Highly conserved s2m element of SARS-CoV-2 dimerizes via a kissing complex and interacts with host miRNA-1307-3p.

The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the genome 3' untranslated region is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the SARS-CoV-2 s2m element dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular microRNA (miRNA) 1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.

Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering.

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Structural Fingerprinting of Antisense Oligonucleotide Therapeutics by Solution NMR Spectroscopy.

PURPOSE: Antisense oligonucleotide (ASO) therapeutics are an emerging class of biopharmaceuticals to treat and prevent diseases, particularly those involving "undruggable" protein targets. Impurities generated throughout the ASO drug manufacturing and formulation pipeline can be detrimental to drug safety and efficacy. Therefore, analytical techniques are needed to rigorously characterize these molecules for quality assurance purposes. METHODS: We demonstrate 1D and 2D nuclear magnetic resonance (NMR) spectroscopy methods that can generate high-resolution structural "fingerprints" of ASOs. RESULTS AND CONCLUSIONS: 1D 1H and 31P measurements are shown to provide rapid initial assessment of the ASO integrity. In particular, a well-resolved pair of 31P signals arising from the 5´-end of the phosphorodiamidate morpholino oligomer (PMO) are sensitive to complex formation and oligomerization state. 2D 1H-1H, 1H-13C, and 1H-15 N experiments, although less sensitive, are further shown to enable resonance assignment, which will allow the tracking of structural changes at high-resolution during the drug development and manufacturing processes. We further anticipate that the described NMR approaches will be broadly applicable to fully formulated ASO therapeutics, including modalities other than PMOs.

Grand Challenges in Pharmaceutical Research Series: Ridding the Cold Chain for Biologics.

Biologics are complex pharmaceuticals that include formulated proteins, plasma products, vaccines, cell and gene therapy products, and biological tissues. These products are fragile and typically require cold chain for their delivery and storage. Delivering biologics, while maintaining the cold chain, whether standard (2°C to 8°C) or deepfreeze (as cold as -70°C), requires extensive infrastructure that is expensive to build and maintain. This poses a huge challenge to equitable healthcare delivery, especially during a global pandemic. Even when the infrastructure is in place, breaches of the cold chain are common. Such breaches may damage the product, making therapeutics and vaccines ineffective or even harmful. Rather than strengthening the cold chain through building more infrastructure and imposing more stringent guidelines, we suggest that money and effort are best spent on making the cold chain unnecessary for biologics delivery and storage. To meet this grand challenge in pharmaceutical research, we highlight areas where innovations are needed in the design, formulation and biomanufacturing of biologics, including point-of-care manufacturing and inspection. These technological innovations would rely on fundamental advances in our understanding of biomolecules and cells.

Practical Applications of NMR to Solve Real-World Problems.

Nuclear magnetic resonance spectroscopy (NMR) is known to be a powerful technique for the characterization of small molecules and structural and dynamics studies of biomolecules [...].

Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli.

Monoclonal antibodies (mAbs) represent an important platform for the development of biotherapeutic products. Most mAbs are produced in mammalian cells, but several mAbs are made in Escherichia coli, including therapeutic fragments. The NISTmAb is a well-characterized reference material made widely available to facilitate the development of both originator biologics and biosimilars. Here, when expressing NISTmAb from codon-optimized constructs in E. coli (eNISTmAb), a truncated variant of its heavy chain was observed. N-terminal protein sequencing and mutagenesis analyses indicated that the truncation resulted from an internal translation initiation from a GTG codon (encoding Val) within eNISTmAb. Using computational and biochemical approaches, we demonstrate that this translation initiates from a weak Shine-Dalgarno sequence and is facilitated by a putative ribosomal protein S1-binding site. We also observed similar internal initiation in the mAb adalimumab (the amino acid sequence of the drug Humira) when expressed in E. coli Of note, these internal initiation regions were likely an unintended result of the codon optimization for E. coli expression, and the amino acid pattern from which it is derived was identified as a Pro-Ser-X-X-X-Val motif. We discuss the implications of our findings for E. coli protein expression and codon optimization and outline possible strategies for reducing the likelihood of internal translation initiation and truncated product formation.

