Overview

The COVID-19 pandemic in recent years markedly propelled the nucleic acid detection industry in China. However, popular techniques, notably those based on CRISPR-Cas9, had patents held internationally. This situation escalated the production costs and curtailed the flexibility of using such technologies. Consequently, there's a pressing need to establish a nucleic acid thermostatic detection technology grounded in foundational research and pioneering innovations, ensuring independent intellectual property rights.

DNA phosphorothionylation, or sulfur modification, is a distinct backbone alteration where non-bridging oxygen atoms on the DNA's phosphodiester bond are replaced by sulfur atoms. Restriction enzymes that are specific to this sulfur modification can precisely target and cleave this uniquely modified DNA. Given their strong affinity for nucleic acids, understanding the binding and cleavage mechanisms of these enzymes can pave the way for their incorporation into thermostable nucleic acid detection systems.

In our project, we aim to synthesize these potential sulfur-modification-dependent restriction enzymes. By constructing them in an expression vector, we intend to purify the resulting protein. Our subsequent focus will be on the exploration of the protein's binding and cleavage capabilities using EMSA assays and cleavage assays.

Round1 

Design

DNA phosphorothionylation modification (also known as sulfur modification) is a backbone modification that replaces non-bridging oxygen atoms on the phosphodiester bond of DNA with sulfur atoms, and sulfur modification-dependent restriction enzymes can target cleavage of this type of modified DNA. Through bioinformatics analysis, our project identified three potential sulfur modification-dependent restriction enzymes: Sga, Sva, and Asp.

Then, we chose pET28a as the vector to carry three target DNA segments, respectively, since the T7 promoter and T7 RNA polymerase greatly benefit protein induction [1]. The next step was enzyme digestion with Nde1 and Xho1, acting on template plasmids of three enzymes and vectors, allowing the subsequent protein DNA insertion to vectors (ligation with T4 DNA ligase, T4 ligase binds preferentially to phosphorylated nicks and catalyzes the sealing reaction ) and transformation [2]. Double enzyme digestion was also necessary since the bands on the gel represented by different DNA fragments could help us confirm if the preview construction was booming.

 

Figure 1 Plasmids construction overview

After that, we induced our proteins with IPTG, a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce protein expression where the gene is under the control of the lac operator. [4] Since the proteins remained in the E. coli after induction, we first changed the pH of the protein system. We carried out ultrasonication  to break  the cell wall of bacteria, managing to get target proteins. As another centrifuge was done, proteins that we needed were suspended in the supernat of the solution. The extracted proteins were not pure and couldn’t be used directly for function analysis, so we decided to do Ni-chelating affinity chromatography, Q column affinity  chromatography, an anion exchange chromatography to remove impure  proteins, DNA attachment on proteins and imidazole in solution, which may disturb subsequent experiments respectively. Then, we sent the pure proteins to concentrate with a centrifuge. During the process, two SDS-PAGE[5] were done to assess protein purity. One was done after Ni-chelating affinity chromatography, and the other was after concentration.

Finally, three pur ified  extracted target proteins were tested for their binding ability with phosphorothioated DNA (EMSA) and the cleavage ability (nucleic cleavage test) of cutting specific DNA sequences to see if they could be applied to nucleic acid testing.

 

Figure 2 Diagrammatic drawing  of target enzymes’ cleavage ability to PT DNA

Build

We first transformed the template plasmids of Sga, Sva, and Asp into E. coli competent cells for conservation and thawed the cells containing pET28a plasmid. After that, we prepared a system to lyse these cells and extracted plasmids. To create the sticky ends on cutting sites, we used two endonucleases, Nde1 and Xho1, to digest four plasmids with PCR amplifier. Then, AGE and gel extraction were performed to separate DNA segments in each plasmid. After purification and concentration, we used T4 DNA ligase to ligate the sticky ends of each enzyme DNA segment and vector pET28a; the recombinant plasmids were finally constructed.

 

Figure 3 Plasmids profiles of pET28a-Asp, pET28a-Sva, pET28a-Sga

To completely ligate enzyme DNA and vector and temporarily conserve the recombinant plasmids, the transformation into DH5ɑ competent cell through heatshock was needed. After double enzyme digestion confirming we got the ideal plasmids, we could transform three plasmids into BL21 (an E.coli used for high-level protein expression [3] to do protein induction.)

Test

1.  Enzymatic validation of recombinant plasmids

We use nanodrop to measure the concentration and purity of plasmids to ensure we extracted enough of them to carry out digestion. After gel extraction, a concentration test of each DNA segment (Sga, Sva, Asp, and pET28a) was conducted to see if they reached the minimum amount to move to the next step --- ligation.

To verify that we successfully constructed the recombinant plasmids, we performed the enzymatic digestion again. In the expected result, the gel should appear to have two large and two small bands for each lane under UV light.

