Part:BBa_K5334004
CU2#4 aptamer for Bevacizumab
Notice: Functional DNA
This part is a sequence of a functional ssDNA. It is only active as single-stranded DNA. It can not be cloned into a plasmid. For use order it as a DNA oligo.
Aptamers are single-stranded nucleic acid ligands with high affinity and specificity to target molecules[1]. This is an anti-idiotype aptamer (Figure 1) that recognizes the CDRs of bevacizumab, an anti-vascular endothelial growth factor (VEGF) mAb, which is used in lateral-flow detection system for bevacizumab. The aptasensor allows the detection of bevacizumab in anti-cancer drugs (Avastin) in a wide concentration range, comprised between 0.05 and 5.0 μg/mL, with the limit of detection was 2.09 ng/mL
Usage and Biology
Aptamers, which include RNA, single-stranded DNA (ssDNA), and peptide molecules, exhibit high target specificity and binding strength due to their distinct three-dimensional structures. Research on the binding of nucleic acids with proteins started in the 1980s, largely from studies on HIV and adenovirus, where small structured RNAs were found to bind viral or cellular proteins with precision [6]. For example, the HIV TAR element RNA binds with the viral Tat protein, while adenovirus VA-RNA controls translation [7]. The major breakthrough in aptamer technology came with the 1990 development of SELEX (Systematic Evolution of Ligands by EXponential enrichment), which made it possible to select aptamers in vitro for a range of targets, from small molecules to cells [8].
Since then, aptamers have been explored in fields like diagnostics, therapeutics, biosensors, and drug delivery [8]. They offer advantages over traditional antibodies, such as being highly stable at elevated temperatures, simpler and more cost-effective to produce, and less likely to trigger immune responses [9]. For instance, Macugen, an aptamer targeting VEGF, was approved by the FDA in 2004 for the treatment of wet age-related macular degeneration [9], marking a key milestone for aptamer applications. Given their versatility and superior characteristics, aptamers are seen as a viable alternative to antibodies across various biological and medical fields.
Source of the part
Given the project timeline and efficacy constraints, the SELEX process was deemed infeasible. Consequently, we opted to model an existing aptamer with a demonstrated high affinity for bevacizumab: 5′-GCGGTTGGTGGTAGTTACGTTCGC-3′ [5]. Utilizing the structural ideogram of the previously characterized aptamer [3], we aimed to engineer interactions involving residues that were not previously engaged in antibody binding: T5, T6, G7, G8, T9, G10, G11, T12, A13, G14, T15, T16, A17, C18, G19, T20, T21.
Modeling
Docking
2D Structure
Consequently, we opted to model an existing aptamer with a demonstrated high affinity for bevacizumab: 5′-GCGGTTGGTGGTAGTTACGTTCGC-3′1. Utilizing the structural ideogram of the previously characterized aptamer [3], we aimed to engineer interactions involving T5 via changing to G5 (5′-GCGGGTGGTGGTAGTTACGTTCGC-3′ - aptamer CU2#4) due to T5 not previously engaged in antibody binding. The DNA secondary structures were predicted using the RNAfold web server [16] with the default parameters (Figure 1). Energy parameters were rescaled to 37 °C and 1.021 M salt concentration. The color represents the base pairing/unpairing probabilities with red being the most plausible.
3D structure and docking
The coordinates of the initial complex (7V5N, 1.70 Å) were taken from the Protein Data Bank [3]. To predict aptamer coordinates 3dDNA web server was used for folding [10] (Figure 2).
For virtual screening, an approach from [11] was adopted. Normalized results of 3 different models (Autodock Vina, PatchDock, and Hex), were used. Smina [12] which is a modification of Autodock vina used for docking as it is capable of setting up the simulation box automatically according to the initial ligand position. The grid box was chosen with 8 Å buffer space in each direction to ensure that different folds would fit. The exhaustiveness was 4000 and 10 best docking poses were taken for further evaluation. Hex 8.0 [13] was used with default parameters and consequent minimization using the OPLS force field. PatchDock [14] docking was performed locally with 4.0 Å RMSD clustering threshold. Therefore, we adopted the methodology outlined by Ahirwar et al.[15] For binding affinity predictions, we employed three distinct docking platforms: Autodock Vina, PatchDock, and Hex (Table 1), and took a total score as characteristic of binding.
Table 1. | Z-score of aptamers from three different docking platforms (Autodock Vina, PatchDock, and Hex) and their cumulative sum for aptamers A14#1 and CU2#4.
| # | Subsequence | AutoDock Vina | PatchDock | Hex | Sum |
|---|---|---|---|---|---|
| A14#1 | 5′-GCGGTTGGTGGTAGTTACGTTCGC-3′ | -2.224 | -1.280 | -1.814 | -5,318 |
| CU2#4 | 5′-GCGGGTGGTGGTAGTTACGTTCGC-3′ | -2.317 | -1.260 | -1.746 | -5.323 |
Aptamer CU2#4 showed better results than initial aptamer A14#1. As a result, aptamer CU2#4 was taken for calculation in MD.