Principal component analysis for automated classification of 2D spectra and interferograms of protein therapeutics: influence of noise, reconstruction details, and data preparation.

Protein therapeutics have numerous critical quality attributes (CQA) that must be evaluated to ensure safety and efficacy, including the requirement to adopt and retain the correct three-dimensional fold without forming unintended aggregates. Therefore, the ability to monitor protein higher order structure (HOS) can be valuable throughout the lifecycle of a protein therapeutic, from development to manufacture. 2D NMR has been introduced as a robust and precise tool to assess the HOS of a protein biotherapeutic. A common use case is to decide whether two groups of spectra are substantially different, as an indicator of difference in HOS. We demonstrate a quantitative use of principal component analysis (PCA) scores to perform this decision-making, and demonstrate the effect of acquisition and processing details on class separation using samples of NISTmAb monoclonal antibody Reference Material subjected to two different oxidative stress protocols. The work introduces an approach to computing similarity from PCA scores based upon the technique of histogram intersection, a method originally developed for retrieval of images from large databases. Results show that class separation can be robust with respect to random noise, reconstruction method, and analysis region selection. By contrast, details such as baseline distortion can have a pronounced effect, and so must be controlled carefully. Since the classification approach can be performed without the need to identify peaks, results suggest that it is possible to use even more efficient measurement strategies that do not produce spectra that can be analyzed visually, but nevertheless allow useful decision-making that is objective and automated.

Best Practices in Utilization of 2D-NMR Spectral Data as the Input for Chemometric Analysis in Biopharmaceutical Applications.

Quality attributes (QAs) are measureable parameters of a biologic that impact product safety and efficacy and are essential characteristics that are linked to positive patient health outcomes. One QA, higher order structure (HOS), is directly coupled to the function of protein biologics, and deviations in this QA may cause adverse effects. To address the critical need for HOS assessment, methods for analyzing structural fingerprints from 2D nuclear magnetic resonance spectroscopy (2D-NMR) spectra have been established for drug substances as large as monoclonal antibody therapeutics. Here, chemometric analyses have been applied to 2D 1H,13C-methyl NMR correlation spectra of the IgG1κ NIST monoclonal antibody (NISTmAb), recorded at natural isotopic abundance, to benchmark the performance and robustness of the methods. In particular, a variety of possible spectral input schemes (e.g., chemical shift, peak intensity, and total spectral matrix) into chemometric algorithms are examined using two case studies: (1) a large global 2D-NMR interlaboratory study and (2) a blended series of enzymatically glycan-remodeled NISTmAb isoforms. These case studies demonstrate that the performance of chemometric algorithms using either peak positions or total spectral matrix as the input will depend on the study design and likely be product-specific. In general, peak positions are found to be a more robust spectral parameter for input into chemometric algorithms, whereas the total spectral matrix approach lends itself to easier automation and requires less user intervention. Analysis with different input data also shows differences in sensitivity to certain changes in HOS, highlighting that product knowledge will further guide appropriate method selection based on the fit-for-purpose application in the context of biopharmaceutical development, production, and quality control.

Chemometric Outlier Classification of 2D-NMR Spectra to Enable Higher Order Structure Characterization of Protein Therapeutics.

Protein therapeutics are vitally important clinically and commercially, with monoclonal antibody (mAb) therapeutic sales alone accounting for $115 billion in revenue for 2018.[1] In order for these therapeutics to be safe and efficacious, their protein components must maintain their high order structure (HOS), which includes retaining their three-dimensional fold and not forming aggregates. As demonstrated in the recent NISTmAb Interlaboratory nuclear magnetic resonance (NMR) Study[2], NMR spectroscopy is a robust and precise approach to address this HOS measurement need. Using the NISTmAb study data, we benchmark a procedure for automated outlier detection used to identify spectra that are not of sufficient quality for further automated analysis. When applied to a diverse collection of all 252 1H,13C gHSQC spectra from the study, a recursive version of the automated procedure performed comparably to visual analysis, and identified three outlier cases that were missed by the human analyst. In total, this method represents a distinct advance in chemometric detection of outliers due to variation in both measurement and sample.

