Fluorescence Anisotropy Assay Research Articles

HLA-DM catalytically enhances peptide dissociation by sensing peptide–MHC class II interactions throughout the peptide-binding cleft

Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.

Abstract MS1-2: T cell-based immunotherapy for a “public” neoantigen derived from a PIK3CA hotspot mutation

Abstract Background: “Public” neoantigens represent immunogenic epitopes encompassing hotspot mutations in driver oncogenes that are also restricted by common HLA alleles. In contrast with patient-specific “private” neoantigens, public neoantigens are conceptually attractive because they are tumor-specific, clonally conserved, and shared across patients. Whether PIK3CA, the most common driver oncogene in breast and endometrial cancer, can yield public neoepitopes that may be exploited for cancer immunotherapy is unknown. Methods: We have developed a high-throughput, single-cell functional assay for the discovery and retrieval of TCR α/β gene sequences that confer specific recognition of endogenously processed and presented public neoantigens and not the corresponding wild type (WT) sequence. In this approach, donor-derived T cells are sensitized with autologous antigen presenting cells (APCs) electroporated withRNA encoding PIK3CA hotspot mutations. Expanded T cells are subsequently divided into paired daughter wells for short-term co-culture with APCs electroporated with minigenes containing either mutant or WT PIK3CA sequences. Acutely re-stimulated T cells from paired wells are subject to single-cell α/β TCR VDJ and RNA sequencing. TCR α/β gene sequences associated with selective upregulation of TCR signaling transcripts to mutant but not WT PIK3CA stimulation are subsequently cloned into retroviral vectors to confirm reactivity. Results: Using this method, we have retrieved multiple TCRs that confer specific recognition of a public neoepitope derived from a PIK3CA hotspot mutation. These TCRs are restricted by HLA-A*03:01, an allele present in 20.5% of the North American population. Immune-precipitation/tandem mass spectrometry analysis determined that the endogenously processed and presented public neoepitope is a 9 amino acid sequence containing the hotspot substitution at position 2. To understand the mechanistic basis for the immunogenicity of this public neoepitope, we generated x-ray crystallography structures of mutant and WT epitopes bound to HLA-A*03:01 at ~2Å resolution. These studies revealed significant topologic overlap in the bound peptides. By contrast, the thermal and kinetic stability of the mutant peptide/HLA-A*03:01 complex was significantly enhanced relative to the WT complex, as measured by differential scanning fluorimetry and fluorescence anisotropy assays. Peripheral blood T cells genetically engineered with PIK3CA public neoantigen-specific TCRs cleared target cells in a HLA/mutation-specific manner, leaving HLA-mismatched or WT target cells unperturbed. Conclusions: These findings reveal for the first time the existence of a naturally processed and presented public neoantigen derived from a prevalent PIK3CA hotspot mutation. These results open the possibility of specifically targeting this common driver oncogene using T cell-based cancer immunotherapies, including genetically redirected T cells and therapeutic vaccines. Citation Format: C Klebanoff. T cell-based immunotherapy for a “public” neoantigen derived from a PIK3CA hotspot mutation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr MS1-2.

Aptamer structure switch fluorescence anisotropy assay for aflatoxin B1 using tetramethylrhodamine-guanine interaction to enhance signal change

Fluorescence anisotropy (FA) assay in homogenous solution is simple, sensitive and reproducible. Here, we reported an aptamer structure switch FA assay for detection of aflatoxin B1 (AFB1), one of the most toxic mycotoxins, by using tetramethylrhodamine (TMR)-labeled aptamer probe and its complementary DNA (cDNA) with tandem G bases extension, to meet the demand in sensitive and selective detection of AFB1. The hybridization of aptamer and cDNA drew TMR close to the repeated guanine (G) bases, and a high FA value was induced due to TMR-G interaction and restricted local rotation of TMR. In the presence of AFB1, aptamer bound to AFB1 instead of the cDNA due to competition. Thus, the TMR-G interaction was eliminated, and FA value of TMR decreased. This assay enabled the detection of AFB1 with detection limit of 125 pmol/L and dynamic range from 125 pmol/L to 31.2 nmol/L

DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing

Recently, various inorganic nanomaterials have been used as fluorescence anisotropy (FA) enhancers for biosensing successfully. However, most of them are size-uncontrollable and possess an intensive fluorescence quenching ability, which will seriously reduce the accuracy and sensitivity of FA method. Herein, we report a two-dimensional DNA nanosheet (DNS) without fluorescence quenching effect as a novel FA amplification platform. In our strategy, fluorophore-labeled probe DNA (pDNA) is linked onto the DNS surface through the hybridization with the handle DNA (hDNA) that extended from the DNS, resulting in the significantly enhanced FA value. After the addition of target, the pDNA was released from the DNS surface due to the high affinity between the hDNA and target, and the FA was decreased. Thus, target could be detected by the significantly decreased FA value. The linear range was 10–50 nM and the limit of detection was 8 nM for the single-stranded DNA detection. This new method is general and has been also successfully applied for the detection of ATP and thrombin sensitively. Our method improved the accuracy of FA assay and has great potential to detect series of biological analytes in complex biosensing systems.

The constitutive activity of melanocortin-4 receptors in cAMP pathway is allosterically modulated by zinc and copper ions.

Melanocortin-4 receptors (MC4 R) are unique among G-protein-coupled receptors (GPCRs) as they have endogenous ligands that can exhibit inverse agonistic properties in the case of elevated basal activity. It is known that the constitutive activity of GPCRs strongly affects the ligand-dependent physiological responses, but little is known about these regulatory mechanisms. Since several metal ions have been shown to be important modulators of the signal transduction of GPCRs, we hypothesized that metal ions regulate the basal activity of MC4 Rs. Implementation of a fluorescence anisotropy assay and novel redshifted fluorescent peptides enabled kinetic characterization of ligand binding to MC4 R expressed on budded baculoviruses. We show that Ca2+ is required for high-affinity ligand binding, but Zn2+ and Cu2+ in the presence of Ca2+ behave as negative allosteric modulators of ligand binding to MC4 R. FRET-based cAMP biosensor was used to measure the activation of MC4 R stably expressed in CHO-K1 cells. At low micromolar concentrations, Zn2+ caused MC4 R-dependent activation of the cAMP pathway, whereas Cu2+ reduced the activity of MC4 R even below the basal level. These findings indicate that at physiologically relevant concentrations can Zn2+ and Cu2+ function as MC4 R agonists or inverse agonists, respectively. This means that depending on the level of constitutive activity induced by Zn2+ ions, the pharmacological effect of orthosteric ligands of MC4 R can be switched from a partial to an inverse agonist. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the OpenScience badges can be found at https://cos.io/our-services/open-science-badges/.

Abstract CN01-03: T cell receptor gene therapy for a public neoantigen derived from mutated PIK3CA, a dominant driver oncogene in breast and endometrial cancers

