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Using our computational ASO design pipeline, we developed optimized ASO constructs and RNA aptamers targeting ALS-related proteins, contributing five new parts to iGEM's Parts Catalog for future therapeutic development.

ASO Design Pipeline

Computational pipeline integrating sequence analysis, GC content optimization, and structural prediction to generate 4 chemically-modified 5-10-5 gapmer ASOs targeting DAZAP1 and FAM98A.

RNA Aptamer Development

In silico designed RNA aptamers targeting TDP-43's intrinsically disordered C-terminal domain, validated through docking simulations and in-vitro binding assays.

Validated Results

ASOs achieved 23-49% transcript reduction in SH-SY5Y cells; TDP-43 aptamer showed nuclear localization and stress granule association, validating computational predictions.

Overview

Using our computational antisense oligonucleotide (ASO) design pipeline, our team developed and cataloged a model ASO construct targeting proteins implicated in stress granule formation, DAZAP1 and FAM98A, which are associated with neurodegenerative diseases such as ALS. This pipeline integrates multiple analytical layers, including sequence tiling, GC content optimization, homopolymer detection, conservation scoring, secondary structure prediction, and mapping specificity to systematically score and rank potential ASO candidates. Top-scoring ASOs were chemically modified into 5-10-5 gapmers using 2′-O-methoxyethyl ribose (2MOEr) and iMe-dC (5-methylcytosine) substitutions to improve nuclease resistance, binding affinity, and physiological stability. Through this process, we developed 4 optimized ASO sequences, which were added to iGEM's Parts Catalog for future teams to reference and expand upon.

Furthermore, we designed RNA aptamers, generated in silico with AptaTrans, to bind the intrinsically disordered C-terminal domain of TDP-43 (a key ALS/FTD protein). We used IUPred2A to pinpoint the longest disordered stretch, then optimized aptamer generation (10 MCTS iterations, seed 1004) to balance diversity and score. The top-scoring aptamers were tested for docking and in-vitro binding assays.

Overall, our models significantly reduce the number of unfiltered ASOs and aptamers requiring experimental validation, allowing us to save time and test the most effective therapeutic solutions in vitro with limited resources. Our design pipeline can also be adapted by other scientists to create different parts to target different disease genes.

Table of Parts

BBa_25JFCXC0

Oligo ASO_A targeting DAZAP-1

BBa_2540ICKT

Oligo ASO_B targeting DAZAP-1

Function

The antisense oligonucleotide is designed to bind to complementary mRNA strands that correspond to the targeted region of ALS-related protein DAZAP1 (DAZ), and reduce protein levels. This forms RNA:DNA duplexes that are then cleaved by RNase H1, thereby breaking the target mRNA strand. Since the mRNA strand has been cleaved, the protein is no longer translated, thus lowering its concentration in the cell. It was designed as a 5-10-5 gapmer with chemical modifications to improve stability and binding efficiency in cells.

Result

Following transfection, ASO_A and ASO_B produced a 23% reduction in DAZAP-1 transcript levels in SH-SY5Y cells compared to control, confirming that they successfully bound and degraded some of their target mRNA. The magnitude of degradation suggests that while the ASO mechanism is operating as intended, factors such as RNA accessibility or intracellular uptake may be limiting its full efficiency and efficacy. These findings validate the computational design pipeline's predictive accuracy and show that ASO_A and ASO_B function as active, partially effective gene silencers, highlighting their potential as gene therapeutics.

BBa_253TN03M

Oligo ASO_C targeting FAM98A

BBa_258KP83X

Oligo ASO_D targeting FAM98A

Function

The antisense oligonucleotide is designed to bind to complementary mRNA strands that correspond to the targeted ALS-related protein FAM98A (FAM), and reduce protein levels. This forms RNA:DNA duplexes that are then cleaved by RNase H1, thereby breaking the target mRNA strand. Since the mRNA strand has been cleaved, the protein is no longer translated, thus lowering its concentration in the cell. It was designed as a 5-10-5 gapmer with chemical modifications to improve stability and binding efficiency in cells.

Result

Following transfection, ASO_C produced a 38% reduction in FAM98A transcript levels and ASO_D produced a 49% reduction in FAM98A transcript levels in SH-SY5Y cells compared to control, confirming that they successfully bound and degraded some of their target mRNA. The magnitude of degradation suggests that while the ASO mechanism is operating as intended, factors such as RNA accessibility or intracellular uptake may be limiting its full efficiency and efficacy. These findings validate the computational design pipeline's predictive accuracy and show that ASO_C and ASO_D function as active, partially effective gene silencers, highlighting their potential as gene therapeutics.

BBa_25C3TVF6

RNA Aptamer targeting TDP-43 C-terminal Domain

Function

This RNA aptamer was computationally designed to bind to the C-terminal domain (CTD) of the TDP-43 protein, a key factor implicated in pathological aggregation in ALS. The aptamer's target region was mapped to residues 375-414 of the TDP-43 protein, where the aptamer is predicted to bind and stabilize the residues; this interferes with pathological aggregation or phase separation of TDP-43 and thus mitigates the formation of toxic aggregates linked to ALS progression.

Result

Fluorescence imaging showed that the TDP-43 aptamer localized to the nucleus of stressed SH-SY5Y cells, with partial overlap with the stress granule marker G3BP1. This suggests that the aptamer remains structurally stable and active in vivo, supporting in-silico predictions that the aptamer binds to the disordered C-terminal domain of TDP-43, which aligns with docking simulations that predicted strong affinity and specificity. While direct molecular binding to TDP-43 was not confirmed, the experiment provides proof-of-concept that the computationally designed aptamer can target and engage stress-related structures, validating that computationally designed RNA aptamers can serve as tools to modulate or monitor TDP-43 associated pathways in neurodegenerative disease models.

References

  1. Suk, Terry R., and Maxime W. C. Rousseaux. "The Role of TDP-43 Mislocalization in Amyotrophic Lateral Sclerosis." Molecular Neurodegeneration, vol. 15, no. 45, 2020. Springer Nature, doi:10.1186/s13024-020-00397-1
  2. Dewey, Colleen M., et al. "TDP-43 Aggregation in Neurodegeneration: Are Stress Granules the Key?" Brain Research, vol. 1462, 2012, pp. 16-25. doi:10.1016/j.brainres.2012.02.032
  3. An, Haiyan, et al. "Compositional Analysis of ALS-Linked Stress Granule-Like Structures Reveals Factors and Cellular Pathways Dysregulated by Mutant FUS under Stress." bioRxiv, 2 Mar. 2021. doi:10.1101/2021.03.02.433611
  4. Ozeki, Kanako, et al. "FAM98A Is Localized to Stress Granules and Associates with Multiple Stress Granule-Localized Proteins." Molecular and Cellular Biochemistry, vol. 451, no. 1-2, 2019, pp. 107-115. https://doi.org/10.1007/s11010-018-3397-6
  5. Yeo Lab. GitHub Organization Page. GitHub, github.com/yeolab Accessed 7 Oct. 2025.
  6. "UCSC Genome Browser on Human (GRCh38/hg38), position chr19:1,435,544-1,435,764." UCSC Genome Browser, University of California, Santa Cruz, genome.ucsc.edu, Accessed 7 Oct. 2025.