Journal: Nature Communications

Article Title: RNA-guided RNA silencing by an Asgard archaeal Argonaute

doi: 10.1038/s41467-024-49452-1

Figure Lengend Snippet: a HrAgo1 associates with 5’ phosphorylated (5’ P) small RNAs in vivo from E. coli . Nucleic acids that co-purified with HrAgo1 were [γ- 32 P] labeled, treated with RNase A or DNase I, and resolved on a denaturing gel (15% polyacrylamide 7 M urea). nt: nucleotides. b Length distribution of small RNAs associated with HrAgo1 as determined by small RNA sequencing. c Small RNAs associated with HrAgo1 have a bias for uracil bases at the 5’ end. d Sequences of guide and target oligonucleotides used in in vitro cleavage assays. e HrAgo1 cleaves ssRNA (but not ssDNA) targets with ssRNA guides, and ssDNA guides at lower efficiency, in the presence of Mg 2+ . HrAgo1 was incubated with ssDNA or ssRNA guides and Cy5-labeled ssDNA or RNA targets. Cy5-labeled cleavage products were resolved through denaturing (7 M urea) polyacrylamide gel electrophoresis and visualized by fluorescence imaging. Both ssRNA and ssDNA targets are 45nt. The HrAgo1-bound RNA extraction and digestion was carried out once; the results of the cleavage assays were confirmed by at least three repetitions.

Article Snippet: A plasmid suitable for expression of a HrAgo1 catalytic double-mutant (D585A & E623A; HrAgo1 DM ) was generated by Quikchange Site-Directed Mutagenesis using primers oPB199 and oPB201 for D585A and oPB200 and oPB198 for E623A, using E. coli strain NEB 5-alpha (New England Biolabs) (Table ). pX-sfGFP vector was a kind gift from Prof. Jae-Sung Woo (Korea University, South Korea).

Techniques: In Vivo, Purification, Labeling, RNA Sequencing Assay, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging, RNA Extraction

Journal: Journal of Clinical Microbiology

Article Title: Optimization of a Combined Human Parechovirus-Enterovirus Real-Time Reverse Transcription-PCR Assay and Evaluation of a New Parechovirus 3-Specific Assay for Cerebrospinal Fluid Specimen Testing

doi: 10.1128/JCM.01982-12

Figure Lengend Snippet: Comparison of CT values of EV, pan-HPeV, and HPeV3 assays with CSF clinical specimens tested in one-step and two-step RT-PCR reactions. (A) Two-step EV assay with the AGP kit enzyme versus one-step Cepheid enterovirus ASR (year 2008; n = 25); (B) two-step EV assay with the AGP kit enzyme versus one-step Argene enterovirus RUO assay (year 2009; n = 25); (C) two-step versus one-step EV assay with the AGP kit enzyme (year 2012; n = 30); (D) two-step versus one-step pan-HPeV assay with the AGP kit enzyme and historic two-step pan-HPeV assay with the ABI enzyme; (E) two-step versus one-step HPeV3 assay with the AGP kit enzyme and historic two-step HPeV3 assay with the ABI enzyme.

Article Snippet: The analytical specificity of the pan-HPeV assay reagents was tested against 13 enterovirus strains and other viruses obtained from ATCC: coxsackievirus types A9 (VR-186), B1 (VR-1032), B2 (VR-29), B3 (VR-30), B4 (VR-184), and B5 (VR-185); echovirus types 3 (VR-1040), 4 (VR-1041), 5 (VR-1043), 6 (VR-36), 7 (VR-37), 9 (VR-39), and 11 (VR-41); rhinovirus types 39 (ATCC VR-340), 8 (ATCC VR-488), 13 (ATCC VR-1123), 26 (ATCC VR-1136), and 27 (ATCC VR-1137); herpes simplex virus 1 (HSV-1) (ATCC VR-539) and HSV-2 (ATCC VR-540); human herpesvirus 6 (HHV-6; ATCC VR-1480); and laboratory-grown clinical isolates of cytomegalovirus (CMV) and Epstein-Barr virus (EBV).

Techniques: Comparison, Reverse Transcription Polymerase Chain Reaction