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DOE OSTI2025

Rapid Screening of Liquid Metal Wetting for a Materials Compatibility Library

Wetting behavior of molten metals on solid substrates is a critical phenomenon influencing numerous industrial applications, including welding, anti-corrosion coatings, and metal additive manufacturing (AM). In particular, molten metal jetting (MMJ), an emerging AM technology, requires that the molten metal remain pinned at the nozzle exit. Thus, each new metal requires a specific nozzle material to ensure consistent droplet ejection and deposition, making it important to rapidly identify the appropriate wetting combinations. However, traditional measurements of wetting angles require expensive equipment and only allow one combination of materials to be investigated at a time which can be time consuming. This work introduces a rapid screening method based on sessile droplet experiments to evaluate wetting profiles across multiple metal–substrate combinations simultaneously. This study investigates the wetting interactions of molten Al alloy (Al4008), Cu, and Sn on various ceramic and metal substrates to identify optimal material combinations for MMJ nozzle designs. Results demonstrate that Al4008 achieves wetting on ceramic substrates such as AlN, TiO 2 , and SiC, with varying mechanisms including chemical reactions and weak surface interactions. Additionally, theoretical predictions regarding miscibility gaps and melting point differences were verified for Cu and Sn on refractory metals like Mo and W. Findings from this study contribute to the establishment of a materials compatibility library, enabling the selection of wetting/non-wetting combinations for stable MMJ operation. This resource not only advances MMJ technologies but also provides valuable insights for broader applications such as welding, coating, and printed electronics.

Materials science↗

DOE OSTI2025

Orthogonal chemical genomics approaches reveal genomic targets for increasing anaerobic chemical tolerance in Zymomonas mobilis

Genetically engineered microbes have the potential to increase efficiency in the bioeconomy by overcoming growth-limiting production stress. Screens of gene perturbation libraries against production stressors can identify high-value engineering targets, but follow-up experiments needed to guard against false positives are slow and resource-intensive. In principle, the use of orthogonal gene perturbation approaches could increase recovery of true positives over false positives because the strengths of one technique compensate for the weaknesses of the other, but, in practice, two parallel screens are rarely performed at the genome scale. Here, we screen genome-scale CRISPRi (CRISPR interference) knockdown and transposon insertion libraries of the bioenergy-relevant Alphaproteobacterium, Zymomonas mobilis, against growth inhibitors commonly found in deconstructed plant material. Integrating data from the two gene perturbation techniques, we established an approach for defining engineering targets with high specificity. This allowed us to identify all known genes in the cytochrome bc1 and cytochrome c synthesis pathway as potential targets for engineering resistance to phenolic acids under anaerobic conditions, a subset of which we validated using precise gene deletions. Strikingly, this finding is specific to the cytochrome bc1 and cytochrome c pathway and does not extend to other branches of the electron transport chain. We further show that exposure of Z. mobilis to ferulic acid causes substantial remodeling of the cell envelope proteome, as well as the downregulation of TonB-dependent transporters. Our work provides a generalizable strategy for identifying high-value engineering targets from gene perturbation screens that is broadly applicable.

CRISPRi↗

DOE OSTI2020

Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library

Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Decoding the protein–ligand interactions using parallel graph neural networks

Abstract Protein–ligand interactions (PLIs) are essential for biochemical functionality and their identification is crucial for estimating biophysical properties for rational therapeutic design. Currently, experimental characterization of these properties is the most accurate method, however, this is very time-consuming and labor-intensive. A number of computational methods have been developed in this context but most of the existing PLI prediction heavily depends on 2D protein sequence data. Here, we present a novel parallel graph neural network (GNN) to integrate knowledge representation and reasoning for PLI prediction to perform deep learning guided by expert knowledge and informed by 3D structural data. We develop two distinct GNN architectures: $$\hbox {GNN}_{\mathrm{F}}$$ GNN F is the base implementation that employs distinct featurization to enhance domain-awareness, while $$\hbox {GNN}_{\mathrm{P}}$$ GNN P is a novel implementation that can predict with no prior knowledge of the intermolecular interactions. The comprehensive evaluation demonstrated that GNN can successfully capture the binary interactions between ligand and protein’s 3D structure with 0.979 test accuracy for $$\hbox {GNN}_{\mathrm{F}}$$ GNN F and 0.958 for $$\hbox {GNN}_{\mathrm{P}}$$ GNN P for predicting activity of a protein–ligand complex. These models are further adapted for regression tasks to predict experimental binding affinities and $$\hbox {pIC}_{\mathrm{50}}$$ pIC 50 crucial for compound’s potency and efficacy. We achieve a Pearson correlation coefficient of 0.66 and 0.65 on experimental affinity and 0.50 and 0.51 on $$\hbox {pIC}_{\mathrm{50}}$$ pIC 50 with $$\hbox {GNN}_{\mathrm{F}}$$ GNN F and $$\hbox {GNN}_{\mathrm{P}}$$ GNN P , respectively, outperforming similar 2D sequence based models. Our method can serve as an interpretable and explainable artificial intelligence (AI) tool for predicted activity, potency, and biophysical properties of lead candidates. To this end, we show the utility of $$\hbox {GNN}_{\mathrm{P}}$$ GNN P on SARS-Cov-2 protein targets by screening a large compound library and comparing the prediction with the experimentally measured data.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1986