Structural insights into DNA-stabilized silver clusters.

Despite their great promise as fluorescent biological probes and sensors, the structure and dynamics of Ag complexes derived from single stranded DNA (ssDNA) are less understood than their double stranded counterparts. In this work, we seek new insights into the structure of single AgNssDNA clusters using analytical ultracentrifugation (AUC), nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular dynamics simulations (MD) of a fluorescent (AgNssDNA)8+ nanocluster. The results suggest that the purified (AgNssDNA)8+ nanocluster is a mixture of predominantly Ag15 and Ag16 species that prefer two distinct long-lived conformational states: one extended, the other approaching spherical. However, the ssDNA strands within these clusters are highly mobile. Ag(i) interacts preferentially with the nucleobase rather than the phosphate backbone, causing a restructuring of the DNA strand relative to the bare DNA. Infrared spectroscopy and MD simulations of (AgNssDNA)8+ and model nucleic acid homopolymers suggest that Ag(i) has a higher affinity for cytosine over guanine bases, little interaction with adenine, and virtually none with thymine. Ag(i) shows a tendency to interact with cytosine N3 and O2 and guanine N7 and O6, opening the possibility for a Ag(i)-base bifurcated bond to act as a nanocluster nucleation and strand stabilizing site. This work provides valuable insight into nanocluster structure and dynamics which drive stability and optical properties, and additional studies using these types of characterization techniques are important for the rational design of single stranded AgDNA nanocluster sensors.

2D (1)H(N), (15)N Correlated NMR Methods at Natural Abundance for Obtaining Structural Maps and Statistical Comparability of Monoclonal Antibodies.

PURPOSE: High-resolution nuclear magnetic resonance spectroscopy (NMR) provides a robust approach for producing unique spectral signatures of protein higher order structure at atomic resolution. Such signatures can be used as a tool to establish consistency of protein folding for the assessment of monoclonal antibody (mAb) drug quality and comparability. METHODS: Using the NIST monoclonal antibody (NISTmAb) and a commercial-sourced polyclonal antibody, both IgG1κ isotype, we apply 2D NMR methods at natural abundance for the acquisition and unbiased statistical analysis of (1)H(N) -(15)N correlated spectra of intact antibody (Ab) and protease-cleaved Fab and Fc fragments. RESULTS: The study demonstrates the feasibility of applying 2D NMR techniques to Abs and the precision with which these methods can be used to map structure and establish comparability between samples at atomic resolution. CONCLUSIONS: The statistical analyses suggests that, within the limit of detection, no significant structural differences are observed between the Fab and Fc domains of each respective intact Ab and its corresponding fragments. Discrimination between dissimilar species, such as between the Fab domains of both Abs or between the glycosylated and deglycosylated Fc domains, was further demonstrated. As such, these methods should find general utility for the assessment of mAb higher order structure.

Mapping monoclonal antibody structure by 2D 13C NMR at natural abundance.

Monoclonal antibodies (mAbs) represent an important and rapidly growing class of biotherapeutics. Correct folding of a mAb is critical for drug efficacy, while misfolding can impact safety by eliciting unwanted immune or other off-target responses. Robust methods are therefore needed for the precise measurement of mAb structure for drug quality assessment and comparability. To date, the perception in the field has been that NMR could not be applied practically to mAbs due to the size (∼150 kDa) and complexity of these molecules, as well as the insensitivity of the method. The feasibility of applying NMR methods to stable isotope-labeled, protease-cleaved, mAb domains (Fab and Fc) has been demonstrated from both E. coli and Chinese hamster ovaries (CHO) cell expression platforms; however, isotopic labeling is not typically available when analyzing drug products. Here, we address the issue of feasibility of NMR-based mapping of mAb structure by demonstrating for the first time the application of a 2D (13)C NMR methyl fingerprint method for structural mapping of an intact mAb at natural isotopic abundance. Further, we show that 2D (13)C NMR spectra of protease-cleaved Fc and Fab fragments can provide accurate reporters on the domain structures that can be mapped directly to the intact mAb. Through combined use of rapid acquisition and nonuniform sampling techniques, we show that these Fab and Fc fingerprint spectra can be rapidly acquired in as short as approximately 30 min.

