In vitro selection of structure-switching, self-reporting aptamers.
We describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.
Oh
Seung Soo
SS
Materials Department, University of California, Santa Barbara, CA 93106, USA.
Plakos
Kory
K
Lou
Xinhui
X
Xiao
Yi
Y
Soh
H Tom
HT
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000302:isolation & purification; D021881:DNA, Catalytic / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D015723:Gene Library; D006801:Humans; D008652:Mesoporphyrins; D009690:Nucleic Acid Conformation; D013917:Thrombin / Q000235:genetics
D052157:Aptamers, Nucleotide; D021881:DNA, Catalytic; D005456:Fluorescent Dyes; D008652:Mesoporphyrins; C065420:N-methylmesoporphyrin IX; D013917:Thrombin