Journal: bioRxiv

Article Title: Selection of antibody-binding covalent aptamers

doi: 10.1101/2023.03.09.530504

Figure Lengend Snippet: Step 1) Enzymatic incorporation of commercially available EdU-triphosphate (replacing thymidine triphosphate) along with natural dATP, dCTP, and dGTP. Step 2) Polymerase-based displacement of the EdU-containing strand. Step 3) CuAAC modification with sulfo-HSAB. Step 4) Exposure of Mouse IgG1 monoclonal Ab (100 nM) to the library. Step 5) Magnetic bead capture of IgG1-aptamer conjugates followed by washing, elution with 350 nm light and amplification of eluted conjugates. Step 6) Isolation of the negative PCR strand. Step 7 and 8) Annealing and ligation to a regeneration oligonucleotide to complete a single round of selection

Article Snippet: To the desalted material, 1X thermopol buffer (final concentration), 200 μM each of dATP, dCTP, dGTP and dTTP, 75 pmol strand displacement primer, and 200 μM dNTPs and 8 units of Bst 2.0 warmstart DNA polymerase (NEB) were added and reaction was incubated at 65°C for 2 minutes.

Techniques: Modification, Amplification, Isolation, Ligation, Selection

Journal: Cell

Article Title: Cryo-EM structure of the RADAR supramolecular anti-phage defense complex

doi: 10.1016/j.cell.2023.01.012

Figure Lengend Snippet: RADAR mediates ATP-to-ITP conversion in anti-phage defense (A) Quantitative mass spectrometry of ATP, dATP, ITP, and dITP in lysates extracted from cells containing Ec RADAR or control plasmid. Cells were infected with T4 phage at an MOI of 2 for indicated amount of time before harvesting. Bar graph shows the mean, with error bars representing standard deviation. (B) HPLC analysis of ATP after incubation with purified E. coli RdrB, showing deamination of ATP to ITP. (C) Summary of HPLC analyses of 1 mM different nucleotide substrates incubated with 1 μM RdrB for 30 min, demonstrating robust deamination of tri-phosphorylated substrates, but not monophosphorylated or unphosphorylated substrate. Full HPLC traces shown in Figure S6 D. (D) Mutation of conserved histidine residues within the active site of RdrB are required for deaminase activity, and RdrB H152/154A mutant no longer converts ATP to ITP. (E) HPLC analysis of nucleotides after digestion of hairpin RNA to assess the identity of each base. Incubation of hairpin RNA with RdrB, in conditions where ATP is robustly converted to ITP (B), does not lead to deamination of adenosine within the RNA. (F) Schematic of fstI hairpin RNA used in (E). Hairpin was digested after incubation with RdrB, and mononucleotides were visualized by HPLC. (G) A proposed schematic for the mechanism of action of the RADAR defense system.

Article Snippet: For comparison of ATP and dATP conversion, reactions containing 1 mM ATP and 1 mM dATP were incubated at 37°C for 10 min. Hairpin RNA experiments contained 1 μM hairpin RNA and were at 37°C for 1 h. Reactions were treated with calf intestinal phosphatase (New England Biolabs) and hairpin RNA samples were concurrently treated with P1 nuclease to release monomeric NMPs for analysis, then samples were spun through a 0.2 μm filter.

Techniques: Mass Spectrometry, Plasmid Preparation, Infection, Standard Deviation, Incubation, Purification, Mutagenesis, Activity Assay

Journal: Cell

Article Title: Cryo-EM structure of the RADAR supramolecular anti-phage defense complex

doi: 10.1016/j.cell.2023.01.012

Figure Lengend Snippet: RdrB converts (d)ATP to (d)ITP in vivo and in vitro , related to Figure 6 (A) Ion count (area under curve) of ITP or dITP (respectively) in lysates extracted from WT Ec RADAR containing cells, as well as RADAR mutated in RdrA-K82A or RdrB-E501A, as measured by LC-MS/MS. The x axis represents min after infection, with zero representing non-infected cells. Cells were infected by phage T4 at an MOI of 2 at 37°C. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (B) Ion count (area under curve) of ITP or dITP (respectively) in lysates extracted from WT Cr RADAR containing cells, or control cells, as measured by LC-MS/MS. The x axis represents min after infection, with zero representing non-infected cells. Cells were infected by phage T2 at an MOI of 2 at 37°C. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (C) Quantitative mass spectrometry of ADP, AMP, IDP, and IMP in lysates extracted from cells containing Ec RADAR or control plasmid. Cells were infected with T4 phage at an MOI of 2 for indicated amount of time before harvesting. Bar graph shows the mean, with error bars representing standard deviation. (D) Full HPLC traces of data summarized in Figure 6 C. (E) HPLC analysis of ATP and dATP co-incubated with RdrB, showing similar deamination of both substrates. (F) Growth curves of cells expressing Ec RdrB (orange), the mutant Ec RdrB E501A (blue), and control cells expressing GFP (gray) in the presence of 2% arabinose without phage infection. Results of two experiments are presented as individual curves. (G) Ion count (area under curve) of ITP in lysates extracted from Ec RdrB and EcRdrB E501A expressing cells, as measured by LC-MS/MS. Cells were supplemented with 0.1% arabinose and incubated at 37°C for 100 min. Bar graphs represent the average of two biological replicates, with individual data points overlaid. (H) Phosphate release assay measuring NTP hydrolysis by RdrA. Purified RdrA was incubated with indicated NTP, then phosphate released by hydrolysis was quantified using malachite green. Bar graph shows the mean, with error bars representing standard deviation. (I) ATP-to-ITP conversion by RdrB either alone, in the presence of RdrA, or in the presence of RdrA and hairpin RNA. RdrA alone does not deaminate ATP to ITP. Bar graph shows the mean, with error bars representing standard deviation.

Article Snippet: For comparison of ATP and dATP conversion, reactions containing 1 mM ATP and 1 mM dATP were incubated at 37°C for 10 min. Hairpin RNA experiments contained 1 μM hairpin RNA and were at 37°C for 1 h. Reactions were treated with calf intestinal phosphatase (New England Biolabs) and hairpin RNA samples were concurrently treated with P1 nuclease to release monomeric NMPs for analysis, then samples were spun through a 0.2 μm filter.

Techniques: In Vivo, In Vitro, Liquid Chromatography with Mass Spectroscopy, Infection, Mass Spectrometry, Plasmid Preparation, Standard Deviation, Incubation, Expressing, Mutagenesis, Phosphate Release Assay, Purification