A set of >300 nonredundant high-resolution RNA–protein complexes were rigorously searched for π-contacts between an amino acid side chain (W, H, F, Y, R, E and D) and an RNA nucleobase (denoted π–π interaction) or ribose moiety (denoted sugar–π). The resulting dataset of >1500 RNA–protein π-contacts were visually inspected and classified based on the interaction type, and amino acids and RNA components involved. More than 80% of structures searched contained at least one RNA–protein π-interaction, with π–π contacts making up 59% of the identified interactions. RNA–protein π–π and sugar–π contacts exhibit a range in the RNA and protein components involved, relative monomer orientations and quantum mechanically predicted binding energies. Interestingly, π–π and sugar–π interactions occur more frequently with RNA (4.8 contacts/structure) than DNA (2.6). Moreover, the maximum stability is greater for RNA–protein contacts than DNA–protein interactions. In addition to highlighting distinct differences between RNA and DNA–protein binding, this work has generated the largest dataset of RNA–protein π-interactions to date, thereby underscoring that RNA–protein π-contacts are ubiquitous in nature, and key to the stability and function of RNA–protein complexes.

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17_135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17_135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17_135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17_135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17_135–555 interacting with and unwinding RVFV non-coding regions.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine‐quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody‐based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single‐domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small‐Angle X‐ray Scattering. Lentiviral transduction of S10 into HepG2‐NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next‐generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

Globally, an estimated 254 million people are living with chronic hepatitis B virus (HBV) infection, yet only 10.5% have been diagnosed, underscoring the urgent need to expand testing to meet the World Health Organization’s HBV elimination targets by 2030. Many HBV diagnostic tests remain expensive and inaccessible in resource-limited settings. In this study, we demonstrate how individually sourced, commercially available reagents can be used to develop cost-effective in-house assays for total DNA isolation, HBV viral load quantification by (q)PCR, and qHBsAg and qHBeAg measurement using sandwich ELISA. These assays were validated using known HBV-positive and HBV-negative plasma samples (genotypes A–F) and HepAD38 cells treated with tenofovir disoproxil fumarate (TDF). DNA isolation using a commercial column-based kit was compared to a high-throughput, column-free method, allowing for HBV quantification from 50 µL of plasma with lower limits of detection (LLOD) of 1.8 × 10³ and 1.8 × 10⁴ HBV DNA copies IU/mL, respectively. Both commercial and in-house DNA isolation methods yielded comparable half-maximal effective concentration (EC₅₀) values in TDF-treated HepAD38 cells. Additionally, in-house sandwich ELISA assays were developed for quantitative HBsAg and HBeAg detection, with LLOD values of 0.78 IU/mL and 0.38 PEI U/mL (Paul Ehrlich Institute), respectively. The in-house reagents for DNA isolation, molecular testing, and serological detection of HBV were estimated to be at least 10 times more cost-effective than commercially available kits, highlighting their potential for broader application in resource-limited regions.

Guanine-rich nucleic acids can form a unique secondary structure called Guanine-quadruplexes (G4s). These G4s are conserved across all domains of life and in viruses, where they can play important regulatory roles in the viral lifecycle. In flaviviruses, the (-) strand 3’ untranslated region (UTR) is essential for initiating genomic RNA synthesis. Within the (-) strand 3’ UTR of hepatitis C virus (HCV), a highly conserved G4 is located within stem-loop IIy’ (SLIIy’), spanning nucleotides 110-131—a region that is essential for efficient replication. Using bioinformatics, we demonstrate that nucleotides 110-131 are highly conserved across HCV genotypes 1-7, and this region within SLIIy’ can likely adopt a more energetically favorable G4 conformation rather than the predicted hairpin. From biophysical and cell biology experiments, we show that stabilizing the predicted hairpin structure in SLIIy’ disrupts RNA synthesis, while successive guanine-to-adenine mutations within the G4 sequence impair G4 formation and hinder replication. Combining our findings with prior studies, we propose that the SLIIy’ G4 plays a crucial role in recruiting NS3 helicase, which is necessary for unwinding RNA structures and facilitating access for NS5B polymerase. The G4 may serve as a regulatory switch that modulates helicase binding and replication efficiency. We propose that in the absence of a stable G4, NS3 recruitment or unwinding is impaired, leading to inefficient RNA unwinding and reduced polymerase activity, ultimately hindering viral replication. These results reveal a previously unrecognized role for G4 structures in the HCV replication cycle, highlighting their importance in regulating (+) strand RNA synthesis. Importance: Hepatitis C virus is a positive-sense single-stranded RNA virus that relies on an intermediate negative strand to generate new genomic RNA. While the last 157 nucleotides of the negative-strand 3’ untranslated region are known to be essential for replication, the underlying regulatory mechanisms have remained poorly understood. Our study demonstrates that a highly conserved guanine-rich sequence (107-131 nt) located on the negative strand SLIIy’ forms a guanine-quadruplex secondary structure that plays a pivotal role in orchestrating viral RNA synthesis. This G4 not only promotes efficient replication but likely acts as a molecular target to recruit the NS3 helicase, thereby allowing access for the NS5B polymerase. Disruption of the G4 structure and stabilization of the stem-loop severely impairs viral replication. These findings reveal an unrecognized layer of post-transcriptional regulation in the HCV lifecycle and establish G4 RNA structures as a critical element in HCV genome replication.