DEC Ada interface to Screen Management Guidelines (SMG)

DEC's Screen Management Guidelines are the Run-Time Library procedures that perform terminal-independent screen management functions on a VT100-class terminal. These procedures assist users in designing, composing, and keeping track of complex images on a video screen. There are three fundamental elements in the screen management model: the pasteboard, the virtual display, and the virtual keyboard. The pasteboard is like a two-dimensional area on which a user places and manipulates screen displays. The virtual display is a rectangular part of the terminal screen to which a program writes data with procedure calls. The virtual keyboard is a logical structure for input operation associated with a physical keyboard. SMG can be called by all major VAX languages. Through Ada, predefined language Pragmas are used to interface with SMG. These features and elements of SMG are briefly discussed.

Laomanachareon, Somsak↗

DOE OSTI2020

Smart Microbial Cells Couple Catalysis and Sensing to Provide High-Throughput Selection of an Organophosphate Hydrolase

Enzyme engineering for gain of function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements, while a single “bad” mutation can inactivate the enzyme. To establish high-throughput screening and achieve enhanced resolution between two variants, genetic libraries of the organophosphate hydrolase enzyme paraoxonase 1 (PON 1 ) were rapidly screened via an engineered positive-feedback circuit: a p-nitrophenol (PNP)-specific transcription factor (TF) regulated expression of PON 1 , which catalyzed paraoxon breakdown and PNP production. Rare active mutant colonies, picked by simple visual fluorescence of a PON 1 –green fluorescent protein (GFP) fusion, were characterized. Furthermore, in a single screening round, high (library-scale) throughput enabled the discovery of enhanced paraoxon degradation activity in PON 1 , including structurally unexpected mutations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Speed vs Accuracy: Effect on Ligand Pose Accuracy of Varying Box Size and Exhaustiveness in AutoDock Vina

Abstract Structure‐based virtual high‐throughput screening involves docking chemical libraries to targets of interest. A parameter pertinent to the accuracy of the resulting pose is the root mean square deviation (RMSD) from a known crystallographic structure, i. e., the ‘docking power’. Here, using a popular algorithm, Autodock Vina, as a model program, we evaluate the effects of varying two common docking parameters: the box size (the size of docking search space) and the exhaustiveness of the global search (the number of independent runs starting from random ligand conformations) on the RMSD from the PDBbind v2017 refined dataset of experimental protein‐ligand complexes. Although it is clear that exhaustiveness is an important parameter, there is wide variation in the values used, with variation between 1 and >100. We, therefore, evaluated a combination of cubic boxes of different sizes and five exhaustiveness values (1, 8, 25, 50, 75, 100) within the range of those commonly adopted. The results show that the default exhaustiveness value of 8 performs well overall for most box sizes. In contrast, for all box sizes, but particularly for large boxes, an exhaustiveness value of 1 led to significantly higher median RMSD (mRMSD) values. The docking power was slightly improved with an exhaustiveness of 25, but the mRMSD changes little with values higher than 25. Therefore, although low exhaustiveness is computationally faster, the results are more likely to be far from reality, and, conversely, values >25 led to little improvement at the expense of computational resources. Overall, we recommend users to use at least the default exhaustiveness value of 8 for virtual screening calculations.

Agarwal, Rupesh↗

DOE OSTI2021

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV-2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1–20 μM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer’s therapy, was shown to have antiviral activity between 10 to 66 μM, in Vero 76, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Antigen‐binding fragments with improved crystal lattice packing and enhanced conformational flexibility at the elbow region as crystallization chaperones

It has been shown previously that a set of three modifications—termed S1, Crystal Kappa, and elbow—act synergistically to improve the crystallizability of an antigen-binding fragment (Fab) framework. Here, we prepared a phage-displayed library and performed crystallization screenings to identify additional substitutions—located near the heavy-chain elbow region—which cooperate with the S1, Crystal Kappa, and elbow modifications to increase expression and improve crystallizability of the Fab framework even further. One substitution (K141Q) supports the signature Crystal Kappa-mediated Fab:Fab crystal lattice packing interaction. Another substitution (E172G) improves the compatibility of the elbow modification with the Fab framework by alleviating some of the strain incurred by the shortened and bulkier elbow linker region. A third substitution (F170W) generates a split-Fab conformation, resulting in a powerful crystal lattice packing interaction comprising the biological interaction interface between the variable heavy and light chain domains. In sum, we have used K141Q, E172G, and F170W substitutions—which complement the S1, Crystal Kappa, and elbow modifications—to generate a set of highly crystallizable Fab frameworks that can be used as chaperones to enable facile elucidation of Fab:antigen complex structures by x-ray crystallography.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