Molecular Dynamics
We initially considered the total binding score from docking as a primary characteristic of molecular dynamics (MD). MD simulations of protein-DNA complexes were performed in GROMACS 2021.1 software [16]. The system was described by the amber14sb_ol21 force field [17]. The initial structure was solvated in a rectangular box with a 10 Å margin of TIP3P water. Na+ and Cl- ions were added up to 0.15 M concentration. Non-bonded interactions were computed with a cutoff distance of 10 Å, while long-range electrostatics were addressed using the Particle-Mesh Ewald method. The SHAKE algorithm was employed to constrain all bonds involving hydrogen during the simulations. The time step used was 2 fs. The system was minimized for 8000 steps using the steepest descent algorithm to avoid steric clashes. Then it was gradually heated up from 0 to 300 K in the NVT ensemble during 1 ns and equilibrated at 1 bar and 300 K in NPT for the same time applying constraints on the protein-aptamer complex. The production run was performed for 200 ns and the last 150 ns were used for analysis. The protein-aptamer binding affinity was assessed using MM/PBSA approach implemented in gmx_MMPBSA software [18] which allows using the original MMPBSA.py script implemented in AmberTools in GROMACS. 50 frames were extracted from each of the trajectories for analysis time averaged values and their standard deviation were an estimate of the binding energetics. Subsequently, we employed aptamers with enhanced binding affinity compared to existing ones in GROMACS to determine the time-averaged total energy (Table 2)[19,20]. The P-value was calculated based on the average MM/PBSA, standard deviation (SD), and a number of frames via a two-tailed student's t-test.
Table 1. | Z-score of aptamers from three different docking platforms (Autodock Vina, PatchDock, and Hex) and their cumulative sum for aptamers A14#1 (BBa_K5334000) and CU2#4.
| # | Subsequence | MM/PBSA | Standard deviation | p-value |
|---|---|---|---|---|
| A14#1 | 5′-GCGGTTGGTGGTAGTTACGTTCGC-3′ | -142.260 | 7.150 | - |
| CU2#4 | 5′-GCGGGTGGTGGTAGTTACGTTCGC-3′ | -114.560 | 5.940 | Not significant |
The molecular dynamics results indicated that the initial aptamer A14#1 (BBa_K5334000) exhibits a stronger affinity for bevacizumab compared to CU2#4. Consequently, for wet-lab experiments and the development of the LFA device, the decision was made to focus on other aptamers.
References
[1] Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature, 346(6287):818-22, (1990).
[2] Nonaka, Y., Sode, K., & Ikebukuro, K. Screening and Improvement of an Anti-VEGF DNA Aptamer. Molecules, 15(1), 215–225, (2010).
[3] Saito, T. et al. Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection. Biosens Bioelectron 203, 114027 (2022).
[4] Hasegawa, H., Sode, K., & Ikebukuro, K. Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method. Biotechnology Letters, 30(5), 829–834, (2008).
[5] Yamada, T., Saito, T., Hill, Y., Shimizu, Y., Tsukakoshi, K., Mizuno, H., … Todoroki, K. (2019). High-throughput bioanalysis of bevacizumab in human plasma based on enzyme-linked aptamer assay using anti-idiotype DNA aptamer. Analytical Chemistry (2019).
[6] Dollins CM, Nair S, Sullenger BA. Aptamers in immunotherapy. Hum Gene Ther, 19(5):443-50, (2008).
[7] Sullenger BA, Gallardo HF, Ungers GE, Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell. (1990)
[8] Han K, Liang Z, Zhou N. Design strategies for aptamer-based biosensors. Sensors (Basel). 10(5):4541-57, (2010).
[9] Mascini M. Aptamers and their applications. Anal Bioanal Chem;390(4):987-8, (2008).
[10] Zhang, Y., Xiong, Y. & Xiao, Y. 3dDNA: A Computational Method of Building DNA 3D Structures. Molecules 27, 5936 (2022).
[11] Ahirwar, R. et al. In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules. Sci Rep 6, 21285 (2016).
[12] Koes, D. R., Baumgartner, M. P. & Camacho, C. J. Lessons Learned in Empirical Scoring with smina from the CSAR 2011 Benchmarking Exercise. J Chem Inf Model 53, 1893–1904 (2013).
[13] Ghoorah, A. W., Devignes, M., Smaïl‐Tabbone, M. & Ritchie, D. W. Protein docking using case‐based reasoning. Proteins: Structure, Function, and Bioinformatics 81, 2150–2158 (2013).
[14] Duhovny, D., Nussinov, R. & Wolfson, H. J. Efficient Unbound Docking of Rigid Molecules. in 185–200 (2002).
[15] Ahirwar, R. et al. In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules. Sci Rep 6, 21285 (2016).
[16] Burford, A. et al. Ookami: Deployment and Initial Experiences. in ACM International Conference Proceeding Series (Association for Computing Machinery, 2021).
[17] Zgarbová, M., Luque, F. J., Šponer, J., Otyepka, M. & Jurečka, P. A Novel Approach for Deriving Force Field Torsion Angle Parameters Accounting for Conformation-Dependent Solvation Effects. J Chem Theory Comput 8, 3232–3242 (2012).
[18] Valdés-Tresanco, M. S., Valdés-Tresanco, M. E., Valiente, P. A. & Moreno, E. gmx_MMPBSA: A New Tool to Perform End-State Free Energy Calculations with GROMACS. J Chem Theory Comput 17, 6281–6291 (2021).
[19] Martin, D. R. et al. Molecular modeling and simulation studies of SELEX-derived high-affinity DNA aptamers to the Ebola virus nucleoprotein. J Biomol Struct Dyn 1–18 (2024)
[20] Sabri, M. Z., Abdul Hamid, A. A., Sayed Hitam, S. M. & Abdul Rahim, Mohd. Z. In Silico Screening of Aptamers Configuration against Hepatitis B Surface Antigen. Adv Bioinformatics 2019, 1–12 (2019).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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