Aptamer
Research
Aptamers are short oligonucleotide sequences (either DNA or RNA) that are typically derived from nucleic acid libraries using an in vitro selection technique known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). These oligonucleotide fragments can bind to various target molecules with high specificity and selectivity, making them widely utilized in the field of biosensors. According to previous studies, there are three well-studied aptamers with strong affinity for thrombin, measuring 19nt, 29 nt and 40 nt in length, respectively. We decided to assess the affinity of these three aptamers for thrombin and select two of them as the foundation for subsequent experiments.
There are several methods to assess the binding strength of aptamers to thrombin, among which Electrophoretic Mobility Shift Assay (EMSA) and Surface Plasmon Resonance (SPR) are two commonly used and sensitive techniques. Consequently, we employed these two methods to validate the affinity of the aptamers for thrombin.
Cycle 1: Affinity Verification Through EMSA
Design:
In the first cycle, we aim to use EMSA to validate the affinity of
three different aptamers for thrombin. In order to detect the
single-strand aptamer after electrophoresis, we added a random extension
to the aptamer(Figure 1-1), enhancing GelRed incorporation.
Figure 1-1 Design of aptamer-linker-probe complex. This structure enlarges the DNA molecule without weakening its binding activity.
Build:
The three aptamers were synthesized commercially, and thrombin was sourced online.
Test:
After diluting thrombin and aptamers in the appropriate buffer, we set up reactions with a gradient of aptamer concentrations. The 15-mer, 29-mer and 40-mer aptamers were tested against varying concentrations of thrombin. After a 60-minute co-incubation, a 12% non-denaturing polyacrylamide gel electrophoresis was performed, followed by staining with GelRed. The results are shown below:
Figure 1-2: Native-PAGE results of EMSA. In both figures, lane 1: marker; lane 2: control group with 15-mer, 29-mer and 40-mer aptamers (all at 10 pM concentration) and without thrombin. Figure 1-2A: lane 3-5: 0.45 pM thrombin incubated with 29-mer+40-mer, 15-mer+40-mer, 15-mer+29-mer, respectively; lane 6-8: 15-mer aptamer incubated with thrombin at concentrations of 0.45, 0.9 and 1.8 pM. Figure 1-2B: lane 3-5: 29-mer aptamer incubated with thrombin at concentrations of 0.45, 0.9 and 1.8 pM.; lane 6-8: 40-mer aptamer incubated with thrombin at concentrations of 0.45, 0.9 and 1.8 pM.
Learn:
The results confirm that all three aptamers have strong binding affinity for thrombin. However, the 40-mer aptamer appears to form multimers, as evidenced by a strong band at the loading well and less distinct bands at the target sites.
Cycle 2: Affinity Verification Through SPR
Design:
While EMSA provided preliminary validation of binding interactions, we sought a more quantitative and precise characterization using Surface Plasmon Resonance (SPR) for the 29-mer and 40-mer aptamers.
Build:
To immobilize the aptamers on the SPR chip, we synthesized two biotinylated aptamers through a commercial provider. The experiments were conducted using an SPR instrument.
Test:
After immobilizing the aptamers, gradient-diluted thrombin was flowed over the chip, and the corresponding binding curves were recorded. Partial experimental results were fitted with 1:1 binding kinetic model in order to calculate dissociation constant (KD).
Figure 1-3: Surface Plasmon Resonance (SPR) results of aptamer-thrombin binding.
The sensorgrams illustrate the binding interactions between the 29-mer/40-mer aptamers and thrombin, showing real-time changes in refractive index. Curves represent the association and dissociation phases, providing insights into the binding kinetics and affinity of the aptamers for thrombin. A & C: Thrombin was subjected to binding and dissociation tests by flowing gradient-diluted samples over the chip. B & D: Partial experimental results were fitted with 1:1 binding kinetic model in order to calculate dissociation constant (KD).
| Aptamer | General Kinetics model | Quality Kinetics Chi² (RU²) | 1:1 binding ka (1/Ms) | kd (1/s) | KD (M) | tc |
|---|---|---|---|---|---|---|
| 29 | 1:1 binding | 1.03e0 | 2.14e+5 | 1.74e-4 | 8.16e-10 | 4.81e+7 |
| 40 | 1:1 binding | 4.58e+1 | 1.59e+5 | 1.85e-1 | 1.17e-6 | 5.29e+7 |
Table 1: Parameters from the 1:1 binding kinetic model.
