Proof of Concept

Part 1: Aptamer/Anti-Aptamer Binding

Our fluorescence binding assay showed evidence that our aptamers were binding to progesterone, specifically P5 and P6. 6-FAM-tagged P6 showed the most consistent fluorescence, followed by P5 and P4G03 [3]. P6 and P4G03 showed fluorescent patterns corresponding with increasing aptamer concentration, as shown in Table 1. P5 and P6 were shown to consistently fluoresce, as shown in Table 2 and 3. Due to our abundance of 6-FAM-tagged P5, we proceeded to use P5 to procedurally optimize our heating and washing steps, as shown in Table 4 and 5, and we were able to continue to prove that P5 consistently fluoresced. Consequently, we were also able to see fluorescence with untagged P5 when stained with SYBR Gold, as shown in Table 6 [4]. However, we were unable to replicate conclusive results in a subsequent test that experimented with our entire aptamer library, shown in Table 7. We speculate that due to prolonged freeze-thaw steps over the course of multiple experiments, our progesterone degraded. We determined that overall, the aptamers were able to bind to progesterone consistently, but more testing is required to fully determine whether any of our SYBR Gold-stained untagged aptamers consistently fluoresced.

We also produced evidence that non-target DNA and anti-aptamers do not bind to progesterone, although various other factors shaped our plate results that require further confirmation. We used anti-aptamer fragments as our non-target DNA and placed them in a column of increasing concentrations next to columns containing our aptamer sequences, increasing at the same concentration. Column 2 with P6 containing aptamers fluoresced higher than the column containing non-target DNA, which shared fluorescence intensity with bordering empty wells serving as negative controls, as shown in Table 8. Outliers did occur on some of the wells, specifically 4F, that we suspect could be pipetting error or some other contamination. In Table 9, we combined anti-aptamer and progesterone in the absence of aptamer and found that it appeared to fluoresce lower than aptamer and progesterone. To contextualize these results, we attempted to repeat the experiment (Table 10) but failed to produce anything conclusive for the same reason that we suspected in the experiment prior (Table 9), in that our progesterone degraded.

Part 2: “Seagull”

Using our developed genetic algorithm, NOODL, and the modeling capabilities of Genious Prime [5], we found that the pairing of P4G and P6 was the least likely to interact with each other when compared to other combinations of aptamers separated by a spacer. NOODL utilizes a genetic algorithm that mimics basic cell biology, genetics, and natural selection to create a spacer that is optimized based on user-inputted parameters, such as aptamer flank sequences. It scans these sequences to look for short reverse complementary nucleotides that would allow the spacer and the aptamers to snap together and fold. Genious Prime models spacers concatenated with aptamers and provides a simulation of how they would interact with each other once folding occurs. Using a conjunction of both tools, P4G and P6 consistently provided the best spacers while not folding in a way that compromises the aptamers. All other combinations of aptamers were combined with spacers that had more short nucleotides that allowed them to fold and produced more hairpins that caused cross hybridization between spacers and aptamers. Because of these reasons, we decided to use P6 and P4G for “Seagull” and proceeded to test them experimentally using our fluorescent binding assay.

Our aptamer binding assay showed that “Seagull” was able to bind to progesterone, though further testing is required to confirm its consistency. Our first test (Table 1) “Seagull” stained with SYBR Gold, showed extremely high fluorescence, but ambiguous results in our negative controls required us to reproduce the experiment. On our second test (Table 11) using SYBR Gold staining, “Seagull” fluoresced relatively high compared to other aptamer sequences, but not with the same intensity as our first test. Through these two results, we came up with two potential hypotheses for these confusing results. The individual aptamers are shorter than “Seagull”, and when stained with SYBR Gold, the differences in length between the two were enhanced by fluorescence. Also, because we incubated our plates using a clear cover under light, we suspect that SYBR Gold may have degraded because of its sensitivity to light. This can be corroborated by the intensity differences between the two plates, because our first test was incubated using an opaque silicone cover. Fluorescence indicates that binding does occur, but more testing is required to disprove our other speculations.

To test whether or not Seagull can be properly filtered out of solution, we tested DNA binding affinity to 100% cotton filter paper through direct pipetting and dipping of the filter to capture DNA out of solution, measuring binding capacity through SYBR Safe fluorescence. Samples pipetted directly onto filter paper treated with citric acid showed the most compelling fluorescent results, but not enough testing was done to reject the notion that this was inherent fluorescence of the filter paper (Figure 1). To better model a real-world scenario, we then dipped the cotton filter in the solution in an attempt to capture DNA. Background fluorescence still made it difficult to determine whether the observed fluorescence was due to DNA binding solution or the filter paper itself (Figure 2). While the first experiment showed potential binding of DNA to the cotton material, this filter-binding methodology could not be conclusively determined due to limitations of fluorescence-based testing on this filtration material.

Part 3: Lambda Exonuclease

Using a denaturing dye in an E-Gel and Sanger sequencing results, we conclude that Lambda exonuclease binds and digests at the 5’-phosphorylated end of our testing sequences and may exhibit pausing activity within the “rumble zone” (BBa_25SURMFG). To assess the effect of rumble zones on digestion rate, we analyzed our samples with both E-Gel and traditional gel electrophoresis.