MALDI-MS screening of microbial colonies with isomer resolution to select fatty acid desaturase variants

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid MS assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Lastly, we used the fabA deficient chassis to concomitantly express a non-native acyl- ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP–importin α interactions

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin β and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Å crystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2–ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117–196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2026

Searching for ternary magnetic Zr–Fe–B compounds through deep machine learning

We use deep machine learning (ML) combined with first-principles calculations to search for energetically favorable ternary magnetic zirconium–iron borides. We show that an iterative ML approach enables efficient screening of vast structural libraries, effectively selecting promising candidates for subsequent first-principles investigations. Twenty-two new Fe-rich ternary compounds with formation energies within 60 meV atom −1 above the known ternary convex hull and with magnetic polarization larger than 0.6 T are identified, among which ten structures exhibit significant uniaxial anisotropy with magnetocrystalline anisotropy constant K 1 ⩾ 0.8 MJ m −3 , including a Zr 2 Fe 14 B phase. Such an ML-guided approach dramatically accelerates the discovery of rare-earth-free permanent magnetic materials.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

DOE OSTI2024

Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

ABSTRACT Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans , Rhodobacter sphaeroides , and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis , N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn 7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides . We screen a synthetic promoter library to identify isopropyl β-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides ). Combining Tn 7 integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for N. aromaticivorans and R. sphaeroides (up to ~10-fold knockdown in N. aromaticivorans and R. sphaeroides ) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria. IMPORTANCE It is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for Novosphingobium aromaticivorans and Rhodobacter sphaeroides that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the N. aromaticivorans or R. sphaeroides genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts.

Hall, Ashley N.↗

DOE OSTI2024

An exopolysaccharide pathway from a freshwater Sphingomonas isolate

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties, and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Data for MALDI-MS Screening of Microbial Colonies With Isomer Resolution to Select Fatty Acid Desaturase Variants

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid mass spectrometry (MS) assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Finally, we used the fabA deficient chassis to concomitantly express a non-native acyl-ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

Conversion↗

DOE OSTI2023

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2000

Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis

In this report, we describe an experiment conducted at Kennedy Space Center in the biomass production chamber (BPC) using soybean plants for purification and processing of human hygiene water. Specifically, we tested whether it was possible to detect changes in the root-associated bacterial assemblage of the plants and ultimately to identify the specific microorganism(s) which differed when plants were exposed to hygiene water and other hydroponic media. Plants were grown in hydroponics media corresponding to four different treatments: control (Hoagland's solution), artificial gray water (Hoagland's+surfactant), filtered gray water collected from human subjects on site, and unfiltered gray water. Differences in rhizosphere microbial populations in all experimental treatments were observed when compared to the control treatment using both community level physiological profiles (BIOLOG) and molecular fingerprinting of 16S rRNA genes by terminal restriction fragment length polymorphism analysis (TRFLP). Furthermore, screening of a clonal library of 16S rRNA genes by TRFLP yielded nearly full length SSU genes associated with the various treatments. Most 16S rRNA genes were affiliated with the Klebsiella, Pseudomonas, Variovorax, Burkholderia, Bordetella and Isosphaera groups. This molecular approach demonstrated the ability to rapidly detect and identify microorganisms unique to experimental treatments and provides a means to fingerprint microbial communities in the biosystems being developed at NASA for optimizing advanced life support operations.

NASA Discipline Life Support Systems↗

DOE OSTI2024

SARS-CoV-2 variant nanobodies and constructs comprising such nanobodies

A large and highly diverse nanobody library was constructed and screened against multiple variants of SARS-COV-2 to find nanobodies with high sensitivity and specificity for the variants. Four rounds of positive selection against a panel of six diverse SARS-COV-2 variant RBDs was performed with our high-diversity. At least 59 of these nanobodies were found to work well against Alpha, Beta, Gamma, Delta, Kappa, Lambda and Mu with some overlap efficacy against other variants. These nanobodies have efficacy as stand-alone nanobodies and as a construct comprising nanobodies linked to the human IgG1 constant fragment (Fc) (nanobody-hFc constructions or nb-hFcs) to make enhanced humanized sdAbs with all the attributes of nanobodies with improved half-life and optimized effector functions. Several promising nanobodies that neutralize the original SARS-COV-2 and several of its variants have been identified, including Delta, with high efficacy. In particular, a subset of these nanobodies bind to the Omicron RBD.

Harmon, Brooke Nicole↗