UMBC Chemistry & Biochemistry Department

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Optimized Preparation of Segmentally Labeled RNAs for NMR Structure Determination
(Elsevier, 2025-03-05) Grossman, Brian D.; Beyene, Bethel G.; Tekle, Bersabel; Sakowicz, William; Ji, Xinjie; Camacho, Joshua Miguele; Vaishnav, Nandini; Ahmed, Amina; Bhandari, Naman; Desai, Kush; Hardy, Josiah; Hollman, Nele M.; Marchant, Jan; Summers, Michael F.
RNA structures are significantly underrepresented in public repositories (? 100-fold compared to proteins) despite their importance for mechanistic understanding and for development of structure prediction/validation tools. A substantial portion of deposited RNA structures have been determined by NMR (? 30%), but most comprise fewer than 60 nucleotides due to complications associated with NMR signal overlap. A promising approach for applying NMR to larger RNAs involves use of a mutated DNA polymerase (TGK) that can extend “primer” RNA strands generated independently by synthetic or enzymatic methods [Haslecker et al., Nat. Commun. 2023]. In attempts to employ this technology, we uncovered sequence- and enzyme-dependent complications for most constructs examined that prohibited preparation of homogeneous samples. By using TGK extension efficiency and NMR as guides, we identified non-templated run-on by wild-type T7-RNA polymerase (RNAPWT) as the primary source of product heterogeneity. Use of 2?-O-methylated DNA templates did not prevent RNAPWT run-on for most constructs examined. However, primer RNAs with appropriate 3?-end homogeneity were obtained in high yield using a recently described T7 RNAP mutant designed for improved immunogenic behavior. Minor spectral heterogeneity sometimes observed for 3? residues, caused by partial premature TGK termination, could be moved to sites downstream of the RNA region of interest by employing extended template DNAs that encode additional non-interacting 3? nucleotides. We additionally present an approach for large-scale synthesis of homogeneous template DNA required for TGK extension. With these modifications, segmentally labeled RNAs appropriate for high resolution structural studies are now routinely obtainable.
A 3D Hepatocyte Model with Composite Nanofibers that Reproduced Human in vivo Drug Clearance Profiles
(2025-03-04) Park, Rudolph; Chen, Chengpeng
This study presents a novel in vitro 3D hepatocyte model that contains a nanofibrous scaffold designed to mimic the extracellular matrix (ECM) of the human liver, both structurally and biochemically. A modular 3D-printed device housing the ECM scaffold was also developed, readily fitting in well plates. HepaRG hepatocytes cultured on the scaffold exhibited enhanced metabolic activity compared to traditional 2D cultures, indicating improved hepatocyte functionality. Drug clearance studies with lidocaine, clozapine, and fluoxetine demonstrated significantly faster clearance rates on the scaffold, closely aligning with in vivo results from literature, while 2D cultures showed limited metabolic capacity. This model offers a physiologically relevant platform for hepatocyte studies. The findings underscore the model’s potential to advance preclinical drug development by replicating liver-specific functions in vitro.
3D-Printed Microfluidic-Based Cell Culture System With Analysis to Investigate Macrophage Activation
(Wiley, 2025-2-18) Selemani, Major A.; Kabandana, Giraso Keza Monia; Chen, Chengpeng; Martin, R. Scott
In this paper, we describe the development of 3D-printed microfluidic cell culture devices that can be coupled with a circulation system to study the dynamics of both intracellular and extracellular (release) processes. Key to this approach is the ability to quantitate key analytes on a minutes timescale with either on-line (in this study, quantitating nitric oxide production using an amperometric flow cell) or off-line (in this work, quantitating intracellular itaconate production with LC/MS) analytical measurements. To demonstrate the usefulness of this approach, we chose to study macrophage polarization as a function of the extracellular matrix (silk) fiber size, a major area of research in tissue engineering. It was found that the use of larger fibers (1280 nm vs. smaller 512 nm fibers) led to increases in the production of both nitric oxide and itaconate. These findings set the foundation for future research for the creation of finely tuned microfluidic 3D cell culture approaches in areas where flow and the extracellular matrix play a significant role in barrier transport and where integrated analysis of key markers is needed.
