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Analytical ultracentrifugation (AUC) has emerged as a robust and reliable technique for biomolecular characterization with extraordinary sensitivity. AUC is widely used to study purity, conformational changes, biomolecular interactions, and stoichiometry. Furthermore, AUC is used to determine the molecular weight of biomolecules such as proteins, carbohydrates, and DNA and RNA. Due to the multifaceted role(s) of non-coding RNAs from viruses, prokaryotes, and eukaryotes, research aimed at understanding the structure-function relationships of non-coding RNAs is rapidly increasing. However, due to their large size, flexibility, complicated secondary structures, and conformations, structural studies of non-coding RNAs are challenging. In this review, we are summarizing the application of AUC to evaluate the homogeneity, interactions, and conformational changes of non-coding RNAs from adenovirus as well as from Murray Valley, Powassan, and West Nile viruses. We also discuss the application of AUC to characterize eukaryotic long non-coding RNAs, Xist, and HOTAIR. These examples highlight the significant role AUC can play in facilitating the structural determination of non-coding RNAs and their complexes.

RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA–protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the “bait” and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.

Worldwide, ∼250 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of cirrhosis and hepatocellular carcinoma. The HBV persists as covalently closed circular DNA (cccDNA), which acts as the template for all HBV mRNA transcripts. Nucleos(t)ide analogs do not directly target the HBV cccDNA and cannot eradicate the HBV. We have discovered a unique structural motif, a G-quadruplex in HBV’s pre-core promoter region that is conserved amongst nearly all genotypes, and is central to critical steps in the viral life-cycle including the production of pre-genomic RNA, core and polymerase proteins, and encapsidation. Thus, an increased understanding of the HBV pre-core may lead to the development of novel anti-HBV cccDNA targets. We utilized biophysical methods to characterize the presence of the G-quadruplex, employed assays using a known quadruplex- binding protein (DHX36) to pull-down HBV cccDNA, and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids. This study provides insights into the presence of a G-quadruplex in the HBV pre-core promoter region essential for HBV replication. The evaluation of this critical host-protein interaction site in the HBV cccDNA may ultimately facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5&prime, terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5&prime, TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5&prime, TR of JEV and demonstrated its direct interaction with recombinant DDX3X (Kd of 1.66 &plusmn, 0.21 &micro, M) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5&prime, and 3&prime, TRs of flaviviruses, we investigated if the ZIKV 5&prime, TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5&prime, TR with a Kd of 7.05 &plusmn, 0.75 &micro, M. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5&prime, TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2′-5′-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2′-5′-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.