Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Fcγ receptor-mediated phagocytosis pathway was involved in phagocytosis of mIgM+ B lymphocytes from largemouth bass (Micropterus salmoides)

The potential for phagocytosis has been proven in teleost B cells, but the research on the regulatory mechanism of phagocytosis remains lacking. In this study, three largemouth bass (Micropterus salmoides) (15 ± 5 g) were injected intraperitoneally with Nocardia seriolae (10⁵ CFU/100 μl/fish) in vivo, and their spleen was collected at 72 h post-infection for mRNA-seq. After the de novo assembly of the paired-end reads, 73,622 unigenes were obtained. Gene expression profiling revealed that 2043 unigenes were differentially expressed after N. seriolae infection, comprising 1285 upregulated and 758 downregulated unigenes (q-value <0.05, log2FC > |2|) of which 181 genes were involved in phagocytosis. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis demonstrated that 12 differentially expressed genes (DEG) associated with phagocytosis were enriched in the Fcγ receptor-mediated phagocytosis signalling pathway. In vitro, the phagocytic ability of mIgM⁺ B lymphocytes was validated using indirect immunofluorescence assay (IIFA) and fluorescence activating cell sorter (FACS), and the phagocytosis rates of the mIgM⁺ B lymphocytes incubated with a Lyn inhibitor had decreased from 18.533 ± 6.00% to 11.610 ± 4.236% compared with the unblocked group. These results suggested that the Fcγ receptor-mediated phagocytosis signalling pathway had participated in the phagocytosis of B cells and provide further insight into the role of B cells in innate immunology.