Application of the Rd/w framework to assess Donnan dialysis performance
(Elsevier, 2023-12-01) Chen, Hui; Souizi, Sahar; Stewart, Kaylyn; Blaney, Lee
Donnan dialysis exploits electrochemical potential gradients across ion-exchange membranes to separate ions between feed and draw solutions. This technique has been applied for treatment and recovery of chemicals in water and wastewater. Previous studies have arbitrarily selected the draw solution chemistry, making it difficult to fairly compare experimental outcomes. A universal framework is needed to standardize design and interpretation of Donnan dialysis systems. We calculated the Rd/w parameter, which is related to the draw ion concentrations in the feed and draw solutions at Donnan equilibrium, for previous studies. Rd/w values were used to determine theoretical recoveries and compare them to experimental outcomes. Of the literature data, 57% matched the theoretical recovery, 37% underperformed due to operating time constraints or transport limitations, and 6% outperformed Donnan equilibrium due to use of integrated processes. Ultimately, this work highlights the benefits of the Rd/w framework for standardizing interpretation of Donnan dialysis systems.
Recent Advances in Wearable Sweat Sensor Development
(Wiley, 2025) Zhang, Tao; Kabandana, Giraso Keza Monia; Terrell, John A.; Chen, Hui; Chen, Chengpeng
Wearable sweat sensors for detecting biochemical markers have emerged as a transformative research area, with the potential to revolutionize disease diagnosis and human health monitoring. Since 2016, a substantial body of pioneering and translational work on sweat biochemical sensors has been reported. This review aims to provide a comprehensive summary of the current state-of-the-art in the field, offering insights and perspectives on future developments. The focus is on wearable microfluidic platforms for sweat collection and delivery and the analytical chemistry applicable to wearable devices. Various microfluidic technologies, including those based on synthetic polymers, paper, textiles, and hydrogels, are discussed alongside diverse detection methods such as electrochemistry and colorimetry. Both the advantages and current limitations of these technologies are critically examined. The review concludes with our perspectives on the future of wearable sweat sensors, with the goal of inspiring new ideas, innovations, and technical advancements to further the development and practical application of these devices in promoting human health.
Identification of a macrocyclic compound targeting the lassa virus polymerase
(Elsevier, 2024-08-01) Aida-Ficken, Virginia; Kelly, Jamie A.; Chatterjee, Payel; Jenks, M. Harley; McMullan, Laura K.; Albariño, César G.; Montgomery, Joel M.; Seley-Radtke, Katherine L.; Spiropoulou, Christina F.; Flint, Mike
There are no approved vaccines or therapeutics for Lassa virus (LASV) infections. To identify compounds with anti-LASV activity, we conducted a cell-based screening campaign at biosafety level 4 and tested almost 60,000 compounds for activity against an infectious reporter LASV. Hits from this screen included several structurally related macrocycles. The most potent, Mac128, had a sub-micromolar EC50 against the reporter virus, inhibited wild-type clade IV LASV, and reduced viral titers by 4 orders of magnitude. Mechanistic studies suggested that Mac128 inhibited viral replication at the level of the polymerase.
Meeting report of the 37th International Conference on Antiviral Research in Gold Coast, Australia, May 20?24, 2024, organized by the International Society for Antiviral Research
(Elsevier, 2024-12-01) Welch, Stephen R.; Bilello, John P.; Carter, Kara; Delang, Leen; Dirr, Larissa; Durantel, David; Feng, Joy Y.; Gowen, Brian B.; Herrero, Lara J.; Janeba, Zlatko; Kleymann, Gerald; Lee, Alpha A.; Meier, Chris; Moffat, Jennifer; Schang, Luis M.; Schiffer, Joshua T.; Seley-Radtke, Katherine L.; Sheahan, Timothy P.; Spengler, Jessica R.
The 37th International Conference on Antiviral Research (ICAR) was held in Gold Coast, Australia, May 20–24, 2024. ICAR 2024 featured over 75 presentations along with two poster sessions and special events, including those specifically tailored for trainees and early-career scientists. The meeting served as a platform for the exchange of cutting-edge research, with presentations and discussions covering novel antiviral compounds, vaccine development, clinical trials, and therapeutic advancements. A comprehensive array of topics in antiviral science was covered, from the latest breakthroughs in antiviral drug development to innovative strategies for combating emerging viral threats. The keynote presentations provided fascinating insight into two diverse areas fundamental to medical countermeasure development and use, including virus emergence at the human-animal interface and practical considerations for bringing antivirals to the clinic. Additional sessions addressed a variety of timely post-pandemic topics, such as the hunt for broad spectrum antivirals, combination therapy, pandemic preparedness, application of in silico tools and AI in drug discovery, the virosphere, and more. Here, we summarize all the presentations and special sessions of ICAR 2024 and introduce the 38th ICAR, which will be held in Las Vegas, USA, March 17–21, 2025.