Detection of contaminating enzymatic activity in plant-derived recombinant biotechnology products.

Residual impurities in recombinantly produced protein biologics, such as host cell proteins (HCP), can potentially cause unwanted toxic or immunogenic responses in patients. Additionally, undetected impurities found in recombinant proteins used in cell culture may adversely impact basic research and biotechnology applications. Currently, the enzyme-linked immunosorbent assay (ELISA) is the standard for detection of residual HCP contamination in recombinantly produced biologics. Alternatively, two-dimensional liquid chromatography coupled to mass spectrometry is being developed as a tool for assessing this critical quality attribute. Both of these methods rely on the direct detection of HCPs and some previous knowledge of the contaminant. For contaminating enzymes, the mass level of the impurity may fall below the threshold of detection of these methods and underestimate the true impact. To address this point, here we demonstrate facile detection and characterization of contaminating phytase activity in rice-derived recombinant human serum albumin (rHSA) using a sensitive, label-free nuclear magnetic resonance (NMR) spectroscopy assay. We observed varying degrees of phytase contamination in biotechnology-grade rHSA from various manufacturers by monitoring the degradation of adenosine-5'-triphosphate and myo-inositol-1,2,3,4,5,6-hexakisphosphate by (31)P NMR. The observed lot-to-lot variability may result in irreproducible cell culture results and should be evaluated as a possible critical quality attribute in plant-derived biotherapeutics.

Backbone NMR assignment of the yeast expressed Fab fragment of the NISTmAb reference antibody.

The monoclonal antibody (mAb) protein class has become a primary therapeutic platform for the production of new life saving drug products. MAbs are comprised of two domains: the antigen-binding fragment (Fab) and crystallizable fragment (Fc). Despite the success in the clinic, NMR assignments of the complete Fab domain have been elusive, in part due to problems in production of properly folded, triply-labeled 2H,13C,15N Fab domain. Here, we report the successful recombinant expression of a triply-labeled Fab domain, derived from the standard IgG1κ known as NISTmAb, in yeast. Using the 2H,13C,15N Fab domain, we assigned 94% of the 1H, 13C, and 15N backbone atoms.

Correlated analytical and functional evaluation of higher order structure perturbations from oxidation of NISTmAb.

The clinical efficacy and safety of protein-based drugs such as monoclonal antibodies (mAbs) rely on the integrity of the protein higher order structure (HOS) during product development, manufacturing, storage, and patient administration. As mAb-based drugs are becoming more prevalent in the treatment of many illnesses, the need to establish metrics for quality attributes of mAb therapeutics through high-resolution techniques is also becoming evident. To this end, here we used a forced degradation method, time-dependent oxidation by hydrogen peroxide, on the model biotherapeutic NISTmAb and evaluated the effects on HOS with orthogonal analytical methods and a functional assay. To monitor the oxidation process, the experimental workflow involved incubation of NISTmAb with hydrogen peroxide in a benchtop nuclear magnetic resonance spectrometer (NMR) that followed the reaction kinetics, in real-time through the water proton transverse relaxation rate R2(1H2O). Aliquots taken at defined time points were further analyzed by high-field 2D 1H-13C methyl correlation fingerprint spectra in parallel with other analytical techniques, including thermal unfolding, size-exclusion chromatography, and surface plasmon resonance, to assess changes in stability, heterogeneity, and binding affinities. The complementary measurement outputs from the different techniques demonstrate the utility of combining NMR with other analytical tools to monitor oxidation kinetics and extract the resulting structural changes in mAbs that are functionally relevant, allowing rigorous assessment of HOS attributes relevant to the efficacy and safety of mAb-based drug products.