Abstract Background: “Public” neoantigens represent immunogenic epitopes encompassing hotspot mutations in driver oncogenes that are also restricted by common HLA alleles. In contrast with patient-specific “private” neoantigens, public neoantigens are conceptually attractive because they are tumor-specific, clonally conserved, and shared across patients. Whether PIK3CA, the most common driver oncogene in breast and endometrial cancer, can yield public neoepitopes that may be exploited for cancer immunotherapy is unknown. Methods: We have developed a high-throughput, single-cell functional assay for the discovery and retrieval of TCR a/b gene sequences that confer specific recognition of endogenously processed and presented public neoantigens and not the corresponding wild type (WT) sequence. In this approach, donor-derived T cells are sensitized with autologous antigen presenting cells (APCs) electroporated with RNA encoding PIK3CA hotspot mutations. Expanded T cells are subsequently divided into paired daughter wells for short-term co-culture with APCs electroporated with minigenes containing either mutant or WT PIK3CA sequences. Acutely re-stimulated T cells from paired wells are subject to single-cell a/b TCR VDJ and RNA sequencing. TCR a/b gene sequences associated with selective upregulation of TCR signaling transcripts to mutant but not WT PIK3CA stimulation are subsequently cloned into retroviral vectors to confirm reactivity. Results: Using this method, we have retrieved multiple TCRs that confer specific recognition of a public neoepitope derived from a PIK3CA hotspot mutation (PIK3CAH1047L). These TCRs are restricted by HLA-A*03:01, an allele present in 20.5% of the North American population. Immune-precipitation/tandem mass spectrometry analysis determined that the endogenously processed and presented public neoepitope is a 9 amino acid sequence containing a HisàLeu substitution at position 2. To understand the mechanistic basis for the immunogenicity of this public neoepitope, we generated x-ray crystallography structures of mutant and WT epitopes bound to HLA-A*03:01 at ~2Å resolution. These studies revealed significant topologic overlap in the bound peptides. By contrast, the thermal and kinetic stability of the mutant peptide/HLA-A*03:01 complex was significantly enhanced relative to the WT complex, as measured by differential scanning fluorimetry and fluorescence anisotropy assays. Peripheral blood T cells genetically engineered with PIK3CA public neoantigen-specific TCRs cytolytically cleared target cells in a HLA/mutation-specific manner, leaving HLA-mismatched or WT target cells unperturbed. Conclusions: These findings reveal for the first time the existence of an endogenously processed and presented public neoantigen derived from a PIK3CA hotspot mutation. These results open the possibility of targeting this common driver oncogene using adoptively transferred and genetically redirected T cells. Citation Format: Smita Chandran, Jiaqi Ma, Martin G. Klatt, Friederike Dündar, Paul Zumbo, Matthew R. Femia, Doron Betel, David A. Scheinberg, Brian M. Baker, Christopher A. Klebanoff. T cell receptor gene therapy for a public neoantigen derived from mutated PIK3CA, a dominant driver oncogene in breast and endometrial cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN01-03. doi:10.1158/1535-7163.TARG-19-CN01-03

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Summary Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi‐drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H‐NS‐like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H‐NS, Lsr2 binds DNA in sequence‐dependent and non‐specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA‐binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP‐sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

An Inhibitor of the Interaction of Survivin with Smac in Mitochondria Promotes Apoptosis.

Herein we report the first small molecule that disrupts the survivin-Smac interaction taking place in mitochondria. The inhibitor, PZ-6-QN, was identified by initially screening a phenothiazine library using a fluorescence anisotropy assay and then conducting a structure-activity relationship study. Mutagenesis and molecular docking studies suggest that PZ-6-QN binds to survivin similarly to the known Smac peptide, AVPI. The results of the effort also show that PZ-6-QN exhibits good anticancer activity against various cancer cells. Moreover, cell-based mechanistic studies provide evidence for the proposal that PZ-6-QN enters mitochondria to inhibit the survivin-Smac interaction and promotes release of Smac and cytochrome c from mitochondria into the cytosol, a process that induces apoptosis in cancer cells. Overall, the present study suggests that PZ-6-QN can serve as a novel chemical probe for study of processes associated with the mitochondrial survivin-Smac interaction and it will aid the discovery of novel anticancer agents.

Direct Zinc Finger Protein Persulfidation by H2S Is Facilitated by Zn2+

AbstractH2S is a gaseous signaling molecule that modifies cysteine residues in proteins to form persulfides (P‐SSH). One family of proteins modified by H2S are zinc finger (ZF) proteins, which contain multiple zinc‐coordinating cysteine residues. Herein, we report the reactivity of H2S with a ZF protein called tristetraprolin (TTP). Rapid persulfidation leading to complete thiol oxidation of TTP mediated by H2S was observed by low‐temperature ESI‐MS and fluorescence spectroscopy. Persulfidation of TTP required O2 , which reacts with H2S to form superoxide, as detected by ESI‐MS, a hydroethidine fluorescence assay, and EPR spin trapping. H2S was observed to inhibit TTP function (binding to TNFα mRNA) by an in vitro fluorescence anisotropy assay and to modulate TNFα in vivo. H2S was unreactive towards TTP when the protein was bound to RNA, thus suggesting a protective effect of RNA.

Direct Zinc Finger Protein Persulfidation by H2 S Is Facilitated by Zn2.