Recent advances in microalgae-driven carbon capture, utilization, and storage: Strain engineering through adaptive laboratory evolution and microbiome optimization
(Elsevier, 2024-11-09) He, Zhongshi; Wang, Jing; Li, Yantao
The potential of microalgae as a biological resource for carbon capture, utilization, and storage (CCUS) has been extensively discussed. Although genetic engineering methods have been employed to improve microalgal phenotypes, they often face challenges related to public concerns regarding genetically modified organisms. By contrast, adaptive laboratory evolution (ALE) and microbiome optimization have emerged as promising non-genetic modification strategies, with notable success in bacterial models. In microalgae, ALE has been employed to improve resilience against varying environmental and stress factors and increase carbon capture efficiency, and for the production of valuable bioproducts through gradual accumulation of beneficial mutations following manual or automated selection. Furthermore, advancements in the understanding of microbial symbiotic relationships in the phycosphere have facilitated microbiome optimization in microalgal cultivation systems, significantly improving their functionality and productivity. In this study, we provide a comprehensive overview of the latest advancements in ALE and microbiome optimization of microalgae for CCUS across different carbon emission scenarios, including flue gas, biogas, wastewater, and landfill leachate. We further discuss the current challenges and future directions for the integration of ALE with microbiome optimization, focusing on the potential synergies of these methodologies. Overall, ALE and microbiome optimization are promising approaches to direct microalgae for environmental and industrial CCUS applications, thereby reducing global carbon emissions and addressing climate change challenges.
Direct Detection and Quantification of Aqueous Proteins via a Fluorescent Probe Through the Use of Fluorophore-Induced Plasmonic Current
(MDPI, 2025-02-27) Pierce, Daniel; Geddes, Chris
We report on the recent advancements in the sensing of proteins, both directly and with the use of a fluorescent probe, through the use of Fluorophore-Induced Plasmonic Current (FIPC). FIPC are a phenomenon where a fluorophore or excited state species is in close proximity to a plasmonically active metal nanoparticle film (MNF), and the excited state is able to couple to the particle, ultimately leading to enhanced spectroscopic properties. This phenomenon is similar to the well-reported metal-enhanced fluorescence (MEF) phenomenon, wherein the coupled complex produces an enhanced fluorescence emission and a shorter lifetime. However, if the particles themselves are sufficiently spaced and oriented, an induced current can transfer from each discreet particle to the next, creating a detectable current across the film. This detectable current has a magnitude that is proportional to the fluorescent properties of the species that produced it, and can be affected by the polarization of the excitation source; the spacing and size of the particles on the film; the overlap between the spectral properties of the film and the species; as well as externally applied voltages and currents. In this study, we examined whether it is possible to detect protein species, directly due to both their intrinsic fluorescent and absorptive properties, and how that compares to commercially available protein detection probes, in a similar manner to prior work by our group addressing analyte detection via turn-on fluorescent probes. This FIPC-based detection technique is a novel method that has not been used for the detection of proteins, and the use of this method could expand the dynamic sensing range of first-pass testing, while overcoming some of the physical limitations on the upper limit of detection of both absorption spectroscopy and fluorescence emission spectroscopy. Our experiments sought to highlight the selectivity of FIPC-based detection relative to both fluorescence and absorption spectroscopy, as well as its sensitivity when working with protein analytes. We examined the effects of protein concentration, intrinsic fluorescent properties, and turn-on probes, as well as how these techniques compare to traditional analytical techniques used today.
