title: Truncation and Mutation of a Transferrin Receptor Aptamer Enhances Binding Affinity.
abstract: Aptamers are proving their utility in a number of applications. However, to be easily functionalized, their structure needs to be simplified. Therefore, we sought to truncate a 50-nucleotide aptamer specific to the transferrin receptor to its smallest functional unit using rational engineering of the predicted two-dimensional structure of the longer parent sequence. In addition, mutations were introduced into the binding loop to determine their effect on the selectivity of the aptamers. These base mutations enhanced the binding affinity of the aptamer, while retaining its specificity. The equilibrium dissociation constant (Kd) was reduced sixfold following the substitution of all four bases in the binding region. In addition, these aptamers were efficiently internalized into transferrin receptor-positive cells in a similar manner to the transferrin receptor antibody and demonstrated colocalization with this antibody. This study has shown that the smallest functional unit of this aptamer was 14 nucleotides. This small size will be advantageous for future applications, such as drug delivery or functionalization of other therapeutic modalities.
authors: 1 School of Medicine, Deakin University , Geelong, Victoria, Australia .
authors: Joanna
authors: J
authors: Macdonald
authors: 1 School of Medicine, Deakin University , Geelong, Victoria, Australia .
authors: Patrick
authors: P
authors: Houghton
authors: 1 School of Medicine, Deakin University , Geelong, Victoria, Australia .
authors: Dongxi
authors: D
authors: Xiang
authors: 1 School of Medicine, Deakin University , Geelong, Victoria, Australia .|2 Centre for Molecular and Medical Research, Deakin University , Geelong, Victoria, Australia .
authors: Wei
authors: W
authors: Duan
authors: 1 School of Medicine, Deakin University , Geelong, Victoria, Australia .|2 Centre for Molecular and Medical Research, Deakin University , Geelong, Victoria, Australia .
authors: Sarah
authors: S
authors: Shigdar
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D020029:Base Pairing; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D004705:Endocytosis; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009690:Nucleic Acid Conformation; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism*; D052156:SELEX Aptamer Technique
chemical_list: D052157:Aptamers, Nucleotide; D011990:Receptors, Transferrin
keywords: aptamer; blood-brain-barrier; receptor-mediated transcytosis; transferrin receptor; truncation