 

Figure 4 Gel electrophoresis results of vectors and target gene fragments

 

2. Protein extraction and purification

IPTG was used to induce protein in E. coli cells. Ultrasonication was performed to extract them, and target proteins were suspended in the supernatant of the solution. The next step was purification.

First, we did Ni-chelating affinity chromatography purification to remove impure proteins. SDS-PAGE of three samples of each protein (supernat after ultrasonic ation , effluent of Ni-chelating affinity chromatography, purified proteins) was followed  to assess purification. The results showed no bands in all three lanes  of Sva, so it hadn’t been collected and purified successfully. Then we did Q column affinity chromatography, anion exchange column to remove DNA attachment, and imidazole sending. After one night's concentration of purified proteins, we did another SDS-PAGE; the results showed a loss of Asp during purification, and some impure  proteins still existed in it. The band of Sga was clear, indicating a successful purification. Meanwhile, the ELISA test (Measuring the OD of BSA with known  concentrations to draw a best-fit line showing the math  relationship between protein concentration and OD, using this relationship to calculate  the concentration of unknown proteins) was carried out to measure the exact engagement of Sga and Asp, the concentration of Asp was relatively low (about 0.16ng/µl). Therefore, only Sga could used to do function tests.

 

Figure 5 SDS-PAGE

（ A ） R esults of target protein after purification （ B ） R esults of target protein after concentration

 

3 . Function Test 1 ： Electrophoretic mobility shift assays (EMSA)

After extracting the target proteins, purification (nickel affinity chromatography, Q column chromatography, gravity column) and concentration were done, preparing for two function analyses: EMSA and nucleic acid cleavage test.

AGE conducted EMSA, dyed using SYBR Gold. It could test the binding ability of Sga and Pt-DNA since when Sga binds to Pt-DNA, the molecular weight of DNA increases, and DNA travels at a lower velocity in the gel than regular DNA strands. To further explore the influence of DNA-protein ratio on binding capacity, we also set 6 different balances of DNA and protein (DNA:protein=1:0; 1:1; 1:2; 1:4; 1:8) in each group.

 

Figure 6 EMSA results of  Sga

（ A ） R esult of BL21 EMSA （ B ） Result of B7A EMSA

 

4 . Function Test 2 ： Nucleic acid cleavage test

A nucleic acid cleavage test was also done by AGE. It is used to examine the cutting ability of Sga to related sites on Pt-DNA. We set several comparison groups: different protein concentrations (0; 0.077; 0.15; 0.31; 0.625; 1.25; 2.5; 5; 10; 20; 40; 80; 160ng/µl),

different pH (6.0; 6.5; 7.0), if DNA contain sulfur modification. The severed DNA fragments would travel faster than complete DNA in gel, and the bands would show a luminance trend as the concentration of proteins changes.

 

 

Figure 7 Nucleic acid cleavage result of Sga

Learn :

In effect, we failed enzymatic digestion verification several times. The AGE test either showed the vague bands or the bands with non-ideal base pairs. We concluded that the reasons for this phenomenon include too low a concentration of plasmids and the contamination of restriction enzymes (Nde1 and Xho1).

About the failure in purify ing  Sva and Asp, we deduced that something went wrong when performing protein induction with IPTG; not all E. coli cell bodies precipitate  successfully after centrifuge. Therefore, the concentration of extracted proteins would be too low, and it would lose even more during the subsequent purification. Unfortunately, the function analysis couldn’t be conducted due to the low protein amount.

When we first did EMSA, someone accidentally dropped a marker into the sample lane in the non-pt-DNA group, causing a sample loading error. Besides, bands of all pt-DNA groups were at the bottom of the gel and showed no luminance change. It told us that SBD didn’t successfully bind to Pt-DNA. We thought it was due to the too-short DNA probe bought from the company. So the next time, we used the longer one (BL21 and B7A) as a probe and made the success.

The first-try results showed no differences between the groups of BL21 and B7A. This might be due to improper temperature, improper incubation time, problems during gel making, etc. A sampling loading error also appeared; someone added the opposite protein concentration in one pH group.

 

Round2

Design

Since we didn’t get Asp and Sva in round one, we carried out round two to manage to purify these Asp and Sva. The general process was the same as the previews one. First ly, digesting Asp and Sva template plasmids  and pET28a with two restriction enzymes ,  Nee1 and Xho1. Then ,  the T4 DNA ligase was used to ligate sticky ends on vector and two template plasmids to form recombinant plasmids. After double enzyme digestion verification, we induced  Asp and Sva in E. coli using IPTG. When protein was expressed in the cell body, we used ultrasonication to extract Asp and Sva, purifying them with Ni-chelating affinity chromatography, Q column affinity chromatography, and anion exchange chromatography for the next concentration. SDS-PAGE was done to measure the purities of proteins in each stage; the EMSA and nucleic acid cleavage were done for highly purified proteins.