Enhanced Line Narrowing of Selectively Labeled [9-15N] Guanosine Enables Probing of Large RNAs
(2025-02-04) Attionu, Solomon; Dill, Rita; Summers, Michael; Case, David; Marchant, Jan; Dayie, T. Kwaku
RNA regulates various cellular processes using malleable 3D structures and characterizing their structural dynamics is critical to shedding light on their mechanism of action. To mitigate continuing limitations on studies of large RNA by solution NMR spectroscopy, we have extended a recently described 2H-enhanced, 1H-15N correlation approach by developing a chemoenzymatic labeling technology that grafts selectively labeled [9-15N]-Guanine on to any available labeled ribose to make [9-15N]-GTP. The low CSA of the N9 nucleus (~112 ppm) in combination with extensive ribose deuteration leads to long-lived NMR signals that enable chemical shift assignment, analyze the structure of three biologically relevant large RNA constructs pivotal to viral life cycles [human hepatitis B virus ? RNA (61nt), the HIV-1 primer binding site segment RNA (103 nt), and the HIV-1 Rev response element (232 nt)], observe N9-H8 and N9-H1? correlations, and measure longitudinal and transverse relaxation rates for RNAs as large as 78 kDa. We show CSA dominates both N7 (>99%) and N9 (>90%) relaxation and enables straightforward analysis of dynamics. Taken together, application of these selective labels in conjunction with optimized NMR pulse sequences could help us push the limits of size restrictions in RNA NMR structural biology beyond 100 nt.
Enhanced Line Narrowing of Selectively Labeled [9-15N] Guanosine Enables Probing of Large RNAs
(2025-02-04) Attionu, Solomon K.; Dill, Rita; Summers, Michael F.; Case, David A.; Marchant, Jan; Dayie, Theodore K.
RNA regulates various cellular processes using malleable 3D structures and characterizing their structural dynamics is critical to shedding light on their mechanism of action. To mitigate continuing limitations on studies of large RNA by solution NMR spectroscopy, we have extended a recently described 2H-enhanced, 1H-15N correlation approach by developing a chemoenzymatic labeling technology that grafts selectively labeled [9-15N]-Guanine on to any available labeled ribose to make [9-15N]-GTP. The low CSA of the N9 nucleus (~112 ppm) in combination with extensive ribose deuteration leads to long-lived NMR signals that enable chemical shift assignment, analyze the structure of three biologically relevant large RNA constructs pivotal to viral life cycles [human hepatitis B virus ε RNA (61nt), the HIV-1 primer binding site segment RNA (103 nt), and the HIV-1 Rev response element (232 nt)], observe N9-H8 and N9-H1' correlations, and measure longitudinal and transverse relaxation rates for RNAs as large as 78 kDa. We show CSA dominates both N7 (>99%) and N9 (>90%) relaxation and enables straightforward analysis of dynamics. Taken together, application of these selective labels in conjunction with optimized NMR pulse sequences could push the limits of size restrictions in RNA NMR structural biology beyond 100 nt.
RNA-Puzzles Round V: blind predictions of 23 RNA structures
(Springer Nature, 2024-12-02) Bu, Fan; Adam, Yagoub; Adamiak, Ryszard W.; Antczak, Maciej; de Aquino, Belisa Rebeca H.; Badepally, Nagendar Goud; Batey, Robert T.; Baulin, Eugene F.; Boinski, Pawel; Boniecki, Michal J.; Bujnicki, Janusz M.; Carpenter, Kristy A.; Chacon, Jose; Chen, Shi-Jie; Chiu, Wah; Cordero, Pablo; Das, Naba Krishna; Das, Rhiju; Dawson, Wayne K.; DiMaio, Frank; Ding, Feng; Dock-Bregeon, Anne-Catherine; Dokholyan, Nikolay V.; Dror, Ron O.; Dunin-Horkawicz, Stanisław ; Eismann, Stephan; Ennifar, Eric; Esmaeeli, Reza; Farsani, Masoud Amiri; Ferré-D’Amaré, Adrian R.; Geniesse, Caleb; Ghanim, George E.; Guzman, Horacio V.; Hood, Iris V.; Huang, Lin; Jain, Dharm Skandh; Jaryani, Farhang; Jin, Lei; Joshi, Astha; Karelina, Masha; Kieft, Jeffrey S.; Kladwang, Wipapat; Kmiecik, Sebastian; Koirala, Deepak; Kollmann, Markus; Kretsch, Rachael C.; Kurciński, Mateusz; Li, Jun; Li, Shuang; Magnus, Marcin; Masquida, BenoÎt; Moafinejad, S. Naeim; Mondal, Arup; Mukherjee, Sunandan; Nguyen, Thi Hoang Duong; Nikolaev, Grigory; Nithin, Chandran; Nye, Grace; Pandaranadar Jeyeram, Iswarya P. N.; Perez, Alberto; Pham, Phillip; Piccirilli, Joseph A.; Pilla, Smita Priyadarshini; Pluta, Radosław ; Poblete, Simón; Ponce-Salvatierra, Almudena; Popenda, Mariusz; Popenda, Lukasz; Pucci, Fabrizio; Rangan, Ramya; Ray, Angana; Ren, Aiming; Sarzynska, Joanna; Sha, Congzhou Mike; Stefaniak, Filip; Su, Zhaoming; Suddala, Krishna C.; Szachniuk, Marta; Townshend, Raphael; Trachman, Robert J.; Wang, Jian; Wang, Wenkai; Watkins, Andrew; Wirecki, Tomasz K.; Xiao, Yi; Xiong, Peng; Xiong, Yiduo; Yang, Jianyi; Yesselman, Joseph David; Zhang, Jinwei; Zhang, Yi; Zhang, Zhenzhen; Zhou, Yuanzhe; Zok, Tomasz; Zhang, Dong; Zhang, Sicheng; Żyła, Adriana; Westhof, Eric; Miao, Zhichao
RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA three-dimensional structure prediction. With agreement from structural biologists, RNA structures are predicted by modeling groups before publication of the experimental structures. We report a large-scale set of predictions by 18 groups for 23 RNA-Puzzles: 4 RNA elements, 2 Aptamers, 4 Viral elements, 5 Ribozymes and 8 Riboswitches. We describe automatic assessment protocols for comparisons between prediction and experiment. Our analyses reveal some critical steps to be overcome to achieve good accuracy in modeling RNA structures: identification of helix-forming pairs and of non-Watson–Crick modules, correct coaxial stacking between helices and avoidance of entanglements. Three of the top four modeling groups in this round also ranked among the top four in the CASP15 contest.
Assessing the K₂BO₃ family of materials as multiferroics
(APS, 2024-11-26) Casale, Anthony; Bennett, Joseph
We evaluate the potential of an overlooked family of materials to support both the magnetization and polarization required to be classified as multiferroics. This family of materials has a stoichiometry of A₂BX₃ and was uncovered in the Inorganic Crystal Structure Database (ICSD) while searching for structural platforms that could support low energy polarization switching. The examples here have the general chemical formula of K₂BO₃, where B is a magnetically active cation located within edge-sharing square pyramids that form a 1D chain. Density functional theory with Hubbard U corrections (DFT + U) are used to determine the potential energy landscape of K₂BO₃, which include investigating multiple magnetic and polarization orderings. We analyze the ground state and electronic structures and report on how the choice of Hubbard U will affect both, which is important when predicting functional properties of low-dimensional and potentially exfoliable systems. This family contains a ferromagnetic insulator, K₂VO₃, as well as antiferromagnetic (K₂NbO₃) and nonmagnetic (K₂MoO₃) insulators with antipolar ground state symmetries, and accessible polar metastable states, that we predict to be antiferroelectric. This preliminary assessment of the K₂BO₃ members of the A₂BX₃ family reveals a new class of materials, that with further optimization via compositional tuning, could be multiferroic.
Mononuclear Aluminum–Fluoride Ions, AlFx(⁺/⁻)—Study of Plausible Frameworks of Complexes with Biomolecules and Their In Vitro Toxicity
(MDPI, 2025-1) Pavlovič, Anja; Janžič, Larisa; Sršen, Lucija; Kopitar, Andreja Nataša; Edwards, Kathleen F.; Liebman, Joel F.; Ponikvar-Svet, Maja
The importance of fluorine and aluminum in all aspects of daily life has led to an enormous increase in human exposure to these elements in their various forms. It is therefore important to understand the routes of exposure and to investigate and understand the potential toxicity. Of particular concern are aluminum–fluoride complexes (AlFx), which are able to mimic the natural isostructural phosphate group and influence the activity of numerous essential phosphoryl transferases. Our review of salts of ionic AlFx species, which plausibly form the framework of complexes with biomolecules, revealed that the octahedral configuration of aluminum in the active site of the enzyme is preferred over the trigonal-bipyramidal structure. The effects of varying concentrations of fluoride, aluminum and AlFx—from micromolar to millimolar levels—on the viability and apoptosis rate of THP-1 monocytes were investigated using phosphate buffer solution as a culture media to simulate physiological conditions. Our results suggest that aluminum can reduce the direct toxicity of fluoride through the formation of AlFx. In view of the results found, further in vitro studies are required to clarify the toxicity mechanisms of these species.