H2 S is a gaseous signaling molecule that modifies cysteine residues in proteins to form persulfides (P-SSH). One family of proteins modified by H2 S are zinc finger (ZF) proteins, which contain multiple zinc-coordinating cysteine residues. Herein, we report the reactivity of H2 S with a ZF protein called tristetraprolin (TTP). Rapid persulfidation leading to complete thiol oxidation of TTP mediated by H2 S was observed by low-temperature ESI-MS and fluorescence spectroscopy. Persulfidation of TTP required O2 , which reacts with H2 S to form superoxide, as detected by ESI-MS, a hydroethidine fluorescence assay, and EPR spin trapping. H2 S was observed to inhibit TTP function (binding to TNFα mRNA) by an in vitro fluorescence anisotropy assay and to modulate TNFα in vivo. H2 S was unreactive towards TTP when the protein was bound to RNA, thus suggesting a protective effect of RNA.

Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease in which the formation of extracellular aggregates of amyloid beta (Aβ) peptide, fibrillary tangles of intraneuronal tau and microglial activation are major pathological hallmarks. One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer’s disease) mice and found specifically expressed in microglia associated with Aβ plaques. Single-nucleotide polymorphisms in the LGALS3 gene, which encodes gal3, were associated with an increased risk of AD. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data revealed that gal3 was required to fully activate microglia in response to fibrillar Aβ. Gal3 deletion decreased the Aβ burden in 5xFAD mice and improved cognitive behavior. Interestingly, a single intrahippocampal injection of gal3 along with Aβ monomers in WT mice was sufficient to induce the formation of long-lasting (2 months) insoluble Aβ aggregates, which were absent when gal3 was lacking. High-resolution microscopy (stochastic optical reconstruction microscopy) demonstrated close colocalization of gal3 and TREM2 in microglial processes, and a direct interaction was shown by a fluorescence anisotropy assay involving the gal3 carbohydrate recognition domain. Furthermore, gal3 was shown to stimulate TREM2–DAP12 signaling in a reporter cell line. Overall, our data support the view that gal3 inhibition may be a potential pharmacological approach to counteract AD.

Design of CK2β-Mimicking Peptides as Tools To Study the CK2α/CK2β Interaction in Cancer Cells.

The ubiquitously expressed Ser/Thr kinase CK2 is a key regulator in a variety of key processes in normal and malignant cells. Due to its distinctive anti-apoptotic and tumor-driving properties, elevated levels of CK2 have frequently been found in tumors of different origin. In recent years, development of CK2 inhibitors has largely been focused on ATP-competitive compounds; however, targeting the CK2α/CK2β interface has emerged as a further concept that might avoid selectivity issues. To address the CK2 subunit interaction site, we have synthesized halogenated CK2β-mimicking cyclic peptides modified with the cell-penetrating peptide sC18 to mediate cellular uptake. We investigated the binding of the resulting chimeric peptides to recombinant human CK2α using a recently developed fluorescence anisotropy assay. The iodinated peptide sC18-I-Pc was identified as a potent CK2α ligand (Ki =0.622 μm). It was internalized in cells to a high extent and exhibited significant cytotoxicity toward cancerous HeLa cells (IC50 =37 μm) in contrast to non-cancerous HEK-293 cells. The attractive features and functionalities of sC18-I-Pc offer the opportunity for further improvement.

A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1–JAZ co-receptor in plants

The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine regulates many developmental and stress responses in plants and induces protein-protein interactions between COI1, the F-box component of E3 ubiquitin ligase, and jasmonate ZIM domain (JAZ) repressors. These interactions cause JAZ degradation and activate jasmonate (JA), leading to plant defense responses, growth inhibition, and senescence. Thirteen JAZ subtypes are encoded in the Arabidopsis thaliana genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of JAZ genes. One strategy to elucidate the complex JA signaling pathways is to develop a reliable and comprehensive binding assay system of the ligands with all combinations of the co-receptors. Herein, we report the development of a fluorescence anisotropy-based in vitro binding assay system to screen for the ligands of the COI1-JAZ co-receptors. Our assay enabled the first quantitative analysis of the affinity values and JAZ-subtype selectivity of various endogenous JA derivatives, such as coronatine, jasmonic acid, and 12-hydroxyjasmonoyl-l-isoleucine. Because of its high signal-to-noise ratio and convenient mix-and-read assay system, our screening approach can be used in plate reader-based assays of both agonists and antagonists of COI1-JAZ co-receptors.

PH- and Temperature-Dependent Peptide Binding to the Lactococcus lactis Oligopeptide-Binding Protein A Measured with a Fluorescence Anisotropy Assay.