RU: resonance units; ka: association rate constant (M-1s-1); kd: dissociation rate constant (s-1); KD: equilibrium dissociation constant (M); tc: flow rate-independent component of the mass transfer constant.
Learn:
Aptamer 29 demonstrated a strong affinity for thrombin with a dissociation constant (KD) of 0.816 nM, whereas aptamer 40 exhibited a much lower affinity with a KD of 1170 nM. By accurately determining the association and dissociation constants, we have significantly bolstered our confidence in the results obtained from our Electrophoretic Mobility Shift Assays (EMSA), laying a solid groundwork for the foundational concepts necessary for our subsequent system development.
Cycle 3: Verification of MAGA-Generated Aptamer Sequence
Design:
Accurate and timely detection of disease biomarkers is crucial for effective diagnosis and treatment across various medical conditions. Traditional methods, such as SELEX, used for screening aptamers that bind to specific biomarkers are often slow, costly, and lack the precision needed for diverse applications. To overcome these limitations, we introduce MAGA: Make Aptamer Generally Applied—a universal machine-learning-based platform designed to predict aptamer sequences that can target a wide range of disease biomarkers with high specificity and affinity.
Build:
We conducted validation experiments using three aptamer sequences predicted by MAGA, along with a randomly generated sequence as a control.
Test:
Our predictive model not only identified aptamer sequences that showed high binding affinity to thrombin, but the structural predictions made using AlphaFold3 were also in excellent agreement with experimental data. We employed EMSA to further validate that the predicted aptamer can indeed bind to the target protein, although DNA aptamers were employed in the experiments instead of the predicted RNA aptamers.
Figure 1-4 Native-PAGE results of EMSA. All aptamers were at a concentration of 20 pM. MAGA_A, MAGA_C and MAGA_E are aptamers obtained from MAGA, and MAGA_D is randomly generated.
Learn:
The EMSA results show a shift band for the MAGA_A aptamer at higher thrombin concentrations, indicating that our predicted aptamer sequence is likely capable of binding to thrombin. The success of the MAGA platform not only validates our approach but also sets the stage for its application to other disease biomarkers. This positions MAGA as a powerful, universal tool for biomarker detection, offering a blend of computational efficiency and experimental precision that could revolutionize the field of diagnostic medicine.
SPOC part Engineering Success
2024.9.22 composed by Xiyue Zhang
wiki-Engineering-SPOC（蛋白表达及提取、蛋白纯化：化学法、tag、无细胞翻译体系、酶活测定）
Cycle 1 - Protein Expression and Solubility Testing
Design
Our goal is to extract active cleavage proteases and conjugate them with aptamers that recognize disease biomarkers for diagnostic purposes. To achieve this, we constructed plasmids to express the target protease and its substrate, using the BL21(DE3) strain for expression.
Build
We constructed plasmids to express full-length TEV and PPV proteases and their substrates, as well as two split-PPV proteases. These plasmids were introduced into BL21(DE3) strains for protein expression. The cells were then centrifuged and ultrasonically disrupted for subsequent solubility testing.
Figure 1: Plasmid profiles of TEVp-pET28a, PPVp-pET28a, FKBP_cPPVp-pET28a, FRB_nPPVp-pET28a, and GFP_truncated_linker(PPV)_YFP_truncated-pET28a
Test
We conducted SDS-PAGE analysis on the supernatant and resuspended pellet from the sonicated cell lysates and found that all expressed proteins were located in the pellet, indicating that these proteins were insoluble and existed as inclusion bodies.
See more details in Result.
Learn
As our target proteins are insoluble and form inclusion bodies, further extraction is required to solubilize them for enzyme activity assays.