Two sequences were compared: one with a rumble zone, TriCycleV2 (T), and one without, NonStopV2 (N). Digested samples were run under two conditions. The first used an Invitrogen E-Gel Size Select II 2% agarose gel with standard 10X Sample Loading Buffer (Figure 3) [6]. The second condition used a denaturing 2X RNA Loading Dye on both a 2% E-Gel and a traditional 1% agarose gel to denature the DNA and visualize individual strands (Figure 4) [7].

On the standard E-gel, bands corresponding to digested samples migrated marginally lower and were less intense than the template (Figure 3). Notably, T samples displayed higher intensity than N samples digested for the same duration, thus the pause sequences may have hindered the exonuclease activity. Longer digestions, such as four hours, also produced fainter bands relative to the shorter digestions, consistent with progressive exonuclease activity.These findings exhibit that Lambda exonuclease is binding and digesting our 5’-phosphorylated sequences, with some evidence suggesting pausing or hindrance in the presence of rumble zones.

Sanger sequencing, however, did not provide base-pair resolution sufficient enough to approximate where the exonuclease is halting. When visualizing 4 hour digestion results, there were consistently low peak heights and many ambiguous nucleotide calls across many samples which is substantial evidence of digestion. Nevertheless, when visualizing the 1 hour Sanger sequencing sample from the first E-gel, the 5’ ends of all the samples displayed insignificant digestion. When comparing the 1 hour to a 10 minute digestion, the chromatograms look nearly identical. This leads us to believe rather than digesting strands partially and leaving behind our desired seagull constructs, Lambda is primarily digesting full strands across all digestion times.

For samples run on both the traditional 1% agarose gel and the 2% agarose E-Gel with the formamide-containing 2X RNA Gel Loading dye, multiple bands of varying sizes were observed in each lane, indicating that digestion occurred and that partially digested sequences may also be present (Figure 4).The samples tested on the E-gel suggest that band intensity is negatively correlated to digestion time. These results are consistent with the hypothesis that Lambda exonuclease has the potential to exhibit pausing or at least controlled digestion in the presence of our rumble zone. Further testing to refine digestion times and optimize the gel assay must be performed to more precisely characterize the extent and consistency of pausing behavior.

Part 4: Conclusion

Our aptamer fluorescent binding assay was partially successful in proving that our aptamers were able to bind to progesterone, but further testing is still required to confirm consistent results and to address other factors. The existence of fluorescence indicates that there is some binding, but other factors potentially causing fluorescence need to be investigated. Future experimentation could contextualize the inconsistent results with SYBR Gold staining and verify reasons for progesterone degradation. Furthermore, multiple pieces of evidence support that aptamers are binding to progesterone, and there is an indication that non-target aptamers and anti-aptamers are not. Similarly, fluorescence indicates that the “Seagull” construct binds progesterone. We also showed Lambda exonuclease is able to target and digest the 5’-phosphorylated end of our testing sequences, and has the potential to exhibit controlled digestion.

In future directions, we aim to optimize the plasmid with our insert and better characterize Lambda digestion. We continue to improve the insert design to remediate possible off-target annealing that could have occurred during Golden Gate assembly. To verify if our issue lies within the insert or the protocol itself, an insert control will be utilized. To improve Lambda exonuclease control, we would like to test varying quantities of pause sequences to better characterize how they hinder the processivity of Lambda exonuclease. We intend to continue doing gel assays with denaturing agents to find a time where stark contrast between NonStop and TriCycle occurs. In the future, our system can be interchanged with different aptamer inserts to target other mycotoxins, providing a base for further optimization.

Tables and Figures

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Citations

[1] M. Jauset-Rubio et al., “One-Pot SELEX: Identification of Specific Aptamers against Diverse Steroid Targets in One Selection,” ACS Omega, vol. 4, no. 23, pp. 20188–20196, Dec. 2019, doi: 10.1021/acsomega.9b02412.

[2] G. Contreras Jiménez, S. Eissa, A. Ng, H. Alhadrami, M. Zourob, and M. Siaj, “Aptamer-Based Label-Free Impedimetric Biosensor for Detection of Progesterone,” Anal. Chem., vol. 87, no. 2, pp. 1075–1082, Jan. 2015, doi: 10.1021/ac503639s.

[3] “5' 6-FAM (Fluorescein) modification | IDT.” Accessed: Jul. 25, 2025. [Online]. Available: https://www.idtdna.com/site/Catalog/Modifications/Product/1108

[4] “SYBRTM Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO),” Thermofisher.com, 2022. https://www.thermofisher.com/order/catalog/product/S11494

[5] Geneious Prime 2025.0.(https://www.geneious.com)

[6] “E-GelTM SizeSelectTM II Agarose Gels, 2% 10 gels | Buy Online | InvitrogenTM.” Accessed: Aug. 25, 2025. [Online]. Available: https://www.thermofisher.com/order/catalog/product/G661012

[7] “RNA Loading Dye, (2X)” Accessed: Oct. 07, 2025. [Online]. Available: https://www.neb.com/en-us/products/b0363-rna-loading-dye-2x