Cellulose acetate microwell plates for high-throughput colorimetric assays.

Single use plasticware (SUP) in scientific, diagnostic, and academic laboratories makes a significant contribution to plastic waste generation worldwide. Polystyrene (PS) microwell plates form a part of this waste. These plates are the backbone of high throughput colorimetric measurements in academic, research, and healthcare settings for detection/quantification of wide-ranging analytes including proteins, carbohydrates, nucleic acids, and enzyme activity. Polystyrene (PS) microwell plates serve as a platform for holding samples and reagents, where mixing initiates chemical reaction(s), and the ensuing color changes are quantified using a microplate reader. However, these plates are rarely reused or recycled, contributing to the staggering amounts of plastic waste generated in scientific laboratories. Here, we are reporting the fabrication of cellulose acetate (CA) microwell plates as a greener alternative to non-biodegradable PS plates and we demonstrate their application in colorimetric assays. These easy to fabricate, lighter weight, customizable, and environmentally friendly plates were fabricated in 96- and 384-well formats and made water impermeable through chemical treatment. The plates were tested in three different colorimetric analyses: (i) bicinchoninic acid assay (BCA) for protein quantification; (ii) chymotrypsin (CT) activity assay; and (iii) alkaline phosphatase (AP) activity assay. Color intensities were quantified using a freely available smartphone application, Spotxel® Reader (Sicasys Software GmbH). To benchmark the performance of this platform, the same assays were performed in commercial PS plates too and quantified using a UV/Vis microplate reader. The two systems yielded comparable linear correlation coefficients, LOD and LOQ values, thereby validating the CA plate-cell phone based analytical method. The CA microwell plates, coupled with smart phone optical data capture, provide greener, accessible, and scalable tools for all laboratory settings and are particularly well-suited for resource- and infrastructure-limited environments.

Heterozygous nonsense variants in the ferritin heavy-chain gene FTH1 cause a neuroferritinopathy.

Ferritin, the iron-storage protein, is composed of light- and heavy-chain subunits, encoded by FTL and FTH1, respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole-exome sequencing, with a recurrent variant (p.Phe171∗) identified in four unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia, and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminal variants in FTH1 truncate ferritin's E helix, altering the 4-fold symmetric pores of the heteropolymer, and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a disorder in the spectrum of NBIA. Targeted knockdown of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this pediatric neurodegenerative disorder.

Chelate-functionalized magnetic micelles for sequestration of cisplatin.

Many cancer patients suffer permanent hearing loss due to accumulation of ototoxic cisplatin in the inner ear. In this study, two types of 100 nm magnetic micelles were developed to sequester cisplatin from aqueous solutions, with the goal of eliminating cochlear ototoxins via magnetic microsurgery. The micellar surface was quantitatively functionalized with anionic S-rich ligands and the micelle core encapsulated superparamagnetic iron oxide nanoparticles. Exceptionally effective sequestration is demonstrated, with removal of greater than 95 and 50% of solution Pt, by means of centrifugal filtration and magnetic extraction. Attraction between negatively charged micellar surfaces and cationic Pt-species played a critical role and was only partially screened by physiologic salt solution. Importantly, magnetic micelles introduce negligible impact on the integrity of inner ear hair cells, demonstrating excellent biocompatibility. This study showcases successful magnetic sequestration of Pt-based ototoxins using highly applicable nano-micellar materials. More generally, these examples highlight features of the micelle-water interfacial environment that are important in developing nanomaterials for metallo-medicinal applications.

Local Xenon-Protein Interaction Produces Global Conformational Change and Allosteric Inhibition in Lysozyme.

Noble gases have well-established biological effects, yet their molecular mechanisms remain poorly understood. Here, we investigated, both experimentally and computationally, the molecular modes of xenon (Xe) action in bacteriophage T4 lysozyme (T4L). By combining indirect gassing methods with a colorimetric lysozyme activity assay, a reversible, Xe-specific (20 ± 3)% inhibition effect was observed. Accelerated molecular dynamic simulations revealed that Xe exerts allosteric inhibition on the protein by expanding a C-terminal hydrophobic cavity. Xe-induced cavity expansion results in global conformational changes, with long-range transduction distorting the active site where peptidoglycan binds. Interestingly, the peptide substrate binding site that enables lysozyme specificity does not change conformation. Two T4L mutants designed to reshape the C-terminal Xe cavity established a correlation between cavity expansion and enzyme inhibition. This work also highlights the use of Xe flooding simulations to identify new cryptic binding pockets. These results enrich our understanding of Xe-protein interactions at the molecular level and inspire further biochemical investigations with noble gases.