Cycle 2 - Protein Extraction, Purification and Activity Assay
Design
After confirming that TEV, PPV, and their substrates (including split-PPV) are in inclusion bodies, we designed a denaturation-renaturation extraction method followed by nickel column purification. Enzyme activity assays were then conducted on the final purified protease and substrate solutions.
Figure 2: Workflow for extracting and purifying target proteins from inclusion bodies.
Build
We used a high-concentration urea solution to dissolve the inclusion bodies, followed by gradient urea-NiA solutions for protein refolding and purification. Ultimately, we successfully extracted TEV, full PPV protease and its substrate, as well as split-PPV.
See more details in our Protocol.
Test
We verified the extracted proteins using SDS-PAGE and conducted enzyme activity assays subsequently. SDS-PAGE results confirmed that the extracted proteins were the target proteins. Enzyme activity assays showed that full TEV, PPV, and split-PPV were active and could effectively cleave their substrates. Notably, split-PPV exhibited significant cleavage only after the addition of the inducer rapamycin, validating the feasibility of our SPOC system.
Learn
We successfully extracted active target proteases from inclusion bodies, but the extraction process is complex and time-consuming. Therefore, we plan to optimize the protocol to enhance the solubility of the target protein and simplify extraction steps.
Cycle 3 - Exploring Tagging Systems
Design
To enhance the solubility of the target proteins, we plan to add solubility tags to the protease sequences.
Build
We linked two common solubility tags (MBP and FH8) to each target protein and reconstructed new plasmids with solubility tag-target protein fusions. These plasmids were introduced into BL21(DE3) strains to test whether the solubility of the target proteins had improved.
Figure 3: Plasmid profiles of MBP-PPVp-pET28a, MBP-FKBP_cPPVp-pET28a, MBP-FRB_nPPVp-pET28a, and MBP-GFP_truncated_linker(PPV)_YFP_truncated-pET28a
Figure 4: Plasmid profiles of FH8-PPVp-pET28a, FH8-FKBP_cPPVp-pET28a, FH8-FRB_nPPVp-pET28a, and FH8-GFP_truncated_linker(PPV)_YFP_truncated-pET28a
Test
After inducing the expression of the target protein, we lysed the cells via sonication and performed SDS-PAGE analysis on the supernatant and the resuspended pellet. The results showed a significant increase in the target protein solubility, especially in the MBP-tagged proteins, which were found mostly in the supernatant.
Learn
Our results confirmed that adding a solubility tag significantly improved the solubility of the target protein, greatly simplifying the extraction process. We also believe that by increasing the solubility of the target protease, we can directly purify it from the cell lysate supernatant without going through denaturation and renaturation steps, which will likely enhance the protease’s activity. However, due to time constraints, we haven’t yet completed the de-tagging, purification, and activity validation for the tagged protease. We hope to continue these experiments after the Grand Jamboree.
Cycle 4 - Exploring Cell-Free Expression System
Design
To eliminate the impact of purification on protease activity, we used the ALiCE® cell-free protein expression system (CFPS) and redesigned the plasmids for target protein expression.
Figure 5: Working principle of the ALiCE® cell-free protein expression system.
Build
We constructed plasmids for expressing FRB_nPPV, FKBP_cPPV, and PPV proteins in the ALiCE® cell-free protein expression system and also used the trial kit from the provider to express EYFP protein.
Figure 6: Plasmid profiles of pALiCE01_FKBP_cPPVp, pALiCE01_FRB_nPPVp, and pALiCE01_PPVp.
Test
We performed SDS-PAGE analysis on the EYFP protein expressed using the trial kit, confirming successful protein extraction with minimal contamination. This demonstrated the potential of the cell-free expression system to produce relatively pure proteins without the need for purification steps.
Learn
The cell-free system showed good performance in the extraction of EYFP protein. However, due to the delay in receiving the full cell-free translation kit from the company, we were unable to validate the expression of our target protease. As a result, this part of the work remained at the plasmid construction and preliminary experiment stages. We plan to complete the remaining experiments after the Grand Jamboree.