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Research Article | | Peer-Reviewed

Optimization of Aptamer AS1411: An Effort to Increase Binding Efficiency to Nucleolin, an Overexpressed Protein on Cancer Cells

Henry Mayerfield*,  Jackson Cunningham
Published in Cancer Research Journal (Volume 13, Issue 3)
Received: 29 July 2025     Accepted: 8 August 2025     Published: 21 August 2025
Views:       Downloads:
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Abstract

This project aimed to manipulate DNA (deoxyribonucleic acid) aptamer AS1411, a short single-stranded oligonucleotide currently being developed to improve chemotherapy’s target cell specificity. As this aptamer binds explicitly to nucleolin, an overexpressed protein on the surface of cancer cells, chemotherapy damage to surrounding tissue may be lessened. This study modified the AS1411 DNA aptamer, which was named AS1411-N12, by adding 12 nucleotides to the 3’ and 5’ ends, forming a “flap” structure. The edition of said flap is attributed to theory that the increased mass will allow for tighter binding. This modification was hypothesized to further improve the DNA aptamer’s binding efficiency to the nucleolin protein expressed on cancer cells. Binding reactions occurred between DNA aptamers (AS1411 and AS1411-N12) and nucleolin samples. The resulting solutions were processed using micro-centrifugal filters, which separated small unbound single-stranded DNA aptamers from bigger unbound proteins and the DNA-Nucleolin complexes. Measured absorbance of the unbound filtered DNA aptamers were analyzed to compare binding efficiencies of the modified aptamer vs. the control. The average absorbance through 3 trials of the control AS1411 DNA aptamer was 1.907 at 260 nm, while the average absorbance through 3 trials was 1.364 at 260 nm. Through Beer's Law, the unbound DNA control concentration was 146.6 µM while the modified DNA aptamers was 54.17 µM. This modification was highly effective as it yielded a 63% change in absorbance showing a drastic decrease in the amount of DNA aptamer left in solution. The modified DNA aptamer was significantly more effective in binding to its target protein. When attached to chemotherapy, AS1411-N12 will have a higher affinity to Nucleolin, improving cancer treatment.

Published in Cancer Research Journal (Volume 13, Issue 3)
DOI 10.11648/j.crj.20251303.15
Page(s) 147-151
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2025. Published by Science Publishing Group
Previous article
Keywords

