Week 2 HW: 🧬 DNA Design for a Cell-Free Insulin Aptamer

Designing the DNA for Rolling-Circle Manufacturing of an Insulin-Binding Aptamer (IGA3)

Core idea: My Homework #1 product is an insulin-binding DNA aptamer, made in a cell-free system and used in a disposable electrochemical cartridge that screens insulin after refrigeration failure. In this homework, I design the DNA template that would produce it.

Figure 1. Cartoon representation of human insulin (PDB 1TRZ), the target of the aptamer. Image: “Insulina.png” by Diego Mariano, Wikimedia Commons, licensed under CC BY-SA 4.0.

🎯 Part 1: Define the production target

Molecule: IGA3, a single-stranded DNA aptamer that binds human insulin. It is the recognition element of the sensor: it binds insulin on the gold electrode, and the binding event is read out as an electrochemical signal.

Sequence and source: IGA3 was reported by Yoshida et al. (2009), who selected a family of insulin-binding DNA aptamers by in vitro selection [1]. Its sequence is:

5′-GGTGGTGGGGGGGGTTGGTAGGGTGTCTTC-3′   (30 nt)

This sequence can’t be retrieved from a database like UniPort or NCBI gene as it’s a selected molecule rather than a gene, its source is the literature. The sequence is G-rich, which allows it to form a G-quadruplex-like strucutre that’s the reason why it can recognize insulin, for this reason, I’ll be keeping this sequence unchanged and designing everything around it. IGA3-type aptamers have been used in electrochemical insulin sensors. [2, 3].

Chassis: cell-free rolling-circle amplification (RCA) with phi29 DNA polymerase. Since the product is DNA, it requires no transcription or translation, which is why I’ve decided against using a living host.

I chose RCA because it’s isothermal and can continuously amplify a circular DNA template without the repeated heating and cooling cycles required for PCR. phi29 DNA polymerase is well suited to this because it has strong strand-displacement activity and high processivity, allowing it to continuously copy the circular template and generate long DNA products containing tandem repeats of the template sequence [7]. Which is useful for this project because the starting template can contain the IGA3 sequence, allowing RCA to generate many copies of the aptamer from a small amount of circular template. RCA has also been used for high-level amplification of circular DNA templates with phi29 DNA polymerase [7].

Where the product goes: The route is:

Circular template → phi29 RCA → processing into aptamer units
      → purification + QC → attachment to gold electrode → cartridge

The purified aptamer would be immobilized on the electrode of a disposable cartridge, as in published insulin aptasensors [2, 3].

🧩 Part 2: Design the DNA parts

Most of the standard expression parts do not apply as no protein synthesis is required, so I kept only the required parts of a rolling-circle reaction.

Parts used

I am avoiding using a circularization sequence since I don’t know how additional DNA parts would affect the folding of the aptamer into its required structure. The template would be arranged as a single circle containing the parts in this order:

[ primer-binding ] – [ cleavage element ] – [ IGA3 (reverse complement) ] – [ cleavage element ] → closed circle

🔧 Part 5: From design to build

How I would build it. As the circular template seems to be short, I would order it as a single synthetic oligonucleotide and circularize it enzymatically.

Where the other methods fit:

• Microchip and parallel synthesis become useful if I need to test many variants (for example, many different flanking sequences). Tian et al. made gene-length DNA from oligonucleotides made on programmable microchips [4].Kosuri et al. amplified oligonucleotide pools from high-fidelity microchips selectively, then assembled and error-corrected them, which lowers cost per variant [5].

What synthesis constrains. Chemical synthesis is error-prone, and errors accumulate with length. Carr et al. showed that protein-mediated error correction can substantially improve the accuracy of synthetic DNA [6]. However, for this short template purification and sequence verification seem more reasonable than error correction. Important notes concerning this sequence include:

• IGA3 contains a long run of guanines (eight in a row). G-rich sequences can be harder to synthesize and can fold on themselves, so I would check the purity and identity of the synthesized oligonucleotide (for example by mass spectrometry or sequencing).
• As every turn of the circle copies the same template, a mistake in the circle would be amplified into every unit of the RCA product, so I would need to verify the template and sequence the product to make sure there are no mistakes.

⚠️ Part 6: What don’t I know yet?

1. Will RCA-derived IGA3 stay functional?

I do not know whether aptamer made by RCA and then processed binds insulin as well as chemically synthesized IGA3; additionally, nucleotides left at the ends could affect folding. I would compare the two directly: check sequence and size by gel electrophoresis or sequencing, measure insulin binding in a binding assay, and compare the response on the electrode, referencing this to chemically synthesized IGA3.

2. Which helper sequences preserve IGA3 folding?

The primer-binding, junction and cleavage sequences sit next to a G-rich aptamer, so they might pair with it and interfere with its structure. To test this, I would keep IGA3 unchanged and test several designs that differ only in the flanking sequences, first by computational folding prediction and then by insulin-binding assays, in this situation, the parallel synthesis of variants would be useful.

3. How should the RCA concatemer be processed?

To cut the long product into single aptamer units, I would consider enzymatic cleavage at an engineered site or a self-cleaving element, I would need to test both methods, measuring recovery, purity, sequence integrity (are the cut ends correct?), insulin binding and finally sensor performance.

📚 References

1. Yoshida, W., et al. (2009). Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing. Biosensors and Bioelectronics, 24, 1116–1120. https://doi.org/10.1016/j.bios.2008.06.016
2. Wu, Y., Midinov, B., & White, R. J. (2019). Electrochemical Aptamer-Based Sensor for Real-Time Monitoring of Insulin. ACS Sensors, 4, 498–503. https://doi.org/10.1021/acssensors.8b01573
3. Zhou, L., et al. (2024). Ratiometric electrochemical aptasensor with strand displacement for insulin detection in blood samples. Analytica Chimica Acta, 1317, 342823. https://doi.org/10.1016/j.aca.2024.342823
4. Tian, J., et al. (2004). Accurate multiplex gene synthesis from programmable DNA microchips. Nature, 432, 1050–1054. https://doi.org/10.1038/nature03151
5. Kosuri, S., et al. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology, 28, 1295–1299. https://doi.org/10.1038/nbt.1716
6. Carr, P. A., et al. (2004). Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Research, 32, e162. https://doi.org/10.1093/nar/gnh160
7. Dean, F. B., Nelson, J. R., Giesler, T. L., & Lasken, R. S. (2001). Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research, 11, 1095–1099. https://doi.org/10.1101/gr.180501
8. Blondal, T., et al. (2005). Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage. The CircLigase family is used for splint-free circularization of single-stranded nucleic acids. For the practical substrate requirements, consult the CircLigase II protocol