Keywords

fluorescent light-up aptamers; crystal violet; DAP-10-42; quantum yield; aptamer-ligand interactions; dissociation constant

Abstract

Crystal violet (CV) is an environment-sensitive dye whose fluorescence is strongly influenced by microenvironment polarity and viscosity, increasing markedly when rotational and vibrational motions are restricted. While the fluorescence enhancement of CV by fluorescent light-up aptamers (FLAPs) has been noted in literature, the specific binding interactions between CV and aptamers in the DAP-10 family have remained under-investigated.  The objective of this work is to elucidate the binding site interactions between CV and the DAP-10-42 FLAP. It is hypothesized that positions 34 and 35 of the aptamer’s sequence reside in a proposed quadruplex-duplex junction that form the dye-binding pocket of DAP-10 variants. This study aims to determine their relevance to the structural integrity of the binding pocket by modulating these positions via deletion or substitution with a hexaethylene glycol linker.  Quantum yield of DAP-10-42 and CV was compared to that of free CV, revealing an approximately 118-fold increase. DAP-10-42 also has been observed to induce bathochromic and hyperchromic shifts in CV absorbance, suggesting stabilization of planar structure of CV by its dye-binding pocket. Influence of structural changes on binding affinity of  CV to several DAP-10 variants with modifications next to the proposed dye-binding pocket was investigated.

Kd values for the DAP-10 variants and CV suggest that increased flexibility at the proposed CV-binding site decreases affinity of the aptamer to CV, while narrowing of the proposed dye-binding pocket increases affinity. The sensitivity of the Kd to structural changes in the region further corroborates that this proposed quadruplex-duplex junction likely comprises the dye-binding pocket and provides insight into factors affecting aptamer-dye interactions, providing a basis for future aptamer-dye pair optimization, especially for multiplex biosensing applications.

Thesis Completion Year

2026

Thesis Completion Semester

Spring

Thesis Chair

Gerasimova, Yulia

College

College of Sciences

Department

Chemistry

Thesis Discipline

Biochemistry

Language

English

Access Status

Open Access

Length of Campus Access

None

Campus Location

Orlando (Main) Campus

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Included in

Biochemistry Commons

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