Electrophoretic mobility shift assay (EMSA)

An electrophoretic mobility shift assay (EMSA) is a common affinity electrophoresis technique used to study protein-DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence(Wikipedia). In the present study, EMSA was employed for affinity test of the aptamers.

After thrombin and aptamers were diluted with proper buffer, reaction systems were built with a gradient of aptamers. 15-mer, 29-mer and 40-mer aptamers were tested, and a gradient of concentration of thrombin were applied to reflect the binding affinity. After the aptamers were co-incubated with thrombin for 60 min, an 12% non-denaturing polyacrylamide gel electrophoresis was performed. The gel was then stained by fluorescent dye. GelRed was used as the DNA dye. Random extension was added to aptamer, in order to enhance GelRed incorporation (Figure 1-1).

Figure 1-1: The design of aptamer-linker-probe complex. This structure enlarged the DNA molecule while its binding activity was not weakened.

The electrophoresis showed that the aptamers showed rather strong binding affinity (Figure 1-2). The shift bands became more clear as the concentration of thrombin increased. 15-mer and 29-mer aptamers had a clear shift band and a clear non-shift band. 40-mer aptamer was suspected to form multimers, causing a strong band at the sampling hole and unclear bands at the target sites.

Figure 1-2: Native-PAGE results of EMSA. In the two figures, lane 1, marker; lane 2, control group with 15-mer, 29-mer and 40-mer aptamers and NO thrombin. All aptamers were at a concentration of 10 pM.

Figure 1-2A: lane 3-5, 0.45 pM thrombin incubated with respectively 29-mer+40-mer, 15-mer+40-mer, 15-mer+29-mer; lane 6-8, 15-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM.

Figure 1-2B: lane 3-5, 29-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM.; lane 6-8, 40-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM.

Surface Plasmon Resonance (SPR)

EMSA provided a relatively rudimentary validation of the binding interactions. To achieve a more quantitative and precise characterization of the interactions between the 29-mer/40-mer aptamers and thrombin, Surface Plasmon Resonance (SPR) was employed for testing. Briefly, streptavidin (SA) was amino-conjugated to capture biotinylated aptamers and seal the chip with bovine serum albumin (BSA) to prevent non-specific binding of thrombin. Then gradient diluted thrombin was loaded to obtain the corresponding curves within SPR buffer. Partial experimental results were fitted with 1:1 binding kinetic model in order to calculate dissociation constant (KD). Detailed operational procedures can be found on the Experiments-Surface Plasmon Resonance (SPR) page.

Figure 1: 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 fitted from 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.

As shown above, aptamer 29 demonstrated a strong affinity for thrombin, with a dissociation constant (KD) of 0.816 nM, while aptamer 40 had a much lower affinity, with a KD of 1170 nM. Due to the high affinity between aptamer 29 and thrombin, the dissociation was incomplete when thrombin concentrations were high, this could be improved by increasing the flow rate during the experiment. A 1:1 binding kinetic model was used based on the assumption that each aptamer binds to a single site on thrombin. Surface Plasmon Resonance (SPR) experiments meticulously verified the binding interactions between aptamers and thrombin. 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.

Protein Purification

Because our system is an in vitro detection system, it’s essential for us to express proteins and then purify them. We have chosen three strategies to express proteins. Directly expression with 6xHis tag but without solubility tags, expression with both 6xHis tag and solubility tags and expression with cell-free system. Methodology of ours for purification is affinity chromatography. To be specific, we use nickel affinity chromatography to purify proteins with 6xhis tag (with or without solubility tags), and use glutathione affinity chromatography to purify proteins expressed by cell-free system. For nickel affinity chromatography, we use both ÄKTA system and gravity chromatography. For glutathione affinity chromatography, we use ÄKTA system. Furthermore, for proteins that are not expressed with solubility tags or cell-free system, they usually form inclusion bodies and we need new strategy to tackle this tricky problem. On-column refolding is the very solution applied by us. Finally, to test whether the affinity chromatograhy works as expected, we have done SDS-PAGE analysis to see the purification results.

Figure 1: Elution profile of the nickel affinity chromatography of cPPV, nPPV, PPV, G-PPVcutsite-Y without solubility tags. Method used for dissolving those proteins from inclusion bodies is on-column refolding. Blue line stands for UV curve and Red line stands for conductivity curve.

Figure 1A, elution profile of cPPV. Peak A stands for transmission peak, peak B stands for elution peak of cPPV.

Figure 1B, elution profile of nPPV. Peak A stands for transmission peak, peak B stands for elution peak of nPPV.

Figure 1C, elution profile of PPV. Peak A stands for transmission peak, peak B stands for elution peak of PPV.

Figure 1D, elution profile of G-PPVcutsite-Y. Peak A stands for transmission peak, peak B stands for elution peak of G-PPVcutsite-Y.

Figure 2: SDS-PAGE analysis of the cell lysate, the transmission peak and the elution peak after nickel affinity chromatography through AKTA or gravity chromatography. Those proteins were expressed without solubility tags.

Figure 2A, Lane2-4, total proteins of bacteria expressing cPPV, elution peak, transmission peak of cPPV; Lane5-7, total proteins of bacteria expressing nPPV; Lane9-10, elution peak and transmission peak of nPPV. All the proteins are purified through AKTA. Because of the low resolution of the picture, other bands of low expression proteins can’t be seen.

Figure 2B, Lane2, total proteins of bacteria expressing G-PPVcutsite-Y; Lane3, elution peak of G-PPVcutsite-Y through AKTA; Lane4, total proteins of bacteria expressing PPV; Lane5, elution peak of PPV through AKTA; Lane6, elution peak of G-PPVcutsite-Y through gravity chromatography; Lane7, elution peak of PPV through gravity chromatography. PPV wasn’t purified successfully in this picture.

