Most mosquito‐borne flaviviruses, including Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), produce long non‐coding subgenomic RNAs (sfRNAs) in infected cells that link to pathogenicity and immune evasion. Until now, the structural characterization of these lncRNAs remains limited. Here, we studied the 3D structures of individual and combined subdomains of sfRNAs, and visualized the accessible 3D conformational spaces of complete sfRNAs from DENV2, ZIKV, and WNV by small angle X‐ray scattering (SAXS) and computational modeling. The individual xrRNA1s and xrRNA2s adopt similar structures in solution as the crystal structure of ZIKV xrRNA1, and all xrRNA1‐2s form compact structures with reduced flexibility. While the DB12 of DENV2 is extended, the DB12s of ZIKV and WNV are compact due to the formation of intertwined double pseudoknots. All 3′ stem‐loops (3′SLs) share similar rod‐like structures. Complete sfRNAs are extended and sample a large conformational space in solution. Our work not only provides structural insight into the function of flavivirus sfRNAs, but also highlights strategies of visualizing other lncRNAs in solution by SAXS and computational methods.

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3′SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3′SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy remains challenging up-to-date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs...

Biofilms are communities of microbes embedded in a microbial extracellular matrix. Their formation is considered the main virulence mechanism enabling the opportunistic bacterial pathogen Staphylococcus epidermidis to cause devastating nosocomial, implant-associated infections. Biofilms often contain proteins, and an 18-kDa protein called small basic protein (Sbp) recently was discovered in the S. epidermidis biofilm matrix and may serve as a scaffolding protein in both polysaccharide intercellular adhesion (PIA)-dependent and accumulation-associated protein (Aap)-dependent biofilm formations. In Aap-mediated biofilm formation, Sbp co-localizes with the domain-B of Aap, implying that Sbp directly interacts with Aap's Domain-B. However, the structure of Sbp and its interaction with Aap, as well as the molecular mechanism underlying Sbp's roles in biofilm formation, are incompletely understood. In this work, we used small-angle X-ray scattering (SAXS), NMR, analytical size-exclusion chromatography, and isothermal titration calorimetry analyses to determine the Sbp structure and characterize its interaction with Aap's Domain-B. We found that Sbp is monomeric and partially folded in solution, and, unexpectedly, we observed no direct interactions between Sbp and Aap Domain-B. Instead, we noted that Sbp forms amyloid fibrils both in vitro and in vivo. Atomic force, transmission electron, and confocal fluorescence microscopy methods confirmed the formation of Sbp amyloid fibrils and revealed their morphology. Taken together, the Sbp amyloid fibril structures identified here may account for Sbp's role as a scaffolding protein in the S. epidermidis biofilm matrix. Sbp forms functional amyloid fibrils 2Previously regarded as an innocuous human commensal that normally colonized the human skin and mucous membrane, Staphylococcus epidermidis has emerged as an leading opportunistic pathogen causing nosocomial infection associated with the permanent or intermittent indwelling medical devices (1,2). In contrast with its more virulent cousin Staphylococcus aureus, which produces an abundance of toxins, adherence factors, and cytolysins, the main virulence mechanism through which Staphylococcus epidermidis evade the host immune response relates to its capability of adhering and forming biofilms on biotic and abiotic surfaces (3). Staphylococcal biofilms are adherent multilayered communities of multicellular organisms embedded in self-produced extracellular matrix that are functionally resistant to antibiotics and components of the immune system (4). Therefore, S. epidermidis-mediated infections are difficult to treat and affect millions of patients worldwide annually, causing significant morbidity and mortality and a high burden for public health system (5).Biofilm formation is a complex, multifactorial process and can be divided into at least three stages: initial attachment to abiotic surfaces, protein-coated materials and host cells, and subsequent cell proliferation and accumulation via cell-cell adhesion leading to ...