High-Throughput Discovery of Aptamers for Sandwich Assays.
Sandwich assays are among the most powerful tools in molecular detection. These assays use "pairs" of affinity reagents so that the detection signal is generated only when both reagents bind simultaneously to different sites on the target molecule, enabling highly sensitive and specific measurements in complex samples. Thus, the capability to efficiently screen affinity reagent pairs at a high throughput is critical. In this work, we describe an experimental strategy for screening "aptamer pairs" at a throughput of 106aptamer pairs per hour-which is many orders of magnitude higher than the current state of the art. The key step in our process is the conversion of solution-phase aptamers into "aptamer particles" such that we can directly measure the simultaneous binding of multiple aptamers to a target protein based on fluorescence signals and sort individual particles harboring aptamer pairs via the fluorescence-activated cell-sorter instrument. As proof of principle, we successfully isolated a high-quality DNA aptamer pair for plasminogen activator inhibitor 1 (PAI-1). Within only two rounds of screening, we discovered DNA aptamer pairs with low-nanomolar sensitivity in dilute serum and excellent specificity with minimal off-target binding even to closely related proteins such as PAI-2.
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
Andrew T
AT
Csordas
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
Anna
A
Jørgensen
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
Jinpeng
J
Wang
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
Emily
E
Gruber
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
Qiang
Q
Gong
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
Elizabeth R
ER
Bagley
Department of Microbiology and Immunology, Stanford University School of Medicine , Stanford, California 94305, United States.
Margaret A
MA
Nakamoto
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
Michael
M
Eisenstein
Institute for Collaborative Biotechnologies, University of California at Santa Barbara , Santa Barbara, California 93106, United States.
H Tom
HT
Soh
D052157:Aptamers, Nucleotide / Q000032:analysis*; D001681:Biological Assay; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry
D052157:Aptamers, Nucleotide; D017395:Plasminogen Activator Inhibitor 1