Staining of cell surface human CD4 with 2'-F-pyrimidine-containing RNA aptamers for flow cytometry.
We have used recombinant human CD4 presented on beads as an affinity matrix to screen a 2'-F-pyrimidine-containing RNA library with a complexity of approximately 10(14) molecules. Affinity-selected aptamers bind recombinant CD4 with low nanomolar equilibrium dissociation constants. These high-affinity aptamers conjugated to different fluorophores such as fluorescein and phycoerythrin were used to stain cells, expressing human CD4 on cell surface, for analysis by flow cytometry. Aptamers, conjugated to fluorophores, stained mouse T cells that express human CD4 on the surface, but not the control mouse T cells lacking human CD4. The control cells, however, do express mouse CD4 whose extracellular domain has 55% sequence identity to the human form. These human CD4-specific aptamers selectively stained CD4(+) T cells in a preparation of human peripheral blood mononuclear cells. These results and others suggest that aptamers are emerging as a versatile class of molecules that can be used for various diagnostic applications performed under different formats or platforms.
NeXstar Pharmaceuticals Inc., 2860 Wilderness Place, Boulder, CO 80301, USA and Becton Dickinson Immunocytometry Systems, 2350 Qume Drive, San Jose, CA 95131, USA.
K A
KA
Davis
Y
Y
Lin
B
B
Abrams
S D
SD
Jayasena
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D019921:Biotinylation; D015704:CD4 Antigens / Q000235:genetics / Q000302:isolation & purification*; D002462:Cell Membrane / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D015723:Gene Library; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism*; D011743:Pyrimidines; D012313:RNA / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*; D013601:T-Lymphocytes / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D005456:Fluorescent Dyes; D009843:Oligoribonucleotides; D011743:Pyrimidines; D012313:RNA