Aptamer, Nuclear Proteins, Nucleolin, AS1411, AS1411-N12, Spectrophotometry, DNA

1. Introduction
Cancer treatment faces a critical challenge: delivering therapies with maximum precision and efficacy while minimizing side effects. For example, chemotherapy is a highly nonselective process as a result of a failure of DNA aptamers or other drug delivery systems to bind tightly to the proteins expressed on cancer, causing the chemotherapy to go “rogue” in the bloodstream, leading to intense side effects. These include side effects such as pain, hair loss, fatigue, kidney problems, weight changes, and more
[4]
. This project aims to manipulate aptamer AS1411, a short single-stranded oligonucleotide that is a delivery method for chemotherapy, by addition of certain nucleotides, in order to make a “flap”, improving binding efficiency to nucleolin, an overexpressed protein on the surface of cancerous tumor cells
[1, 11, 12]
, and hopefully minimize the amount of rogue chemotherapy in the bloodstream.
Aptamer: Aptamers are oligomers of artificial ssDNA (single-stranded DNA), RNA (ribonucleic acid), XNA (xenonucleic acid) or peptide that bind to a specific target molecule, or family of target molecules.
2. Materials
This project is centered around the binding reaction of aptamer AS1411 (IDT)
[10]
and Nucleolin (FC Tag: NUL-H5253). Two sets of aptamers are used: the stock AS1411 and a modified version, AS1411-N12
[2]
. The protein is resuspended with deionized water 250 µL. A Microcentrifuge administers the binding reaction. Amicon filters (30 kDa (kilodalton)) separate the bound complex and the unbound proteins from the unbound aptamers
[5]
. A NanoDrop (SKU: 28127 Thermo Fisher Scientific) is used to read and analyze the unbound DNA to determine concentration. Clean, dry, lint-free lab wipes handle the Nanodrop sensor.
3. Methodology
Stage 1: Preparation
The first step is to obtain the AS1411 aptamer (IDT)
[10]
, an ssDNA oligonucleotide, and the Nucleolin protein (Acrobio). This is the control sample, which is an unmodified stock version. The Aptamer will arrive in a 100 μM solution of IDTE. Next, the protein nucleolin, which is overexpressed on cancerous tumors, is obtained, and the aptamers will bind to it
[6]
. The nucleolin arrives lab lyophilized and reconstituted with deionized water 250 µL and then further diluted with 1.25 mL PBS. (phosphate buffered saline).
Stage 2: Modification of AS1411
AS1411 is an aptamer with a unique g-quadruplex structure, which provides an opportunity to improve the surface area-to-volume ratio. 12 Nucleotides are placed at the 3’ and 5’ ends to create a “flap.” This flap aims to increase binding efficiency by adding additional contact points to the biological receptor (Nucleolin). The enhanced aptamer, named AS1411-N12, reflects the original AS1411 with the addition of 12 nucleotides on each end
[2]
. AS1411-N12 is ordered from IDT
[10]
(Integrated DNA Technologies), matching the specifications of the control.
Stage 3: Binding Reaction
Next, 0.25 mL of the protein and DNA are combined and directly incubated at 4°C (degrees celsius) for 1 hour. Two different binding reactions are conducted: a control (AS1411 and Nucleolin) and an experimental (AS1411-N12 and Nucleolin). Both reactions follow the same procedure. Three tests per control and modified DNA aptamer will be carried out.
Stage 4: Filtration
Amicon Ultra centrifugal filters collect filtrate via the microcentrifuge for both the control and experimental with equal volumes. These filters consist of two tubes, one collecting filtrate while the other recovers the concentrated sample. This is used to filter the solution, separating, based on physical size, the small, unbound aptamer from the aptamer-protein-bound complex
[5]
. This was centrifuged at 13,500 RPM (revolutions per minute) for 20 min at 22°C.
Stage 5: NanoDrop/Data
DNA is extracted and the NanoDrop is set to the ssDNA tab-measuring the absorbance of unbound single-stranded DNA (aptamer). 5 μL (microliter) of the blanking solution, derived from the resuspension solute, is placed on the sensor-sanitized to prevent contamination. Trials use 5 μL of the unbound DNA concentrated sample. The NanoDrop provides graphs based on the Ultraviolet-visible spectrum. Using Beer's Law, the concentration of unbound DNA is calculated to assess the aptamer’s binding efficiency
[7]
. The concentration of DNA found in the sample indirectly relates to the binding capability of AS1411. The lower the concentration of DNA found infers that a larger amount of DNA was bound to the protein.
Figure 1. Methodology of collecting unbound DNA Aptamer.
4. Results
The average absorbance through 3 trials of the control AS1411 DNA aptamer was 1.907 at 260 nm, while the average absorbance through 3 trials of AS1411-N12 was 1.364 at 260 nm. Through Beer's Law, the unbound DNA control concentration was 146.6 µM while the modified DNA aptamers was 54.17 µM
[7]
. This modification was highly effective as it yielded a 63% change in absorbance, showing a drastic decrease in the amount of DNA aptamer left in solution.