Bacterial ATP-binding cassette transporters are a superfamily of transport systems involved in the import of various molecules including amino acids, ions, sugars, and peptides. In the lactic acid bacteria Lactococcus lactis, the oligopeptide-binding protein A (OppA) binds peptides for import to support nitrogen metabolism and cell growth. The OppA protein is of great interest because it can bind peptides over a broad variety of lengths and sequences; however, current methods to study peptide binding have employed low throughput, endpoint, or low dynamic range techniques. Therefore, in this study, we developed a fluorescence anisotropy-based peptide-binding assay that can be readily employed to quantify OppA function. To test the utility of our assay, we characterized the pH dependence of oligopeptide binding because L. lactis is commonly used in fermentation and often must survive in low pH environments caused by lactic acid export. We determined that OppA affinity increases as pH or temperature decreases, and circular dichroism spectroscopy further indicated that acidic conditions increase the thermal stability of the protein, increasing the unfolding transition temperature by 10 °C from pH 8 to pH 6. Thus, our fluorescence anisotropy assay provides an easy technique to measure peptide binding, and it can be used to understand molecular aspects of OppA function under stress conditions experienced during fermentation and other biotechnology applications.

Molecular mechanism of poliovirus Sabin vaccine strain attenuation

Recruitment of poliovirus (PV) RNA to the human ribosome requires the coordinated interaction of the viral internal ribosome entry site (IRES) and several host cellular initiation factors and IRES trans-acting factors (ITAFs). Attenuated PV Sabin strains contain point mutations in the PV IRES domain V (dV) that inhibit viral translation. Remarkably, attenuation is most apparent in cells of the central nervous system, but the molecular basis to explain this is poorly understood. The dV contains binding sites for eukaryotic initiation factor 4G (eIF4G) and polypyrimidine tract-binding protein (PTB). Impaired binding of these proteins to the mutant IRESs has been observed, but these effects have not been quantitated. We used a fluorescence anisotropy assay to reveal that the Sabin mutants reduce the equilibrium dissociation constants of eIF4G and PTB to the PV IRES by up to 6-fold. Using the most inhibitory Sabin 3 mutant, we used a real-time fluorescence helicase assay to show that the apparent affinity of an active eIF4G/4A/4B helicase complex for the IRES is reduced by 2.5-fold. The Sabin 3 mutant did not alter the maximum rate of eIF4A-dependent helicase activity, suggesting that this mutant primarily reduces the affinity, rather than activity, of the unwinding complex. To confirm this affinity model of attenuation, we show that eIF4G overexpression in HeLa cells overcomes the attenuation of a Sabin 3 mutant PV-luciferase replicon. Our study provides a quantitative framework for understanding the mechanism of PV Sabin attenuation and provides an explanation for the previously observed cell type-specific translational attenuation.

Identification of inhibitors of the E. coli chaperone SurA using in silico and in vitro techniques

SurA is a gram-negative, periplasmic chaperone protein involved in the proper folding of outer membrane porins (OMPs), which protect bacteria against toxins in the extracellular environment by selectively regulating the passage of nutrients into the cell. Previous studies demonstrated that deletion of SurA renders bacteria more sensitive to toxins that compromise the integrity of the outer membrane. Inhibitors of SurA will perturb the folding of OMPs, leading to disruption of the outer membrane barrier and making the cell more vulnerable to toxic insults. The discovery of novel SurA inhibitors is therefore of great importance for developing alternative strategies to overcome antibiotic resistance. Our laboratory has screened over 10,000,000 compoundsin silicoby computationally docking these compounds onto the crystal structure of SurA. Through this screen and a screen of fragment compounds (molecular weight less than 250 g/mol), we found twelve commercially readily available candidate compounds that bind to the putative client binding site of SurA. We confirmed binding to SurA by developing and employing a competitive fluorescence anisotropy-based binding assay. Our results show that one of these compounds, Fmoc-β-(2-quinolyl)-d-alanine, binds the client binding site with high micromolar affinity. Using this compound as a lead, we also discovered that Fmoc-l-tryptophan and Fmoc-l-phenylalanine, but not Fmoc-l-tyrosine, bind SurA with similar micromolar affinity. To our knowledge, this is the first report of a competitive fluorescence anisotropy assay developed for the identification of inhibitors of the chaperone SurA, and the identification of three small molecules that bind SurA at its client binding site.

