensing accumulated intracellular citrate. CcpE comprises an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD) and senses citrate with conserved arginine residues in the RD. Although the crystal structure of the apo SaCcpE-RD has been reported, the citrate-responsive and DNA-binding mechanisms by which CcpE regulates TCA activity remain unclear. Here, we report the crystal structure of the apo and citrate-bound SaCcpE-RDs. The SaCcpE-RD exhibits conformational changes between the two subdomains via hinge motion of the central β4 and β10 strands. The citrate molecule is located in a positively charged cavity between the two subdomains and interacts with the highly conserved Ser98, Leu100, Arg145, and Arg256 residues. Compared with that of the apo SaCcpE-RD, the distance between the two subdomains of the citrate-bound SaCcpE-RD is more than ∼3 Å due to the binding of the citrate molecule, and this form exhibits a closed structure. The SaCcpE-RD exhibits various citrate-binding-independent conformational changes at the contacting interface. The SaCcpE-RD prefers the dimeric state in solution, whereas the SaCcpE-FL prefers the tetrameric state. Our results provide insight into the molecular function of SaCcpE.

Bioorthogonal DNA Adsorption on Polydopamine Nanoparticles Mediated by Metal Coordination for Highly Robust Sensing in Serum and Living Cells.

DNA-functionalized nanomaterials, such as various 2D materials, metal oxides, and gold nanoparticles, have been extensively explored as biosensors. However, their practical applications for selective sensing and imaging in biological samples remain challenging due to interference from the sample matrix. Bioorthogonal chemistry has allowed specific reactions in cells, and we want to employ this concept to design nanomaterials that can selectively adsorb DNA but not proteins or other abundant biomolecules. In this work, DNA oligonucleotides were found to be adsorbed on polydopamine nanoparticles (PDANs) via polyvalent metal-mediated coordination, and such adsorption bioorthogonally resisted DNA displacement by various biological ligands, showing better performance compared to graphene oxide and metal oxide nanoparticles for DNA detection. Using DNA/PDANs as biosensors, a detection limit of <1 nM target DNA was achieved in serum and other biological samples, and imaging of cancer-related microRNA in cells was demonstrated. The DNA binding mechanism on PDAN was further studied by ligand displacement experiments and X-ray photoelectron spectroscopy characterization, which demonstrated the critical role of polyvalent metal ions to bridge DNA with PDANs. This work provides fundamental insights into the biointerface science of PDANs with DNA, which can benefit applications in biosensor design, directed assembly of nanomaterials, bioimaging, and drug delivery.