Quantitation of Per- and Polyfluoroalkyl Substances (PFAS) in Aquaculture Systems
(2024-01-01) Belunis, Amanda; LaCourse, William R; Chemistry & Biochemistry; Chemistry
Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals that have been used since the 1940s in a wide variety of applications including water-repellant clothing, stain-resistant sprays, food packaging, and aqueous film forming foam (AFFF). Due to their prevalent use in consumer products, and their persistence in the environment, PFAS have leached into the air, soil, and water, leaving almost no ecosystem untouched. PFAS are known to be bioaccumulative and studies have shown potential links between exposure and several negative human health effects. A large focus has been placed on understanding and regulating PFAS exposure. Contaminated food and water are believed to be the main routes of exposure for the general population. It has been hypothesized that fish and seafood are one of the main dietary sources of exposure, as associations between consumption and PFAS serum concentrations have been observed globally. With efforts to improve environmental conservation and sustainability while keeping up with increasing demands for fish and seafood, aquaculture has grown rapidly since the 1980s. While numerous studies have shown the presence of PFAS in various environments, data regarding the compounds in aquaculture environments is scarce. In recent years, a large focus has been to reduce the potential for environmental interactions with coastal aquaculture facilities, leading to the increase in land-based marine and freshwater systems. There is currently a lack of understanding with regards to the presence of PFAS in aquaculture environments. As farmed fish continues to account for a large portion of fish for consumption, it is important to understand the fate and transport of PFAS in these environments, requiring proper analytical techniques to be developed. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the separation and detection of 40 target PFAS. Various solid phase extraction (SPE) cartridges were selected and tested to determine the best extraction technique for the target analytes. Automated and manual SPE setups were compared, and the automated setup shows a statistically significant increase in average recovery and reproducibility. Instrument and method figures of merit show the sensitivity, reproducibility, and robustness of the developed method. The method was applied to a proof-of-concept study testing various types of aquaculture tanks across two facilities and PFAS were found in each of the samples tested.
Hybrid Gold Nanostructures with Unique Architectures: From Potential Applications in Drug Delivery to Photonic Devices
(2024-01-01) Baradaran Kayyal, Tohid; Daniel, Marie-Christine; Chemistry & Biochemistry; Chemistry
The domain of nanotechnology has experienced remarkable progress through the engineering of hybrid nanostructures. These structures integrate diverse materials to create synergistic properties that are unachievable or less efficient when using individual components alone. Central to this progress are gold-based hybrid nanostructures, favored for their inherent advantages ranging from structural diversity and biocompatibility to remarkable optical properties. The present study focuses on the creation of gold-based hybrid systems with special nanostructures (rattle and bipyramid shapes), which have potential for medical and optical applications. The first project focuses on the design of a porous gold nanoarchitecture, termed nanorattles. These structures consist of a porous gold cage surrounding a core, offering two accessible surfaces within a single system. This design can enhance the surface area available for cargo loading, which is advantageous for drug delivery applications. Incorporated within these nanorattles are dendrons—highly branched nanopolymers—that play a crucial role in forming a hybrid structure. Dendrons not only act as catalysts, facilitating the formation of the porous cage and the overall nanorattle structure, but also serve as a nanosystem capable of covalently carrying cargo. Cisplatin, a widely used anticancer drug, is employed as a model cargo to demonstrate the potential of gold nanorattles in targeted drug delivery applications. The other project explores the directed assemblies of gold nanoparticles and quantum dots (QDs) to form a novel hybrid nanostructure yielding unique optical characteristics through plasmon-exciton coupling. The gold used in this assembly is in the form of bipyramids, chosen for their distinctive asymmetrical shape and sharp end tips. The sharp end tips of the gold nano bipyramids (AuBPs) are the designated locations for placing the QDs. Upon light irradiation, the excitons from the QDs couple with the plasmons resonating from the ends of the AuBPs, facilitating efficient plasmon-exciton coupling. The resulting coupling occurs through a selective copper-free click reaction between surface modified QDs and AuBPs. The significance of this plasmon-exciton coupling extends particularly to photonic devices, where it can greatly enhance device performance and efficiency in light manipulation, driving next-generation photonic technologies. The PhD dissertation comprehensively explains the foundational concepts, intrinsic properties, and importance of hybrid nanomaterials as outlined in the literature review. It details the experimental procedures for synthesizing the aforementioned gold nanostructures, presents characterizations and analyses to validate their structure, size, and properties, and discusses the results, interprets data, and explores potential applications.
