Multicomponent Hybridization Probe with a Label-Free Reporter Based on a DNA Light-Up Aptamer

Author

AnnaMarie J. Knowles, University of Central FloridaFollow

Keywords

Multicomponent Probe System; Split Light-Up Aptameric Sensor; DAP-10-42 Aptamer; Label-Free Fluorescence Detection; Sequence-Selective Hybridization Probe; Fluorescent Light-Up Aptamer

Abstract

Hybridization probes are valuable tools for detecting nucleic acids and have applications in diagnostics and research. However, traditional monolithic probes like molecular beacons have some drawbacks, such as structural interference and difficulty working with folded RNA targets. To help address these problems, this project focuses on developing Split Light-Up Aptameric Sensors (SLAS), which is based on splitting the fluorogenic aptamer DAP-10-42 into two parts: SLAS-U (universal) and SLAS-S (selective). These two strands only combine and produce a fluorescence signal when a fully matching target is present.

This project aimed to determine the most effective site to split the DAP-10-42 aptamer without losing its ability to bind dye and activate fluorescence. To compare their performance, six different split sites were tested across the aptamer’s hairpin loop. Two of these—27U/28S and 29U/30S—produced the strongest fluorescence signals and highest sequence selectivity. Differentiation factor (DF) analysis confirmed that these probes could reliably distinguish between a perfect match and a single-base mismatch. Additionally, both probes achieved detection limits in the nanomolar range.

The optimized SLAS design was applied to detect a single-nucleotide difference in the NANOGP8 pseudogene linked to glioblastoma. Since this system uses a label-free dye and works with simple equipment like UV lights or portable fluorometers, it could be helpful for low-cost or point-of-care testing. Future work will focus on testing SLAS in more complex biological samples and real-world settings.

Thesis Completion Year

2025

Thesis Completion Semester

Spring

Thesis Chair

Gerasimova, Yulia

College

College of Sciences

Department

Department of Chemistry

Thesis Discipline

Chemistry

Language

English

Access Status

Campus Access

Length of Campus Access

1 year

Campus Location

Orlando (Main) Campus

STARS Citation

Knowles, AnnaMarie J., "Multicomponent Hybridization Probe with a Label-Free Reporter Based on a DNA Light-Up Aptamer" (2025). Honors Undergraduate Theses. 274.
https://stars.library.ucf.edu/hut2024/274