Rational design of a genetically encoded NMR zinc sensor.

Elucidating the biochemical roles of the essential metal ion, Zn2+, motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn2+ in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive 129Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn2+, we converted the MBP salt bridge into a Zn2+ binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 µM achieved 'turn-on' detection of Zn2+ with pronounced hyper-CEST contrast. This made it possible to determine different Zn2+ levels in a biological fluid via hyper-CEST. ZS was responsive to low-micromolar Zn2+, only modestly responsive to Cu2+, and nonresponsive to other biologically important metal ions, according to hyper-CEST NMR spectroscopy and isothermal titration calorimetry (ITC). Protein X-ray crystallography confirmed the identity of the bound Zn2+ ion using anomalous scattering: Zn2+ was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn2+ forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, 129Xe NMR chemical shift, and exchange rate are tunable parameters via protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.

Ratiometric, pH-Sensitive Probe for Monitoring siRNA Delivery.

Many RNA delivery strategies require efficient endosomal uptake and release. To monitor this process, we developed a 2'-OMe RNA-based ratiometric pH probe with a pH-invariant 3'-Cy5 and 5'-FAM whose pH sensitivity is enhanced by proximal guanines. The probe, in duplex with a DNA complement, exhibits a 48.9-fold FAM fluorescence enhancement going from pH 4.5 to pH 8.0 and reports on both endosomal entrapment and release when delivered to HeLa cells. In complex with an antisense RNA complement, the probe constitutes an siRNA mimic capable of protein knockdown in HEK293T cells. This illustrates a general approach for measuring the localization and pH microenvironment of any oligonucleotide.

Heterozygous Nonsense Variants in the Ferritin Heavy Chain Gene FTH1 Cause a Novel Pediatric Neuroferritinopathy.

Ferritin, the iron storage protein, is composed of light and heavy chain subunits, encoded by FTL and FTH1 , respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole exome sequencing, with a recurrent de novo variant (p.F171*) identified in three unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminus variants in FTH1 truncate ferritin's E-helix, altering the four-fold symmetric pores of the heteropolymer and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a novel disorder in the spectrum of NBIA. Targeted knock-down of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this novel pediatric neurodegenerative disorder.

Turn-on fluorescent capsule for selective fluoride detection and water purification.

It has been a long-standing challenge to develop organic molecular capsules for selective anion binding in water. Here, selective recognition of aqueous fluoride was achieved through triple protonation of a hemicryptophane (L), which is composed of a fluorescent cyclotriveratrylene (CTV) cap and tris(2-aminoethyl)amine (tren) as the anion binding site. Fluoride encapsulation by [3H-L]3+ was evidenced by 1H NMR, 19F NMR, LC-MS, and X-ray crystallography. In addition, [3H-L]3+ exhibited a 'turn-on' fluorescence signal (λ em = 324 nm) upon fluoride addition. An apparent association constant K A = (7.5 ± 0.4) × 104 M-1 and a detection limit of 570 nM fluoride were extracted from the fluorescence titration experiments in citrate buffer at pH 4.1. To the best of our knowledge, [3H-L]3+ is the first example of a metal-free molecular capsule that reports on fluoride binding in purely aqueous solutions with a fluorescence response. Finally, the protonated capsule was supported on silica gel, which enabled adsorptive removal of stoichiometric fluoride from water and highlights real-world applications of this organic host-guest chemistry.

Structure-Guided Design of a Potent and Specific Inhibitor against the Genomic Mutator APOBEC3A.

Nucleic acid structure plays a critical role in governing the selectivity of DNA- and RNA-modifying enzymes. In the case of the APOBEC3 family of cytidine deaminases, these enzymes catalyze the conversion of cytosine (C) to uracil (U) in single-stranded DNA, primarily in the context of innate immunity. DNA deamination can also have pathological consequences, accelerating the evolution of viral genomes or, when the host genome is targeted by either APOBEC3A (A3A) or APOBEC3B (A3B), promoting tumor evolution leading to worse patient prognosis and chemotherapeutic resistance. For A3A, nucleic acid secondary structure has emerged as a critical determinant of substrate targeting, with a predilection for DNA that can form stem loop hairpins. Here, we report the development of a specific nanomolar-level, nucleic acid-based inhibitor of A3A. Our strategy relies on embedding the nucleobase 5-methylzebularine, a mechanism-based inhibitor, into a DNA dumbbell structure, which mimics the ideal substrate secondary structure for A3A. Structure-activity relationship studies using a panel of diverse inhibitors reveal a critical role for the stem and position of the inhibitor moiety in achieving potent inhibition. Moreover, we demonstrate that DNA dumbbell inhibitors, but not nonstructured inhibitors, show specificity against A3A relative to the closely related catalytic domain of A3B. Overall, our work demonstrates the feasibility of leveraging secondary structural preferences in inhibitor design, offering a blueprint for further development of modulators of DNA-modifying enzymes and potential therapeutics to circumvent APOBEC-driven viral and tumor evolution.

