Keywords

Fluorescent light-up DNA aptamer, G-quadruplex, Label-free biosensing, Thioflavin T

Abstract

Fluorescent light-up aptamers (FLAPs) are promising label-free signal reporters for biosensing, as they enhance the fluorescence of weakly emissive fluorogenic dyes through noncovalent binding. In this study, a DNA aptamer, DAP-1 previously selected to bind a fluorogenic dye dapoxyl sulfonyl ethylenediamine, was repurposed as a modular fluorescent signal transducer utilizing Thioflavin T (ThT), a fluorogen known for its enhanced fluorescence upon binding to β-sheet-rich amyloids and structured nucleic acids such as G-quadruplex DNA and RNA. Systematic truncation combined with photophysical characterization identified DAP-1-47R as the shortest sequence of the original aptamer required for efficient fluorescence activation while preserving nanomolar binding affinity to ThT.

Structural and spectroscopic analyses demonstrated that DAP-1-47R adopts an antiparallel G-quadruplex stabilized by the adjacent duplex domain, which creates a binding environment for ThT that results in about 290-fold fluorescence enhancement and a quantum yield of 0.395. The dye binds to the aptamer in a 1:1 binding stoichiometry and causes structural stabilization with an increase in melting temperature of about 8 °C.

Leveraging its ability to enhance ThT fluorescence, DAP-1-47R was engineered into a split transducer and fused with the caffeine-binding aptamer Caff203 to design a caffeine-sensing fluorescent aptaswitch. In the absence of caffeine, the split DAP-1-47R fragments are mostly misaligned, resulting in background ThT fluorescence. Upon caffeine binding, a target-induced conformational change promotes the reassembly of the ThT-binding core, which results in a concentration-dependent fluorescence increase. This modular design enables signal transduction without covalent labeling, thereby simplifying sensor construction and operation. The resulting biosensor exhibited selective caffeine detection over other members of the xanthine family of alkaloids, with a limit of detection (LOD) of 0.85 µM and a limit of quantification (LOQ) of 2.6 µM.

Overall, this study highlights the potential of DAP-1-47R as a versatile ThT-based signal transducer for label-free biosensing applications.

Completion Date

2026

Semester

Spring

Committee Chair

Yulia Gerasimova

Degree

Master of Science (M.S.)

College

College of Sciences

Department

Department of Chemistry

Format

PDF

Document Type

Thesis

Identifier

DP0053134

Release Date

5-15-2027

Download

Available for download on Saturday, May 15, 2027

Share

COinS
 

Accessibility Statement

This item was created or digitized prior to April 24, 2027, or is a reproduction of legacy media created before that date. It is preserved in its original, unmodified state specifically for research, reference, or historical recordkeeping. In accordance with the ADA Title II Final Rule, the University Libraries provides accessible versions of archival materials upon request. To request an accommodation for this item, please submit an accessibility request form.