The efficient cell-SELEX strategy, Icell-SELEX, using isogenic cell lines for selection and counter-selection to generate RNA aptamers to cell surface proteins.
Aptamers are short single-stranded nucleic acid molecules that are selected in vitro from a large random sequence library based on their high and specific affinity to a target molecule by a process known as SELEX. Cell-SELEX that employs whole living cells overexpressing the defined cell surface proteins (for selection) and appropriate mock cells (for counter-selection) has been widely used as a valid and feasible method for generating aptamers against specific cell surface proteins. However, the endogenous expression of target proteins in mock cells or the heterogeneity of surface proteins between selection and counter-selection cells often impeded the isolation of proper aptamers against target proteins. To solve this problem, we developed "Isogenic cell-SELEX" (Icell SELEX in short) method, in which isogenic cell lines were manipulated for counter-selection by microRNA-mediated silencing and for selection by overexpression of target proteins. As a model experiment, we targeted integrin alpha V (ITGAV), which is a major transmembrane receptor expressed in almost all the cells, and established ITGAV-overexpressed and -downregulated HEK293 cells for selection and counter-selection, respectively. By taking advantage of a hundred-fold difference in the expression level of ITGAV between these two isogenic cell lines, we easily isolated several anti-ITGAV aptamers, whose binding to the cell-surface ITGAV was confirmed by flow cytometry with the dissociation constant of 300-400 nM range. We assume that Icell-SELEX could be applicable to a wide range of cell-surface proteins including various transmembrane proteins of biological and pharmacological significance.
The institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Masaki
M
Takahashi
The institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Eri
E
Sakota
The institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan; RIBOMIC Inc., Minato-ku, Tokyo 108-0071, Japan. Electronic address: nak@ims.u-tokyo.ac.jp.
Yoshikazu
Y
Nakamura
D052157:Aptamers, Nucleotide / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D005434:Flow Cytometry; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D039564:Integrin alphaV / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D015203:Reproducibility of Results; D052156:SELEX Aptamer Technique / Q000379:methods*; D020349:Surface Plasmon Resonance
D052157:Aptamers, Nucleotide; D039564:Integrin alphaV; D008565:Membrane Proteins; D035683:MicroRNAs; D049452:Green Fluorescent Proteins
Cell surface proteins; Integrin alpha V; Isogenic cell-SELEX; RNA aptamer; RNA silencing