Harnessing a 4-Formyl-Aniline Handle to Tune the Stability of a DNA Aptamer-Protein Complex via Fluorescent Surrogates.
Ryan E JohnsonMakay T MurrayLucas J BycraftPeter MylerStacey D WetmoreRichard A MandervillePublished in: Bioconjugate chemistry (2023)
Interactions between DNA aptamers and protein targets hold promise for the development of pharmaceuticals and diagnostics. As such, the utilization of fluorescent nucleobase surrogates in studying aptamer-protein interactions is a powerful tool due to their ability to provide site-specific information through turn-on fluorescence. Unfortunately, previously described turn-on probes serving as nucleobase replacements have only been strongly disruptive to the affinity of aptamer-protein interactions. Herein, we present a modified TBA15 aptamer for thrombin containing a fluorescent surrogate that provides site-specific turn-on emission with low nanomolar affinity. The modification, referred to as AnBtz, was substituted at position T3 and provided strong turn-on emission ( I rel ≈ 4) and brightness (ε·Φ > 20 000 cm -1 M -1 ) with an apparent dissociation constant ( K d ) of 15 nM to afford a limit of detection (LOD) of 10 nM for thrombin in 20% human serum. The probe was selected through a modular "on-strand" synthesis process that utilized a 4-formyl-aniline (4FA) handle. Using this platform, we were able to enhance the affinity of the final aptamer conjugate by ∼30-fold in comparison with the initial conjugate design. Molecular dynamics simulations provide insight into the structural basis for this phenomenon and highlight the importance of targeting hydrophobic protein binding sites with fluorescent nucleobase surrogates to create new contacts with protein targets.
Keyphrases
- living cells
- sensitive detection
- label free
- quantum dots
- fluorescent probe
- gold nanoparticles
- molecular dynamics simulations
- protein protein
- single molecule
- binding protein
- amino acid
- small molecule
- photodynamic therapy
- magnetic resonance imaging
- magnetic nanoparticles
- cancer therapy
- machine learning
- mass spectrometry
- drug delivery
- high throughput
- circulating tumor cells
- ionic liquid
- social media
- cell free
- deep learning
- single cell
- real time pcr
- artificial intelligence
- diffusion weighted imaging