Session: 17-01-01: Research Posters

Paper Number: 149735

149735 - Rapid Screening of New Dna Fluorogenic Aptamers With the Selex-Ngs Platform 

Fluorogenic aptamers, or FAPs, are gaining significant importance in the fields of cellular sensing and pathogen diagnostics. However, among all the current FAPs, R-FAPs—specially designed RNA structures that can bind and light up a specific chromophore—have been much more extensively reported over the past decade. As counterparts to R-FAPs, D-FAPs composed of DNA are distinguished by their inherently more chemically stable structure. This structural stability not only enables the production of mass quantities with higher purity at reduced costs but also positions D-FAPs as promising alternatives in various applications. However, despite these advantages, current D-FAPs often fall short in comparison to R-FAPs primarily due to their lower brightness levels. This disparity underscores the ongoing challenge in optimizing D-FAPs to achieve comparable or superior luminosity while leveraging their cost-effectiveness and stability advantages.

 

Here, we have developed an innovative platform, the Systematic Evolution of Ligands by Exponential Enrichment-Next-Generation Sequencing (SELEX-NGS) combinatorial screening platform, aimed at the development and optimization of a new DNA fluorogenic aptamer that surpasses existing ones in performance. Our approach focused on leveraging TO1-biotin, a fluorophore known for its robust fluorescence signal upon binding to the widely used R-FAP Mango, as the target ligand for the aptamer.

 

The SELEX process was initiated using magnetic beads, allowing for the screening of a vast DNA pool ranging from 10^12 to 10^14 sequences. Through multiple iterative rounds of SELEX and counter SELEX, we systematically enhanced the specificity of candidate aptamers towards TO1-biotin. Each round involved binding the DNA library to TO1-biotin immobilized on magnetic beads, followed by stringent washing to remove non-specific binders. The bound sequences were then eluted, amplified via PCR, and subjected to subsequent rounds of selection to enrich for aptamers with improved binding affinity and specificity.

 

After that, an artificial library will be developed based on the selected sequences. We then utilized our previously devised D-FAP/CHAMP (DNA-based fluorogenic aptamers on chip-hybridized association-mapping platform) workflow to study the complex’s photophysical properties in a massively parallel fashion. Our NGS platform is adapted to characterize over >40,000 DNA materials in a single run by monitoring distinct colored fluorescence signals corresponding to each specific protein bound. This innovative approach effectively eliminates the bottlenecks associated with traditional SELEX methods.

 

Presently, we have demonstrated the feasibility of the screening platform with published FAPs and have seen promising preliminary results from the ongoing beads-SELEX. The new optimized D-FAP can be used to develop stable and low-cost biosensors for versatile in-vitro purposes, including biomedical or environmental sensing and monitoring.

Presenting Author: Kyle Le University of Texas at Austin

Presenting Author Biography: My name is Kyle, and I am an undergraduate research assistant at the University of Texas at Austin. I am passionate about exploring innovative methods in biomedical research, particularly in the field of small molecule detection. Currently, I am part of the Yeh lab, where I contribute to the development of novel aptamers using SELEX (Systematic Evolution of Ligands by Exponential enrichment). SELEX allows us to identify and optimize ligands that bind specifically to small molecules, advancing our understanding and applications in biochemistry. I am excited to be at the forefront of research that could have significant implications for healthcare and beyond.

Authors:

Kyle Le University of Texas at Austin
Yujie He Univeristy of Texas at Austin
Zhenglin Yang University of Texas at Austin
Yu-An Kuo University of Texas at Austin
Yuting Wu University of Texas at Austin
Yi Lu University of Texas at Austin
Tim Yeh University of Texas at Austin