Figure 2. Concentration of unbound DNA Aptamer.
Figure 3. Molecular weight comparison chart.
Figure 4. Measured absorbance and concentration of control and experimental groups.
5. Analysis
The results show that the AS1411-N12 had a higher binding affinity, which would lead to fewer total unbound strands. This modification proved to exponentially increase the binding efficiency to the protein. Clearly, since significantly less DNA aptamer is left in the filtered solution, this means that a significantly greater amount has remained bound to the protein, and not filtered through. These non-deviating results very conclusively and clearly demonstrate how the new, modified, DNA aptamer is significantly more effective and efficient in binding to the nucleolin protein found on cancer cells, improving treatment for patients indirectly by 63%(the percent change in concentration between the original and modified DNA aptamer)
[1, 2]
.
6. Discussion
The results clearly show how adding the “flap” enhanced binding efficiency. The increased surface area to volume ratio has created additional points of contact for the aptamer to latch onto the protein
[2, 15]
. Just looking at absorbance, the average of the control was 1.907 (10 mm) at 260 nm (nanometer) while the modified was 1.364 (10 mm) at 260 nm. This modification reduced the amount of unbound DNA strands by 63%. Through this lowered concentration due to the higher binding affinity of AS1411-N12, treatment in the future can be less physically demanding, reducing the volume of side effects. Through the six tests performed, 3 control and 3 experimental, the data stayed consistent. The control had a standard deviation of 5.98, while the experimental had a standard deviation of 8.91. This shows how the control data stayed extremely close to the average concentration which was 146.6 µM. Similarly the experimental data stayed consistent with the average concentration of 54.17 µM. This consistency exemplifies how the modified flap, consisting of 12 nucleotides on each end, was not a fluke result and improved the concentration of bound complexes. This concentration was lower than the control by 92.43 µM. With these consistent results, further testing should be conducted in different environments to monitor performance
[8]
. Clearly, since significantly less DNA aptamer is left in the filtered solution, this means that a significantly greater amount has remained bound to the protein, and not filtered through. These non-deviating results very conclusively and clearly demonstrate how the new, modified, DNA aptamer is significantly more effective and efficient in binding to the nucleolin protein found on cancer cells
[13, 14]
, improving treatment for patients indirectly by 63%(the percent change in concentration).
7. Possible Errors
DNA and protein have specific ranges including pH, temperature, etc., which will alter their structure, disabling the intended function
[9]
. When Nucleolin is exposed to uninhabitable conditions, hydrogen bonds will break, leading to a changed quaternary structure- disabling the function. If the AS1411 DNA aptamer is exposed to uninhabitable conditions, the phosphodiester bonds connecting the pentose sugar and the phosphate group from one nucleotide to another would break. No hydrogen bonds would break because it is a single strand of DNA. In the PCR (polymerase chain reaction) process at IDT
[10]
, they could have introduced a mutated version and replicated that, which would have heavily skewed results. There could be errors in the nanodrop reading, including cross-contamination, a sample size that was too small, particulate matter in the sample, unequal volumes between tests, and more. Additionally, when the chemo drug is attached there may be errors in delivery as it is a much more complex process with the flap and binding to the receptor.
8. Uncontrollable Events
Our results were affected by many uncontrolled events, including shipping, our DNA passing quality control, a broken NanoDrop, and more, which delayed our project. These events created a time crunch but did not affect our project’s outcome.
9. Conclusion
Our hypothesis was proven correct based on the results shown above. The average concentration left in solution of the modified DNA aptamer was 54.17 µM while the average concentration left in solution of the original DNA aptamer was 146.6 µM. This means that more of the modified DNA aptamer has bound to the protein and not been filtered through into the collected solution
[1, 3]
.
10. Practical Applications
This project is a precursor to curing cancer with high specificity and efficiency without killing as many healthy cells as possible. Aptamer AS1411- N12 is responsible for selectively binding to overexpressed proteins in cancer cells on the outer membrane
[6]
. AS1411- N12 serves as a biological ligand and binds to receptors with extreme levels of specificity as visualized with our results. The development of this new DNA aptamer, can improve cancer therapy and help many who are currently struggling
[1, 2]
.
Abbreviations