Structural characterization of the viral and cRNA panhandle motifs from the infectious salmon anemia virus.

Infectious salmon anemia virus (ISAV) has emerged as a virus of great concern to the aquaculture industry since it can lead to highly contagious and lethal infections in farm-raised salmon populations. While little is known about the transcription/replication cycle of ISAV, initial evidence suggests that it follows molecular mechanisms similar to those found in other orthomyxoviruses, which include the highly pathogenic influenza A (inf A) virus. During the life cycle of orthomyxoviruses, a panhandle structure is formed by the pairing of the conserved 5' and 3' ends of each genomic RNA. This structural motif serves both as a promoter of the viral RNA (vRNA)-dependent RNA polymerase and as a regulatory element in the transcription/replication cycle. As a first step toward characterizing the structure of the ISAV panhandle, here we have determined the secondary structures of the vRNA and the cRNA panhandles on the basis of solution nuclear magnetic resonance (NMR) and thermal melting data. The vRNA panhandle is distinguished by three noncanonical U · G pairs and one U · U pair in two stem helices that are linked by a highly stacked internal loop. For the cRNA panhandle, a contiguous stem helix with a protonated C · A pair near the terminus and tandem downstream U · U pairs was found. The observed noncanonical base pairs and base stacking features of the ISAV RNA panhandle motif provide the first insight into structural features that may govern recognition by the viral RNA polymerase.

Structural Fingerprinting of siRNA Therapeutics by Solution NMR Spectroscopy.

Nucleic acids are an increasingly popular platform for the development of biotherapeutics to treat a wide variety of illnesses, including diseases where traditional drug development efforts have failed. To date, there are 14 short oligonucleotide therapeutics and 2 messenger RNA (mRNA) vaccines approved by the U.S. Food and Drug Administration (FDA), which demonstrates the potential of nucleic acids as a platform for the development of safe and effective medicines and vaccines. Despite the increasing popularity of nucleic acid-based drugs, there has been a paucity of high-resolution structural techniques applied to rigorously characterize these molecules during drug development. Here, we present application of nuclear magnetic resonance (NMR) methods to structurally "fingerprint" short oligonucleotide therapeutics at natural isotope abundance under full formulation conditions. The NMR methods described herein leverage signals arising from the native structural features of nucleic acids, including imino, aromatic, and ribose resonances, in addition to non-native chemistries, such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and phosphorothioate (PS) modifications, introduced during drug development. We demonstrate the utility of the NMR methods to structurally "fingerprint" a model short interfering RNA (siRNA) and a sample that simulated the drug product Givosiran. We anticipate broad applicability of the NMR methods to other nucleic acid-based therapeutics due to the generalized nature of the approach and ability to monitor many quality attributes simultaneously.

Interlaboratory Studies Using the NISTmAb to Advance Biopharmaceutical Structural Analytics.

Biopharmaceuticals such as monoclonal antibodies are required to be rigorously characterized using a wide range of analytical methods. Various material properties must be characterized and well controlled to assure that clinically relevant features and critical quality attributes are maintained. A thorough understanding of analytical method performance metrics, particularly emerging methods designed to address measurement gaps, is required to assure methods are appropriate for their intended use in assuring drug safety, stability, and functional activity. To this end, a series of interlaboratory studies have been conducted using NISTmAb, a biopharmaceutical-representative and publicly available monoclonal antibody test material, to report on state-of-the-art method performance, harmonize best practices, and inform on potential gaps in the analytical measurement infrastructure. Reported here is a summary of the study designs, results, and future perspectives revealed from these interlaboratory studies which focused on primary structure, post-translational modifications, and higher order structure measurements currently employed during biopharmaceutical development.

SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2'-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)-containing RNA/DNA hybrids, their size (20-25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M(r) = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2'-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.