Harnessing a 4-Formyl-Aniline Handle to Tune the Stability of a DNA Aptamer–Protein Complex via Fluorescent Surrogates
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".