DNA

Deoxyribonucleic Acid

ssDNA

Single-stranded DNA

RNA

Ribonucleic Acid

XNA

Xenonucleic Acid

PBS

Phosphate Buffered Saline

IDT

Integrated DNA Technologies

IDTE

(IDT Tris EDTA Buffer)

µM

Micromolar

PCR

Polymerase Chain Reaction

kDa

Kilodalton

RPM

Revolutions per Minute

°C

Degrees Celsius

nm

Nanometer
Conflicts of Interest
The authors declare no conflicts of interest.
References
[1] Bates, P. J., Laber, D. A., Miller, D. M., Thomas, S. D., & Trent, J. O. (2009). Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Experimental and Molecular Pathology, 86(3), 151-164.

https://doi.org/10.1016/j.yexmp.2009.01.004

[2] Chanda, K., Motilal Balamurali, M., & Venkatesan, S. (2023). Recent Advancements of Aptamers in Cancer Therapy. ACS Omega, 8, 12034-12047.

https://doi.org/10.1021/acsomega.3c04345

[3] Wang, H., Zheng, X., Jiang, Z., et al. (2023). Aptamer-drug conjugates for targeted leukemia therapy. ACS Omega, 8(4), 12345-12356.

https://doi.org/10.1021/acsomega.3c04345

[4] Nimjee, S. M., Rusconi, C. P., & Sullenger, B. A. (2005). Aptamers: An emerging class of therapeutics. Annual Review of Medicine, 56, 555-583.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2640431/

[5] Schmidt, C., Kammel, A., Tanner, J. A., et al. (2022). A multiparametric fluorescence assay for screening aptamer-protein interactions based on microbeads. Scientific Reports, 12, 2961.

https://doi.org/10.1038/s41598-022-06817-0

[6] Thongchot, S., Aksonnam, K., Thuwajit, P., Yenchitsomanus, P. T., & Thuwajit, C. (2023). Nucleolin-based targeting strategies in cancer treatment: Focus on cancer immunotherapy (Review). International Journal of Molecular Medicine, 52(3), 81.

https://doi.org/10.3892/ijmm.2023.5284

[7] Wilchek, M., & Bayer, E. A. (1988). The avidin-biotin complex in bioanalytical applications. Analytical Biochemistry, 171(1), 1-32.

https://doi.org/10.1016/0003-2697(88)90422-8

[8] News Medical. (n.d.). Limitations of aptamers. Retrieved October 29, 2024, from.

https://www.news-medical.net/life-sciences/Limitations-of-Aptamers.aspx

[9] Tipping, A. J., Deininger, M. W., Goldman, J. M., & Melo, J. V. (2003). Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate. Science of The Total Environment, 313(1-3), 243-253.

https://www.sciencedirect.com/science/article/pii/S0301472X03002650

[10] Earnest, K. G., Merino, E. J., McConnell, E. M., et al. (2021). Development and characterization of a DNA aptamer for MLL-AF9 expressing acute myeloid leukemia cells using whole cell-SELEX. Scientific Reports, 11, 19350.

https://www.nature.com/articles/s41598-021-98676-4

[11] Editorial Content Team, American Cancer Society. (2024). Immunotherapy for Acute Lymphocytic Leukemia. Retrieved October 23, 2024.

https://www.cancer.org/cancer/types/acute-lymphocytic-leukemia/treating/monoclonal-antibodies.html

[12] American Cancer Society. (n.d.). Monoclonal antibodies for acute lymphocytic leukemia. Retrieved October 29, 2024, from.

https://www.cancer.org/cancer/types/acute-lymphocytic-leukemia/treating/monoclonal-antibodies.html

[13] GeneMedSyn. (n. d.). Acute myeloid leukemia cells (28KH1C1229) - DNA aptamer (AD-102). Retrieved October 29, 2024, from

https://www.genemedsyn.com/commerce/ccpc1103-11796-acute-myeloid-leukemia-cells-28kh1c1229--dna-aptame-ad-102-u.htm#

[14] GeneMedSyn. (n.d.). AD-102 aptamer product brochure [PDF]. Retrieved October 29, 2024, from

https://www.genemedsyn.com/objects/catalog/product/extras/AD-102.pdf

[15] Alhabbab, R. Y. (2021). DNA aptamers targeting leukemia and lymphoma cells: A review. Scientific Reports, 11, Article 19350.

https://doi.org/10.1038/s41598-021-98676-4

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    Mayerfield, H., Cunningham, J. (2025). Optimization of Aptamer AS1411: An Effort to Increase Binding Efficiency to Nucleolin, an Overexpressed Protein on Cancer Cells. Cancer Research Journal, 13(3), 147-151. https://doi.org/10.11648/j.crj.20251303.15

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    Mayerfield, H.; Cunningham, J. Optimization of Aptamer AS1411: An Effort to Increase Binding Efficiency to Nucleolin, an Overexpressed Protein on Cancer Cells. Cancer Res. J. 2025, 13(3), 147-151. doi: 10.11648/j.crj.20251303.15

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    AMA Style

    Mayerfield H, Cunningham J. Optimization of Aptamer AS1411: An Effort to Increase Binding Efficiency to Nucleolin, an Overexpressed Protein on Cancer Cells. Cancer Res J. 2025;13(3):147-151. doi: 10.11648/j.crj.20251303.15

