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

• Published in: Biochimica et biophysica acta. General subjects 2019-01, Vol.1863 (1), p.232-240
• Main Authors: Batool, Sana, Argyropoulos, Kimon V., Azad, Roksana, Okeoma, Precious, Zumrut, Hasan, Bhandari, Sanam, Dekhang, Rigzin, Mallikaratchy, Prabodhika R.
• Format: Article
• Language: English
• Subjects: antibodies; Aptamer; Aptamers, Nucleotide - chemistry; B-cell lymphoma; B-lymphocytes; B-Lymphocytes - metabolism; Burkitt Lymphoma - metabolism; Cells, Cultured; confocal microscopy; CRISPR-Cas Systems; Dimerization; DNA; Epitopes - chemistry; HEK293 Cells; Humans; image analysis; immunoglobulin M; Immunoglobulin M - chemistry; Lentivirus - genetics; Leukocytes, Mononuclear - cytology; Ligand-guided selection; Ligands; Lymphoma, B-Cell - metabolism; mIgM; neoplasm cells; neoplasms; oligonucleotides; Plasmids - metabolism; Protein Binding; Protein Engineering; Puromycin - chemistry; RNA; SELEX Aptamer Technique; Temperature; therapeutics; Waldenstrom Macroglobulinemia - metabolism
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2018.10.006

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. •Dimerization of an aptamer selected by Ligand-guided selection (LIGS) significantly improves the affinity.•Dimerization of LIGS-generated aptamer does not alter specificity against B-cells.•Dimerized LIGS-generated aptamer recognizes same epitope expressed on clinical B-cell samples.