Part:BBa_K5334000
A14#1 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
The aptamer sequence used in this study was selected by Saito et al. through a specific SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process. A library of single-stranded DNA (ssDNA) containing a 24-mer randomized region was created, flanked by 18-mer primer-binding sequences linked by a 3-mer thymine spacer. To prevent interactions between the randomized sequence and the primer-binding region, complementary strands of the primer region were added. This library was then heated to 95°C, gradually cooled to 25°C, and incubated with bevacizumab, an anti-VEGF antibody, to form aptamer-bevacizumab complexes. After incubation, the complexes were captured using Protein A-coated magnetic beads, which bind to the Fc-region of bevacizumab. The bound DNAs were eluted with Proteinase K, purified, and amplified by PCR. A library of ssDNAs was prepared for the next round of selection from the amplified DNAs by removing biotinylated reverse strands using streptavidin agarose [2]. After five selection rounds, the process was refined by adding VEGF165 to target aptamers specific to bevacizumab's CDR, and human IgG1 was used to remove non-specific DNAs. This selection process continued for 14 rounds, with DNA quantities monitored by real-time PCR (figure 2) [3].
Characterization
Enzyme-Linked Oligonucleotide Assay (ELONA) and Western-Blotting
To evaluate the binding efficiency of aptamer to immunoglobulin G (bevacizumab), the following experiments were conducted by Yamada et al.: Enzyme-linked oligonucleotide assay (ELONA)(Figure 3)[5] and Aptamer blotting assay(Figure 4) [4]. The aptamer A14 demonstrated strong binding to bevacizumab with a dissociation constant (Kₐ) of 12 nM. In addition to its strong binding capacity, the aptamer A141 demonstrated high specificity for bevacizumab, showing no binding to human IgG1 or other anti-VEGF antibodies [2]. This indicates that A141 can effectively distinguish bevacizumab from human IgG, despite the similarities in their amino acid sequences
→ PCR conditions and possible storage in vector are described on Design page.
MicroScale Thermophoresis (MST)
To evaluate the binding affinity, we performed the binding of bevacizumab to the initial aptamer А14#1 (5′-GCGGTTGGTGGTAGTTACGTTCGC-3′) via MST (Fig. 5). The dissociation constant Kd was determined to be 11.0 ± 3.7 nM under pH 6.3, mean standard deviations were calculated based on at least three independent experiments. The results are similar to the results of Saito et al with Kd 12 nM for pH 7.4 obtained from the ELONA assay [3]. For our MST experiments, we selected a pH to closely mimic the clinical conditions of bevacizumab solutions – pH 6.2 [10].
Enzyme-Linked Oligonucleotide Assay (ELONA) Evaluation
To evaluate the binding affinity, we performed the binding of bevacizumab to the initial aptamer А14#1 via ELONA (Fig. 6). We conducted a series of experiments to validate the method of chromogenic signal detection and establish a lower limit of Bevacizumab detection.
In our results, we observed that DNA aptamers, unlike monoclonal antibodies (mAbs) commonly used in ELISA methods, demonstrate high stability against factors such as heat, organic solvents, and acids. The chemical modification of the DNA aptamers on the microplate surface remained stable as well. By maintaining proper washing protocols and preserving the three-dimensional structure of the aptamers, strong linearity of the calibration curves was achieved (r² > 0.9847) (Fig.6)
However, lowering the concentration of bevacizumab in samples (Fig. 7) resulted in a reduction of the calibration curve's slope and increased variance in absorbance between bevacizumab samples. These issues likely stemmed from nonspecific adsorption of IgG or other components to the plate surface, as well as working below the recommended detection limit of TMB. Despite this, the CV (coefficient of variation) values across all concentrations remained below 30%, demonstrating that some accuracy was maintained. The lowest detectable concentration was determined to be 0,005 μg/ml, which equals 0,5 ng.
References
[1] Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990 Aug 30;346(6287):818-22.
[2]Nonaka, Y., Sode, K., & Ikebukuro, K. (2010). Screening and Improvement of an Anti-VEGF DNA Aptamer. Molecules, 15(1), 215–225.
[3] Saito T, Shimizu Y, Tsukakoshi K, Abe K, Lee J, Ueno K, Asano R, Jones BV, Yamada T, Nakano T, Tong J, Hishiki A, Hara K, Hashimoto H, Sode K, Toyo'oka T, Todoroki K, Ikebukuro K. 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. (2022).
[4] Hasegawa, H., Sode, K., & Ikebukuro, K. (2008). Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method. Biotechnology Letters, 30(5), 829–834.
[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.
[6] Dollins CM, Nair S, Sullenger BA. Aptamers in immunotherapy. Hum Gene Ther. 2008 May;19(5):443-50..
[7] Sullenger BA, Gallardo HF, Ungers GE, Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell. 1990 Nov 2;63(3):601-8.
[8] Han K, Liang Z, Zhou N. Design strategies for aptamer-based biosensors. Sensors (Basel). 2010;10(5):4541-57. Epub 2010 May 4.
[9] Mascini M. Aptamers and their applications. Anal Bioanal Chem. 2008 Feb;390(4):987-8.
[10] Ingram, R. L. & Weiser, S. E. Development of the Drug Product Formulation of the Bevacizumab Biosimilar PF-06439535 (Bevacizumab-bvzr). Drugs R D 23, 55–64 (2023).
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]
| None |