Enhanced fluorescence via DNA aptamer optimization

This invention improves the fluorescence of DNA-based aptamers by substituting the original fluorophore with TO1-biotin and identifying a critical mutation that further enhances fluorescence.

Background

Fluorogenic DNA aptamers (FAPs) are a crucial tool in molecular biology, particularly for applications like live-cell imaging and highly specific molecular detection. These aptamers, which are essentially short, single-stranded DNA sequences, exhibit a significant increase in fluorescence upon binding to specific target molecules. This unique property makes them ideal for visualizing and tracking molecules within living cells, as well as developing highly sensitive diagnostic tools.

However, optimizing FAPs for enhanced fluorescence and binding affinity poses a significant challenge. Traditional methods, such as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), are effective for initial identification but often fall short in fine-tuning the aptamer’s properties. Structure-guided optimization via X-ray crystallography provides valuable insights, but it requires specialized equipment and expertise, limiting its accessibility to many research groups. Furthermore, current approaches often struggle to systematically analyze many aptamer variants, hindering the identification of subtle but crucial modifications that can significantly impact fluorescence and binding.

Technology description

This technology describes a novel method for optimizing fluorogenic DNA aptamers (FAPs) using a high-throughput screening process on Illumina next-generation sequencing (NGS) chips. The process involves substituting the original fluorophore with TO1-biotin, leading to a significant increase in fluorescence enhancement. By screening thousands of aptamer variants, including mutations, insertions, and deletions, the researchers identified modifications that affect fluorescence. Notably, the C14T mutation was found to further increase fluorescence by 50% due to stronger binding affinity and higher quantum yield.

This technology differentiates itself by providing a robust method for optimizing DNA-based FAPs without requiring prior knowledge of their crystal structure. This is a significant advantage as it eliminates the need for time-consuming and expensive structure determination techniques. Molecular dynamic simulations revealed that the TO1-biotin fluorophore interacts with the Lettuce aptamer primarily through π-π stacking interactions, which are crucial for the observed fluorescence enhancement.

This platform offers valuable insights into aptamer-fluorophore interactions and has the potential to facilitate the development of improved FAPs for various applications, including in vivo sensing and in vitro diagnostics.

Benefits

Optimizes fluorogenic DNA aptamers (FAPs) for enhanced fluorescence, useful in diagnostics and sensing
Utilizes a high-throughput screening method on Illumina next-generation sequencing (NGS) chips, enabling rapid analysis of thousands of aptamer variants
Identifies specific mutations, like C14T, that significantly increase fluorescence intensity and binding affinity
Provides insights into aptamer-fluorophore interactions, particularly the role of π-π stacking interactions in fluorescence enhancement
Offers a robust method for optimizing DNA-based FAPs without requiring prior knowledge of crystal structures, making it widely accessible for research and development