Direct fluorescence anisotropy approach for aflatoxin B1 detection and affinity binding study by using single tetramethylrhodamine labeled aptamer

The discovery of aptamers for aflatoxin B1 (AFB1), one of toxic carcinogens, has allowed to develop aptamer-based sensors and assays for aflatoxin. In this work, we reported a direct fluorescence anisotropy (FA) assay for investigation of aptamer-AFB1 binding and detection of AFB1 with the aptamer having single tetramethylrhodamine (TMR) label on a specific site. From a series of labeling sites of a 50-mer aptamer, we screened out the aptamer with TMR labeling at the 26th T, capable of generating good and large FA-decreasing response to AFB1. By using the T26-labeled 50-mer aptamer probe in FA analysis, we determined the affinity and selectivity of aptamer, and identified the crucial region of aptamer and optimum experimental conditions for strong binding. The aptamer could be further truncated to as short as 26 nucleotides in length, and this shorter aptamer possessed a simple stem-loop secondary structure and retained good binding affinity. Nucleotides in the loop region of the aptamer were conserved and important for affinity recognition. We achieved FA detection of AFB1 with a detection limit about 2 nM by using the TMR-labeled aptamer probe. The cross reactivity of aflatoxin B1, aflatoxin B2, aflatoxin M1, aflatoxin M2, aflatoxin G1, and aflatoxin G2 with aptamer were estimated to be 100%, 61%, 23%, 21%, 6.3%, 6.5%, respectively. The aptamer probe presented good selectivity over other mycotoxins and showed potential in complex sample analysis. This study of affinity binding between aptamer and aflatoxins will be helpful for developing other aptamer-based assays and sensors for aflatoxins.

Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins

Herein, we report for the first time the isolation of DNA aptamers directed against the whole tau protein, an important Alzheimer's disease (AD) biomarker. Non-SELEX approach based on the capillary electrophoresis partitioning technique was employed to isolate a high-affinity DNA sequence pool towards the target in only three rounds and one working day. High-throughput sequencing was next performed and the recognition ability of five selected aptamers was preliminary evaluated by surface plasmon resonance using the protein target immobilized on the chip. Finally, the analytical potential of the most affine aptamer was demonstrated through the design of a homogeneous-phase fluorescence anisotropy assay. This DNA aptamer was found to be able to recognize not only the whole τ-441 but also the τ-381, τ-352, τ-383 isoforms. The sensing platform allowed the determination of these four targets with a detection limit of 28 nM, 3.2 nM, 6.3 nM and 22 nM, respectively.

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine\xa0site of tubulin

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Precision Targeting of Tumor Macrophages with a CD206 Binding Peptide

Tumor-associated macrophages (TAMs) expressing the multi-ligand endocytic receptor mannose receptor (CD206/MRC1) contribute to tumor immunosuppression, angiogenesis, metastasis, and relapse. Here, we describe a peptide that selectively targets MRC1-expressing TAMs (MEMs). We performed in vivo peptide phage display screens in mice bearing 4T1 metastatic breast tumors to identify peptides that target peritoneal macrophages. Deep sequencing of the peptide-encoding inserts in the selected phage pool revealed enrichment of the peptide CSPGAKVRC (codenamed “UNO”). Intravenously injected FAM-labeled UNO (FAM-UNO) homed to tumor and sentinel lymph node MEMs in different cancer models: 4T1 and MCF-7 breast carcinoma, B16F10 melanoma, WT-GBM glioma and MKN45-P gastric carcinoma. Fluorescence anisotropy assay showed that FAM-UNO interacts with recombinant CD206 when subjected to reducing conditions. Interestingly, the GSPGAK motif is present in all CD206-binding collagens. FAM-UNO was able to transport drug-loaded nanoparticles into MEMs, whereas particles without the peptide were not taken up by MEMs. In ex vivo organ imaging, FAM-UNO showed significantly higher accumulation in sentinel lymph nodes than a control peptide. This study suggests applications for UNO peptide in diagnostic imaging and therapeutic targeting of MEMs in solid tumors.