Prism version 5.0c

Data are presented as averages with 95% confidence intervals. All data were statistically analyzed using PRISM version 5.0c software (GraphPad Software, La Jolla, CA, USA). Statistical significance was assigned at values of p < 0.05 (95% confidence interval).

G4 DNA containing a 3′ FAM modification (F-G4) was solubilized to 50 µM in G4 folding buffer [10 mM Tris·HCl (pH 8.0), 100 mM KCl]. Using a heat block, the DNA was heated to 95 °C for 5 min, after which the block was removed from heat and allowed to cool to room temperature over approximately 4 h. Folded DNA was then stored at 4 °C. RecQ proteins were serially diluted from 20,000 to 0.6 nM in G4 binding buffer [20 mM Tris·HCl (pH 8.0), 100 mM NaCl, 1 mM MgCl₂, 1 mM β-mercaptoethanol, 0.1 mg/mL bovine serum albumin, 4% (vol/vol) glycerol], then incubated with 5 nM F-G4 for 30 min at room temperature in a total volume of 100 µL. The fluorescence anisotropy of each sample was measured at 25 °C with a Beacon 2000 fluorescence polarization system. Measurements are reported in duplicate and error bars represent 1 SEM. Binding affinities and uncertainties were determined using Prism version 5.0c (GraphPad Software, La Jolla, CA, USA). Duplex binding assays were performed as the G4 binding assays using a 3ʹ FAM-labeled ssDNA (duplex 1) annealed to an unlabeled 18mer (duplex 2) to create a substrate with an 18-bp duplex with 3ʹ overhang of 12 nucleotides.^{21 (link)} Duplex binding assays were performed in triplicate and error bars represent 1 SEM.

Bone marrow or splenocytes were enriched for B cells via EasySep B-cell enrichment kit (Stemcell Technologies) and CD138⁺B220⁻ PCs were FACS sorted from C57BL/6 mice 2 months post immunization. Marrow or splenic PCs (10⁵) were then transferred via tail-vein injection into cohorts of sex-matched μMT mice or were measured for NP₂₂CGG specificity via ELISPOT. Serum antibody titres were measured by ELISA at days 7, 14, 21, 30 and 60. Plates were coated with 20 μg ml⁻¹ NP₂₂CGG (or 2 μg ml⁻¹ anti-IgG or -IgM for standard wells) and blocked with 3% BSA. Sera from recipient μMT mice was serially diluted in PBS and incubated for 2 h. Plates were then treated with either anti-IgG–horseradish peroxidase or -IgM-biotin, followed by incubation with streptavidin–horseradish peroxidase for IgM plates (Southern Biotechnology). TMB substrate (Pierce) was added for 30 s and then the reaction stopped with 0.16 M sulfuric acid. Plates were read at 450 nm. PC numbers in the bone marrow and spleen were assayed by ELISPOT at days 7, 14, 30 and at 2 months post adoptive transfer as previously described, or at 2 months for bone marrow transfer cohorts. Half-lives were determined by linear regression of transferred cells, comparing the slopes for statistically significant differences (Graphpad Prism Version 5.0c).