control rna motif plasmid cloning backbone  (New England Biolabs)

Journal: bioRxiv

Article Title: UCHL3 regulates subgenomic flaviviral RNA condensates to promote virus propagation

doi: 10.1101/2025.09.05.674517

Figure Lengend Snippet: (A) Co-immunoprecipitation analysis of Flag-HA-UCHL3 from 293 cells transfected with either empty vector control (pcDNA3.1) or Flag-HA-UCHL3 construct and infected with ZIKV or mock-infected. Input lysates and immunoprecipitated (IP) fractions were analysed by immunoblotting for viral non-structural proteins NS5, NS3, and NS4A, viral structural protein prM, and Flag-HA-UCHL3. (B) Schematic representation of the biotinylated sfRNA-interactome capture strategy. The engineered sfRNA construct contains flanking BoxB stem-loop structures that specifically recruit the BirA-λN fusion protein (BASU). Upon biotin supplementation, BASU biotinylates proteins in close proximity to sfRNA, enabling streptavidin-mediated affinity purification of sfRNA-associated ribonucleoprotein complexes. (C) Silver staining analysis of sfRNA-interacting protein complexes. Total protein profiles from streptavidin pulldowns using control RNA motif, ZIKV sfRNA, or DENV sfRNA constructs in the presence or absence of ZIKV infection for infection-dependent recruitment of cellular factors to sfRNA assemblies. (D) Streptavidin-affinity capture followed by immunoblot analysis showing selective enrichment of UCHL3 and G3BP1 in the presence of ZIKV or DENV sfRNA constructs. Cell lysates and streptavidin pulldown (SA-Pulldown) fractions show specific association of these proteins with sfRNA-containing complexes. BASU (HA-tagged) serves as an internal control for the capture system. (E-F) Quantitative RT-PCR analysis of (E) NS5 genomic RNA and (F) sfRNA levels in cells transfected with either pcDNA3.1 empty vector or Flag-HA-UCHL3 at 24 and 48 hours post-ZIKV infection. Ectopic UCHL3 expression increases both viral genomic and subgenomic RNA species compared to vector controls. Data represent mean ± SD from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The resulting amplicon and a control RNA motif plasmid cloning backbone were digested with the BsmBI-v2 restriction enzyme (NEB) for 1h at 55°C.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Control, Construct, Infection, Western Blot, Affinity Purification, Silver Staining, Quantitative RT-PCR, Expressing