Programming xenon diffusion in maltose-binding protein.

Protein interiors contain void space that can bind small gas molecules. Determination of gas pathways and kinetics in proteins has been an intriguing and challenging task. Here, we combined computational methods and the hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR technique to investigate xenon (Xe) exchange kinetics in maltose-binding protein (MBP). A salt bridge ∼9 Å from the Xe-binding site formed upon maltose binding and slowed the Xe exchange rate, leading to a hyper-CEST 129Xe signal from maltose-bound MBP. Xe dissociation occurred faster than dissociation of the salt bridge, as shown by 13C NMR spectroscopy and variable-B1 hyper-CEST experiments. "Xe flooding" molecular dynamics simulations identified a surface hydrophobic site, V23, that has good Xe binding affinity. Mutations at this site confirmed its role as a secondary exchange pathway in modulating Xe diffusion. This shows the possibility for site-specifically controlling xenon protein-solvent exchange. Analysis of the available MBP structures suggests a biological role of MBP's large hydrophobic cavity to accommodate structural changes associated with ligand binding and protein-protein interactions.

Counteranions at Peripheral Sites Tune Guest Affinity for a Protonated Hemicryptophane.

The affinity of small molecules for biomolecular cavities is tuned through a combination of primary and secondary interactions. It has been challenging to mimic these features in organic synthetic host molecules, however, where the cavities tend to be highly symmetric and nonpolar, and less amenable to chemical manipulation. Here, a host molecule composed of a TREN ligand and cyclotriveratrylene moiety was investigated. Size-matched polar guests were encapsulated within the cavity via triple protonation of the TREN moiety with various sulfonic acids. X-ray crystallography confirmed guest encapsulation and identified three methanesulfonates, p-toluenesulfonates, or 2-naphthalenesulfonates hydrogen-bonded with H3TREN at the periphery of the cavity. These structurally diverse counteranions were shown by 1H NMR spectroscopy to differentially regulate guest access at the three portals, and to undergo competitive displacement in solution. This work reveals "counteranion tuning" to be a simple and powerful strategy for modulating host-guest affinity, as applied here in a TREN-hemicryptophane.

Cryptophane-xenon complexes for 129Xe MRI applications.

The use of magnetic resonance imaging (MRI) and spectroscopy (MRS) in the clinical setting enables the acquisition of valuable anatomical information in a rapid, non-invasive fashion. However, MRI applications for identifying disease-related biomarkers are limited due to low sensitivity at clinical magnetic field strengths. The development of hyperpolarized (hp) 129Xe MRI/MRS techniques as complements to traditional 1H-based imaging has been a burgeoning area of research over the past two decades. Pioneering experiments have shown that hp 129Xe can be encapsulated within host molecules to generate ultrasensitive biosensors. In particular, xenon has high affinity for cryptophanes, which are small organic cages that can be functionalized with affinity tags, fluorophores, solubilizing groups, and other moieties to identify biomedically relevant analytes. Cryptophane sensors designed for proteins, metal ions, nucleic acids, pH, and temperature have achieved nanomolar-to-femtomolar limits of detection via a combination of 129Xe hyperpolarization and chemical exchange saturation transfer (CEST) techniques. This review aims to summarize the development of cryptophane biosensors for 129Xe MRI applications, while highlighting innovative biosensor designs and the consequent enhancements in detection sensitivity, which will be invaluable in expanding the scope of 129Xe MRI.

Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization.

Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.

Conditionally Activated ("Caged") Oligonucleotides.

Conditionally activated ("caged") oligonucleotides provide useful spatiotemporal control for studying dynamic biological processes, e.g., regulating in vivo gene expression or probing specific oligonucleotide targets. This review summarizes recent advances in caging strategies, which involve different stimuli in the activation step. Oligo cyclization is a particularly attractive caging strategy, which simplifies the probe design and affords oligo stabilization. Our laboratory developed an efficient synthesis for circular caged oligos, and a circular caged antisense DNA oligo was successfully applied in gene regulation. A second technology is Transcriptome In Vivo Analysis (TIVA), where caged oligos enable mRNA isolation from single cells in living tissue. We highlight our development of TIVA probes with improved caging stability. Finally, we illustrate the first protease-activated oligo probe, which was designed for caspase-3. This expands the toolkit for investigating the transcriptome under a specific physiologic condition (e.g., apoptosis), particularly in specimens where light activation is impractical.