 

Build

As in the first round, we constructed Sva-pET28a and Asp-pET28a plasmids. To obtain Sva-pET28a and Asp-pET28a plasmids, we used Nde1 and Xho1 to digest vector and template plasmids. After purification and recovery of the vector and target fragments, we ligated them using T4 DNA ligase. To conserve, stabilize, and completely ligate enzyme DNA, transformation into DH5ɑ competent cell through a heatshock was needed. When double enzyme digestion confirmed we got the ideal plasmids, we transformed two plasmids into BL21 (an E.coli used for high-level protein expression to do protein induction.)

Test

In the second round of our experiment, the basic methodology of protein extraction and purification, EMSA, and nucleic acid cleavage tests are all kept the same as usual to test our newly synthesized enzymes.

 

1. Protein extraction and purification

IPTG was used to induce protein in E. coli cells. Ultrasonication was performed to extract them, and target proteins were suspended in the supernatant of the solution. The next step was purification. Our protein purification has two steps. The first is nickel column purification and the second is anion chromatography.

 

Figure 8 SDS PAGE r esults of the target protein

(A) SDS PAGE of Asp ;(B) SDS PAGE of Sva

“S”: Protein from the supernate after ultrasonication

“P”: Protein from the precipitate after ultrasonication

“Ni”: Protein obtained after nickel column purification

“Q”: Protein obtained after ion-exchange chromatography

“M”: Marker

Protein extraction, purification and concentration are all in preparation for subsequent functional testing. We similarly performed the following two functional tests on Sva and Asp - EMSA and nucleic acid cleavage.

2. Function Test 1 ： Electrophoretic mobility shift assays (EMSA)

We performed EMSA using vertical DNA gels, using SYBR Gold for dyeing. It could test the binding ability of Sga and Pt-DNA since when Sga binds to Pt-DNA, the molecular weight of DNA increases, and DNA travels at a lower velocity in the gel than regular DNA strands. To further explore the influence of DNA-protein ratio on binding capacity, we also set 6 different balances of DNA and protein (DNA:protein=1:0; 1:1; 1:2; 1:4; 1:8) in each group.

 

 

Figure 9 EMSA result s  of BL21 and B7A

(A) EMSA result of Asp ;(B) EMSA result of Sva

3. Function Test 2 ： Nucleic acid cleavage test

A nucleic acid cleavage test was also done by AGE. It is used to examine the cutting ability of Sga to related sites on Pt-DNA. We set several comparison groups: different protein concentrations (0; 0.077; 0.15; 0.31; 0.625; 1.25; 2.5; 5; 10; 20; 40; 80; 160ng/µl), different pH (6.0; 6.5; 7.0), if DNA contain sulfur modification. The severed DNA fragments would travel faster than complete DNA in gel, and the bands would show a luminance trend as the concentration of proteins changes.

 

 

Figure 10 Nucleic acid cleavage result s  of BL21 & B7A

(A)Result of Asp ;(B) Result of Sva

Learn

We generally considered our second round of experiments more successful than in round one. In the protein extraction and purification test, it is clear enough to identify that all the irrelevant proteins disappeared gradually as the experiment continued, eventually revealing a clear, bright, and uniform band at 45 and 35 for SVA and ASP, respectively; this result corresponded with our previous estimation of the mass of the two proteins. In the EMSA binding experiment, it is also straightforward to observe that the binding of both proteins - SVA and ASP – turned out to be successful inside the B7A bacteria, where phosphorothioated-DNA strands occur naturally, as the concentration of the prokaryotic DNA increases. Finally, in the nucleic acid cleavage test, although it is clearer to identify the cleavage results of ASP on the B7A gDNA than those results of SVA on the B7A gDNA, both results proved an increase in cleavage level and success rate with the increase of gDNA concentration. In conclusion, our second round of protein expression and verifications turned out to be a great success that consolidated the practicality  and functionality of our three enzymes as potential raw materials for nucleic acid testing and POCT technologies.

 

 

Reference :

[1]  Addgene: PET28-MHL. (n.d.). https://www.addgene.org/26096/

[2]  Doherty, A. J., & Dafforn, T. R. (2000). Nick recognition by DNA ligases one 1Edited by K. Nagai. Journal of Molecular Biology, 296(1), 43–56. https://doi.org/10.1006/jmbi.1999.3423

[3]  BL21(DE3) competent cells. (n.d.). https://www.thermofisher.cn/order/catalog/product/EC0114

[4]  Wikipedia contributors. (2023). Isopropyl Β-D-1-thiogalactopyranoside. Wikipedia. https://en.wikipedia.org/wiki/Isopropyl_%CE%B2-D-1-thiogalactopyranoside

[5]  Protein electrophoresis using SDS-PAGE: A detailed overview | GoldBio. (n.d.). https://goldbio.com/articles/article/Protein-Electrophoresis-SDS-PAGE-Overview