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  • @article{10.11648/j.crj.20251303.15,
  author = {Henry Mayerfield and Jackson Cunningham},
  title = {Optimization of Aptamer AS1411: An Effort to Increase Binding Efficiency to Nucleolin, an Overexpressed Protein on Cancer Cells
},
  journal = {Cancer Research Journal},
  volume = {13},
  number = {3},
  pages = {147-151},
  doi = {10.11648/j.crj.20251303.15},
  url = {https://doi.org/10.11648/j.crj.20251303.15},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.crj.20251303.15},
  abstract = {This project aimed to manipulate DNA (deoxyribonucleic acid) aptamer AS1411, a short single-stranded oligonucleotide currently being developed to improve chemotherapy’s target cell specificity. As this aptamer binds explicitly to nucleolin, an overexpressed protein on the surface of cancer cells, chemotherapy damage to surrounding tissue may be lessened. This study modified the AS1411 DNA aptamer, which was named AS1411-N12, by adding 12 nucleotides to the 3’ and 5’ ends, forming a “flap” structure. The edition of said flap is attributed to theory that the increased mass will allow for tighter binding. This modification was hypothesized to further improve the DNA aptamer’s binding efficiency to the nucleolin protein expressed on cancer cells. Binding reactions occurred between DNA aptamers (AS1411 and AS1411-N12) and nucleolin samples. The resulting solutions were processed using micro-centrifugal filters, which separated small unbound single-stranded DNA aptamers from bigger unbound proteins and the DNA-Nucleolin complexes. Measured absorbance of the unbound filtered DNA aptamers were analyzed to compare binding efficiencies of the modified aptamer vs. the control. The average absorbance through 3 trials of the control AS1411 DNA aptamer was 1.907 at 260 nm, while the average absorbance through 3 trials was 1.364 at 260 nm. Through Beer's Law, the unbound DNA control concentration was 146.6 µM while the modified DNA aptamers was 54.17 µM. This modification was highly effective as it yielded a 63% change in absorbance showing a drastic decrease in the amount of DNA aptamer left in solution. The modified DNA aptamer was significantly more effective in binding to its target protein. When attached to chemotherapy, AS1411-N12 will have a higher affinity to Nucleolin, improving cancer treatment.},
 year = {2025}
}

    Copy | Download 

  • TY  - JOUR
T1  - Optimization of Aptamer AS1411: An Effort to Increase Binding Efficiency to Nucleolin, an Overexpressed Protein on Cancer Cells

AU  - Henry Mayerfield
AU  - Jackson Cunningham
Y1  - 2025/08/21
PY  - 2025
N1  - https://doi.org/10.11648/j.crj.20251303.15
DO  - 10.11648/j.crj.20251303.15
T2  - Cancer Research Journal
JF  - Cancer Research Journal
JO  - Cancer Research Journal
SP  - 147
EP  - 151
PB  - Science Publishing Group
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AB  - This project aimed to manipulate DNA (deoxyribonucleic acid) aptamer AS1411, a short single-stranded oligonucleotide currently being developed to improve chemotherapy’s target cell specificity. As this aptamer binds explicitly to nucleolin, an overexpressed protein on the surface of cancer cells, chemotherapy damage to surrounding tissue may be lessened. This study modified the AS1411 DNA aptamer, which was named AS1411-N12, by adding 12 nucleotides to the 3’ and 5’ ends, forming a “flap” structure. The edition of said flap is attributed to theory that the increased mass will allow for tighter binding. This modification was hypothesized to further improve the DNA aptamer’s binding efficiency to the nucleolin protein expressed on cancer cells. Binding reactions occurred between DNA aptamers (AS1411 and AS1411-N12) and nucleolin samples. The resulting solutions were processed using micro-centrifugal filters, which separated small unbound single-stranded DNA aptamers from bigger unbound proteins and the DNA-Nucleolin complexes. Measured absorbance of the unbound filtered DNA aptamers were analyzed to compare binding efficiencies of the modified aptamer vs. the control. The average absorbance through 3 trials of the control AS1411 DNA aptamer was 1.907 at 260 nm, while the average absorbance through 3 trials was 1.364 at 260 nm. Through Beer's Law, the unbound DNA control concentration was 146.6 µM while the modified DNA aptamers was 54.17 µM. This modification was highly effective as it yielded a 63% change in absorbance showing a drastic decrease in the amount of DNA aptamer left in solution. The modified DNA aptamer was significantly more effective in binding to its target protein. When attached to chemotherapy, AS1411-N12 will have a higher affinity to Nucleolin, improving cancer treatment.
VL  - 13
IS  - 3
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