title: Isolation and optimization of murine IL-10 receptor blocking oligonucleotide aptamers using high-throughput sequencing.
abstract: Interleukin-10 (IL-10) is a key suppressor of inflammation in chronic infections and in cancer. In mice, the inability of the immune system to clear viral infections or inhibit tumor growth can be reversed by antibody-mediated blockade of IL-10 action. We used a modified selection protocol to isolate RNA-based, nuclease-resistant, aptamers that bind to the murine IL-10 receptor. After 5 rounds of selection high-throughput sequencing (HTS) was used to analyze the library. Using distribution statistics on about 11 million sequences, aptamers were identified which bound to IL-10 receptor in solution with low K(d). After 12 rounds of selection the predominant IL-10 receptor-binding aptamer identified in the earlier rounds remained, whereas other high-affinity aptamers were not detected. Prevalence of certain nucleotide (nt) substitutions in the sequence of a high-affinity aptamer present in round 5 was used to deduce its secondary structure and guide the truncation of the aptamer resulting in a shortened 48-nt long aptamer with increased affinity. The aptamer also bound to IL-10 receptor on the cell surface and blocked IL-10 function in vitro. Systemic administration of the truncated aptamer was capable of inhibiting tumor growth in mice to an extent comparable to that of an anti- IL-10 receptor antibody.
authors.affiliation: Department of Microbiology and Immunology, Dodson Interdisciplinary Immunotherapy Institute and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida 33136, USA.
authors.forename: Alexey
authors.initials: A
authors.lastname: Berezhnoy
authors.forename: C Andrew
authors.initials: CA
authors.lastname: Stewart
authors.forename: James O
authors.initials: JO
authors.lastname: Mcnamara
authors.forename: William
authors.initials: W
authors.lastname: Thiel
authors.forename: Paloma
authors.initials: P
authors.lastname: Giangrande
authors.forename: Giorgio
authors.initials: G
authors.lastname: Trinchieri
authors.forename: Eli
authors.initials: E
authors.lastname: Gilboa
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D057166:High-Throughput Screening Assays; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D053701:Receptors, Interleukin-10 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D052157:Aptamers, Nucleotide; D053701:Receptors, Interleukin-10