eIF4E-Dependent Capture of the Monomeric HIV-1 RNA Genome
(2024-01-01) Singh, Karndeep; Summers, Michael F; Chemistry & Biochemistry; Biochemistry
The human immunodeficiency virus type-1 (HIV-1) RNA genome’s 5'- untranslated region (5'-Leader, 5'-L) serves as a master regulator for numerous viral replication processes within a cell such as genome dimerization, splicing, packaging, and translation initiation. This 5'-Leader adopts two conformations dependent on the transcriptional start site usage: 5'-capped RNAs beginning with one guanosine (Cap1G) adopt the dimeric conformation whereas 5'-capped RNAs beginning with two or three guanosines (Cap2G and Cap3G, respectively) adopt a monomeric conformation. Published work from our laboratory revealed that Cap1G leader RNAs sequester the 5'-cap through a coaxial stacking of two 5'-hairpins – the TAR and polyA hairpin. This prevents binding to the human eukaryotic translation initiation factor 4E (eIF4E) cap binding protein – the initial recognition step in cap/eIF4E dependent translation of HIV-1 mRNAs. For the monomeric RNA transcripts, the 5'-cap is exposed and accessible for the recruitment and binding of eIF4E. Therefore, the exposure and sequestration of the 5'-cap (a 7- methylguanosine triphosphate) within the HIV-1 5'-Leader dictates the capture of the RNAtranscripts by cap-dependent translation machinery for translation of viral proteins or genome packaging by the Gag polyprotein, respectively. While it is well-established that cap-dependent translation serves as the primary mechanism of HIV-1 genome translation in eukaryotes, the molecular nature of interactions between Cap3G RNAs and eIF4E remain unknown. Electrophoretic mobility shift assays (EMSAs) and isothermal titration calorimetry (ITC) experiments reveal that the human eIF4E binds to the HIV-1MAL Cap3G RNAs at least 2.5-fold tighter than the 5'-cap, suggesting that RNA elements influence binding of the human eIF4E. Using nuclear magnetic resonance (NMR) spectroscopy with a variety of selectively labeled 1H-, 15N-, and 13C-labeling schemes, we worked to characterize the first three-dimensional structure of a structured 5'-capped RNA – a HIV- 1MAL Cap3G RNA oligo (~13 kDa) – bound to the human eIF4E (~22 kDa). The 5'-cap forms a pi-pi stacking interaction with two tryptophan residues (W56 and W102) while residues of the TAR hairpin forming electrostatic interactions with eIF4E’s exposed charged residues to orient the RNA oligo out of the 5'-cap binding pocket. Surprisingly, the unstructured residues of the polyA region at the 3'-end interact with lysine residues of eIF4E. Our findings reveal that HIV-1 5'-Leader’s structural elements influence the recruitment and binding of eIF4E, suggesting an additional quality control mechanism that HIV-1 uses to ensure the translation of its Cap3G mRNA transcripts into viral proteins.
