Structural considerations for a DNA light-up aptamer

Harun Kapidzic, Ryan Connelly, Nicholas Raddi, Yulia Gerasimova

University of Central Florida.

Fluorescent light-up aptamers (FLAPs) are nucleic acid strands that bind fluorogenic dyes to enhance they fluorescence manyfold. RNA-based FLAPs have been suggested for intracellular monitoring of RNA and small molecules and for studying RNA-RNA interactions.¹ DNA FLAPs have an advantage of a greater chemical stability than their RNA counterparts as reporters in bioassay outside of the cell. One of the most efficient DNA FLAPS is dapoxyl-binding aptamer DAP-10-42.² The fluorescence enhancement observed for dapoxyl sulfonyl dyes in the presence of DAP-10-42 exceeds 700-fold.^2,3 In addition, it exhibits promiscuity in fluorogen binding, with hundred-fold fluorescent enhancement recently demonstrated for other fluorogenic dyes.³ These properties make DAP-10-42 a promising scaffold for nucleic acid-based biosensors. In this work, we aimed to shed light on structural characteristics of the aptamer and its complexes with dyes to assist in its use for bioanalytical applications.

The primary structure of the aptamer is guanine-rich, which suggest a guanine quadruplex (G4) motif as part of the aptamer. Previously, based on circular dichroism (CD) spectroscopy, we proposed a mix G4-duplex architecture of the aptamer. Moreover, we observed as conformational change upon interaction of DAP-10-based construct with either dapoxyl sulfonyl fluoride or a molecular rotor auramine O. Here, we study dependence of light-up properties of DAP-10-42 and its affinity to auramine O on monovalent ion concentration. Our data provide additional evidence for G4 motif requirement for DAP-10-42 activity.

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