Figure 2C, Lane2-3, elution peak of PPV through gravity chromatography; Lane4-5, elution peak of PPV through AKTA after ultrafiltration inside the ultrafiltration membrane; Lane6-7, elution peak of PPV through AKTA after ultrafiltration outside the ultrafiltration membrane. Lane4-5&Lane6-7 showed that the ultrafiltration membrane was integrated and leak of proteins did not happen.

As shown above, we successfully purified nPPV, cPPV, PPV and their substrate G-PPVcutsite-Y without solubility tags.

Figure 3: SDS-PAGE analysis of proteins expressed by the ALiCE ® Cell-Free Protein Expression System.

Figure 3A, SDS-PAGE analysis of positive control EYFP expressed by cell-free system. Lane2, negative control (ALiCE reaction mixture only) ; Lane3, reaction mixture with EYFP expression; Lane4,reaction mixture diluted two times with EYFP expression; Lane5, reaction mixture diluted twenty times with EYFP expression. We proved that this cell-free protein expression system was feasible.

Protease Activity Verification

Since our system relies on the protease both amplifying the signal and triggering the release of the final colloidal gold output, it is crucial to verify the target protease activity to ensure that the enzymes used in our experiments are active and functioning as expected. To achieve this, we designed a experiment to verify the enzyme activity under controlled conditions (you can find more detailed information about this experiment in our protocol). We validated the activity of two intact proteases and one split protease. For the intact TEV and PPV proteases, we mixed a calculated amount of the enzyme with its corresponding substrate and added the appropriate amount of reaction buffer. The mixture was incubated at 30°C, and samples were taken at different time points. The reaction was stopped with SDS loading buffer, followed by electrophoresis. Enzyme activity was confirmed by observing the reduction in substrate and the presence of cleavage product bands. For the split PPV protease, we additionally added a fixed amount of rapamycin to induce protease dimerization and activation.

Figure 1: Enzymatic activity assay of TEV protease extracted from inclusion bodies.

The figure shows the SDS-PAGE analysis of the enzymatic activity of TEV protease on its substrate, under two different reaction buffer conditions (Tris-HCl: 20 mM Tris-HCI+10 mM NaCl+10 mM KCI+1 mM DTT and HEPES: 20 mM HEPES+10 mM NaCI+10 mM KCI+1 mM DTT). The top band corresponds to the intact substrate (tGFP-tevS-tYFP), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products (tGFP and tYFP), which increase over time. Samples were taken at 0, 5, 30, 120, and 240 minutes, with the reaction stopped by adding SDS loading buffer and heating at 95°C for 5-10 minutes.The results confirm that the TEV protease extracted from inclusion bodies is active.

Figure 2: SDS-PAGE analysis of protease activity verification in split and full-length PPV constructs.

Figure 2A：Lane M: Marker; Lane 0: Split-1 at 0 min; Lane Split-1: Split PPVp with 350 nM rapamycin at 10, 30, and 120 min; Lane Split-N: Split PPVp without rapamycin at 10, 30, and 120 min; Lane cPPV: Full-length FKBP-cPPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane nPPV: Full-length FRB-nPPVp with 1400 nM rapamycin at 10, 30, and 120 min.

Figure 2B：Lane M: Marker; Lane Split-2: Split PPVp with 700 nM rapamycin at 10, 30, and 120 min; Lane Split-3: Split PPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane PPV: Full-length PPVp with its substrate at 10, 30, and 120 min.

The results show that both the full-length PPVp and the split PPVp (when induced by rapamycin) exhibit protease activity, as evidenced by the reduction in substrate over time. In contrast, a single split PPVp cannot cleave the substrate. However, when two split PPVs are combined without rapamycin induction, there is minimal cleavage activity, as indicated by the slight reduction in substrate from 10 to 30 and 120 minutes. The amount of substrate reduction is significantly less than that observed with rapamycin induction, leading to the conclusion that rapamycin is necessary for the optimal activity of the split PPVp.

Protein Solubility Analysis

Since our attempt of purifying PPVp, FKBP-cPPVp, FRB-nPPVp, and G-PPV-Y(the substrate of PPVp) without soluble tags ended up in failure, we further examined the solubility of our target proteins. It is achieved by 3s ultrasonication on ice + 10s interval, power 300W, for 40 minutes to completely destruct bacterial structure. Then the sample is centrifuged. The soluble and insoluble components will appear in the supernatant and the precipitate respectively. With 5×SDS loading buffer treated, the two parts can be used for downstream SDS-PAGE analysis. As a control, EGFP, which is soluble in E.coli, is also expressed and analyzed with the same protocol.

Figure 1: Solubility analysis of PPV, its split form and substrate.

SU: supernatant after ultrasonication; PU: precipitate after ultrasonication

Figure 1A: Solubility analysis of FKBP-cPPV and FRB-nPPV

The figure shows solubility analysis of the two split enzymes. MW: FRB-nPPV: 24.9kDa; FKBP-cPPV: 27.3kDa. The reult shows that almost all of the FRB-nPPV and FKBP-cPPV exist in PU but not SU, which indicates the insoluble state(inclusion body) of the proteins. In contrast, the EGFP control mainly appears in the soluble component(SU), proving the correctness of our protocol. The unlabeled electrophoresis bands are due to sample loading mistakes.

Figure 1B: Solubility analysis of PPVp and G-PPVs-Y(GFP-PPV cut site-YFP)

The result is similar to figure 1A. MW: PPVp: 27.7kDa; G-PPVs-Y: 22.3kDa. Both of the proteins are insoluble. The unlabeled electrophoresis bands are due to sample loading mistakes.