STRUCTURAL & BIOPHYSICAL CHARACTERIZATIONS OF MEMBRANE-BOUND AND MEMBRANE-ASSOCIATED COMPONENTS OF THE FERROUS IRON TRANSPORT (FEO) SYSTEM
(2024-01-01) Lee, Mark; Smith, Aaron T.; Chemistry & Biochemistry; Chemistry
Iron (Fe) is a vital transition metal for virtually all living organisms. In its ionic form, this element functions as a powerful cofactor within a multitude of enzymes responsible for a wide range of complex chemical reactions including, but not limited to, de novo DNA biosynthesis, central carbon metabolism, vitamin biogenesis, and even N2 fixation. Due to its critical necessity in these essential metabolic processes, organisms such as bacteria have a high demand for the uptake of iron. However, the mode of iron acquisition is linked to the oxidation state of the iron ion, which is a product of the prevailing environmental conditions. In acidic and reducing conditions commonly encountered by pathogenic bacteria within the human gut or locations within biofilms, ferrous iron (Fe2+) dominates the environment, but the precise mechanism by prokaryotes acquire Fe2+ remains unclear. In this dissertation, the structural and biophysical characteristics of the membrane-bound and membrane-associated components of the ferrous iron transport (Feo) system, the most widely distributed and the primary Fe2+ transport system utilized by bacteria, are investigated. First, structures of the N-terminal domain of FeoB (NFeoB) from Vibrio cholerae (VcNFeoB), the causative agent of the disease cholera, are presented for the first time and give critical insight into the surprising nucleotide promiscuity of FeoB. Next, in an attempt to provide homogeneous, near-native samples of full-length, intact FeoB for future structural characterization, V. cholerae FeoB (VcFeoB) was extracted directly from bacterial membranes in near native-like lipid environments through the use of styrene-maleic acid (SMA)-copolymers. SMA-extracted VcFeoB showed high purity, good monodispersity, and displayed differences in NTP-dependent activity in the presence of lipids compared to detergent-solubilized VcFeoB. Finally, a newly discovered single-pass transmembrane protein of the Feo system termed FeoD (formally FeoI) from Streptococcus thermophilus (StFeoD) was first modeled using AlphaFold3 before being cloned, expressed, purified, and partially characterized. Contrary to previous reports, we show that the Cys-rich StFeoD co-purifies with a partially bound [Fe-S] cluster, similarly to FeoC, and optimization and further characterization is currently underway. When taken all together, this dissertation deepens our understanding of the membrane-bound and membrane-associated components of the Feo system and paves the way for future structural work to understand the precise mechanism of prokaryotic Fe2+ transport.
Broad spectrum antiviral nucleosides—Our best hope for the future
(Elsevier, 2021) Seley-Radtke, Katherine ; Thames, Joy E.; Waters, Charles D.
The current focus for many researchers has turned to the development of therapeutics that have the potential for serving as broad-spectrum inhibitors that can target numerous viruses, both within a particular family, as well as to span across multiple viral families. This will allow us to build an arsenal of therapeutics that could be used for the next outbreak. In that regard, nucleosides have served as the cornerstone for antiviral therapy for many decades. As detailed herein, many nucleosides have been shown to inhibit multiple viruses due to the conserved nature of many viral enzyme binding sites. Thus, it is somewhat surprising that up until very recently, many researchers focused more on “one bug one drug,” rather than trying to target multiple viruses given those similarities. This attitude is now changing due to the realization that we need to be proactive rather than reactive when it comes to combating emerging and reemerging infectious diseases. A brief summary of prominent nucleoside analogues that previously exhibited broad-spectrum activity and are now under renewed interest, as well as new analogues, that are currently under investigation against SARS-CoV-2 and other viruses is discussed herein.
Synthetic anti-RNA antibody derivatives for RNA visualization in mammalian cells
(Oxford, 2024-12-31) Banna, Hasan Al; Berg, Kimberley; Sadat, Tasnia; Das, Naba Krishna; Paudel, Roshan; D'Souza, Victoria; Koirala, Deepak
Although antibody derivatives, such as Fabs and scFvs, have revolutionized the cellular imaging, quantification and tracking of proteins, analogous tools and strategies are unavailable for cellular RNA visualization. Here, we developed four synthetic anti-RNA scFv (sarabody) probes and their green fluorescent protein (GFP) fusions and demonstrated their potential to visualize RNA in live mammalian cells. We expressed these sarabodies and sarabody–GFP modules, purified them as soluble proteins, characterized their binding interactions with their corresponding epitopes and finally employed two of the four modules, sara1-GFP and sara1c-GFP, to visualize a target messenger RNA in live U2OS cells. Our current RNA imaging strategy is analogous to the existing MCP-MS₂ system for RNA visualization, but additionally, our approach provides robust flexibility for developing target RNA-specific imaging modules, as epitope-specific probes can be selected from a library generated by diversifying the sarabody complementarity determining regions. While we continue to optimize these probes, develop new probes for various target RNAs and incorporate other fluorescence proteins like mCherry and HaloTag, our groundwork results demonstrated that these first-of-a-kind immunofluorescent probes will have tremendous potential for tracking mature RNAs and may aid in visualizing and quantifying many cellular processes as well as examining the spatiotemporal dynamics of various RNAs.

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