Monomeric Cryptophane with Record-High Xe Affinity Gives Insights into Aggregation-Dependent Sensing.

Cryptophane host molecules provide ultrasensitive contrast agents for 129Xe NMR/MRI. To investigate key features of cryptophane-Xe sensing behavior, we designed a novel water-soluble cryptophane with a pendant hydrophobic adamantyl moiety, which has good affinity for a model receptor, beta-cyclodextrin (ß-CD). Adamantyl-functionalized cryptophane-A (AFCA) was synthesized and characterized for Xe affinity, 129Xe NMR signal, and aggregation state at varying AFCA and ß-CD concentrations. The Xe-AFCA association constant was determined by fluorescence quenching, KA = 114,000 ± 5000 M-1 at 293 K, which is the highest reported affinity for a cryptophane host in phosphate-buffered saline (pH 7.2). No hyperpolarized (hp) 129Xe NMR peak corresponding to AFCA-bound Xe was directly observed at high (100 µM) AFCA concentration, where small cryptophane aggregates were observed, and was only detected at low (15 µM) AFCA concentration, where the sensor remained fully monomeric in solution. Additionally, we observed no change in the chemical shift of AFCA-encapsulated 129Xe after ß-CD binding to the adamantyl moiety and a concomitant lack of change in the size distribution of the complex, suggesting that a change in the aggregation state is necessary to elicit a 129Xe NMR chemical shift in cryptophane-based sensing. These results aid in further elucidating the recently discovered aggregation phenomenon, highlight limitations of cryptophane-based Xe sensing, and offer insights into the design of monomeric, high-affinity Xe sensors.

Photoactivatable Circular Caged Oligonucleotides for Transcriptome In Vivo Analysis (TIVA).

Light activation is an effective way to impart spatiotemporal control over oligonucleotide probes that are widely applied for gene expression regulation and target function investigation. Among the major oligonucleotide caging strategies, cyclization with a photocleavable linker is an elegant design, which affords both atom efficiency and stability in many biological environments. Here, we introduce an improved protocol for circular oligonucleotide synthesis requiring only one round of HPLC purification. With a series of poly-U oligonucleotide strands of different sizes and backbone modifications, the pre-photolysis caging stability and post-photolysis target binding affinity were studied through a denaturing gel assay and melting temperature measurements. A 14U 2'-OMe RNA probe was selected, with strong potential application in transcriptome in vivo analysis (TIVA) for mRNA isolation.

129Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.

Oligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background.

Messenger RNA (mRNA) isolated from single cells can generate powerful biological insights, including the discovery of new cell types with unique functions as well as markers potentially predicting a cell's response to various therapeutic agents. We previously introduced an oligonucleotide-based technique for site-selective, photoinduced biotinylation and capture of mRNA within a living cell called transcriptome in vivo analysis (TIVA). Successful application of the TIVA technique hinges upon its oligonucleotide probe remaining completely inert (or "caged") to mRNA unless photoactivated. To improve the reliability of TIVA probe caging in diverse and challenging biological conditions, we applied a rational design process involving iterative modifications to the oligonucleotide construct. In this work, we discuss these design motivations and present an optimized probe with minimal background binding to mRNA prior to photolysis. We assess its caging performance through multiple in vitro assays including FRET analysis, native gel comigration, and pull down with model mRNA transcripts. Finally, we demonstrate that this improved probe can also isolate mRNA from single living neurons in brain tissue slices with excellent caging control.

Detecting protein-protein interactions by Xe-129 NMR.

Detection of protein-protein interactions (PPIs) is limited by current bioanalytical methods. A protein complementation assay (PCA), split TEM-1 ß-lactamase, interacts with xenon at the interface of the TEM-1 fragments. Reconstitution of TEM-1-promoted here by cFos/cJun leucine zipper interaction-gives rise to sensitive 129Xe NMR signal in bacterial cells.

Caspase-Activated Oligonucleotide Probe.

Light-activated ("caged") oligonucleotides provide a strategy for modulating the activity of antisense oligos, siRNA, miRNA, aptamers, DNAzymes, and mRNA-capturing probes with high spatiotemporal resolution. However, the near-UV and visible wavelengths that promote these bond-breaking reactions poorly penetrate living tissue, which limits some biological applications. To address this issue, we describe the first example of a protease-activated oligonucleotide probe, capable of reporting on caspase-3 during cellular apoptosis. The 2'-F RNA-peptide substrate-peptide nucleic acid (PNA) hairpin structure was generated in 30% yield in a single bioconjugation step.