In order to reduce the detection threshold, the first step of the detection process is to amplify a specific DNA fragment needed to be detected. The RPA is a technique that can amplify DNA fragments at a constant temperature. After experimental verification, the RPA system used by us can detect nucleic acid with a very low copy number in the sample, and produce a large amount of the templates for later targeted cleavage, leading to improved detection sensitivity. We diluted the DNA template into different concentrations for RPA reaction, and the following results were obtained.
Figure 1. Agarose gel electrophoresis of the RPA products with different starting template copy numbers in the samples. | 1. Positive control using the template and primers provided in the commercial kit. 2. Negative control of a reaction mix without any template or primer. 3. RPA products diluted to 1 copy of the template. 4. RPA products diluted to 1×103 copies of the template.
The results showed that the DNA template as low as 1 copy/µl could still produce a visible amplified band by the RPA for 30 min (Figure 1). This indicated that the RPA had both high sensitivity, and rapid response with simple designs. We can also selectively amplify the nucleic acid fragments that need to be detected by designing different primers. Therefore, if a new infectious disease breaks out, we can quickly design corresponding primers and detect the new pathogen using our detection platform.
Figure 2. Agarose gel electrophoresis of the RPA products from the reaction at different temperatures. | 1. 25℃ 2. 31℃ 3. 37℃ 4. Negative control of a reaction mix without any template or primer. 5. Positive control using the template and primers provided in the commercial kit.
After comparing the product levels at three temperatures, we concluded that the RPA could reach the highest efficiency at a temperature about 30℃ (Figure 2). We also concluded that the RPA system could produce detectable products with a sample containing as low as 1 copy/µl of the template DNA at 30℃.
To ensure that the detection is highly specific, we planned to use a specifically targeted cleaving enzyme to produce nicks at specific sites of a double-stranded DNA (dsDNA) fragment amplified in the previous step. After extensively exploring literature, we identified two targeting tools, nCas9 and Nb.BbvCI, and obtained the part required for their functional studies through plasmid construction and protein expression.
We used site-directed mutagenesis to create a Cas9 mutant that could only cleave one strand of dsDNA and was designated as nCas9. A plasmid was constructed to in vitro transcribe sgRNAs that could guide nCas9 to find a specific site on a sequence derived from the M13 geneIII.
Purification of nCas9 protein
Figure 3. SDS-PAGE analysis of purified recombinant nCas9 protein. | 1 and 2. Eluent of His×6-tagged nCas from Ni-NTA agarose beads.
We successfully expressed and purified the nCas9 protein from E. coli (Figure 3).
Acquisition of sgRNA
Purification of T7 RNA polymerase
Figure 4. SDS-PAGE analysis of purified recombinant T7 RNA polymerase. | 1 and 2. Eluent of His×6 tagged T7 RNA polymerase from Ni-NTA agarose beads.
We successfully expressed and purified the recombinant T7 RNA polymerase (Figure 4). In the following experiment, we used T7 RNA polymerase to conduct in vitro transcription to produce sgRNAs that could guide the nCas9 to cleave single-stranded DNA (ssDNA).
Design of sgRNA
In order to make the targeting of sgRNAs highly specific, we used a website-based algorithm (https://crispy.secondarymetabolites.org/#/input) to screen the sequence of the geneIII, a key gene of M13 phage, to identify a suitable gRNA binding site. The M13 geneIII-sgRNA was constructed as a transcriptional template.
Figure 5. The plasmid used to in vitro express the M13 geneIII-sgRNA.
In vitro transcription of sgRNA
Figure 6. Denatured electrophoresis of in vitro transcribed sgRNAs. | M. DNA ladder, 1. The in vitro transcribed sgRNA.
We used the recombinant T7 RNA polymerase to carry out in vitro transcription and obtained a good yield sgRNA that will be used in the reaction of the next step (Figure 6).
nCas9-nickase activity verification
The nickase activity of the nCas9 was determined by its ability of creating nicked DNA within a reaction time of 30 min. The target plasmid carrying the geneIII fragment and streptomycin resistant gene was transformed bacteria, and the constructed plasmid was extracted and analyzed by agarose gel electrophoresis.
Figure 7. Verification of nCas9 nickase activity of target plasmids. | 1-2. Standard plasmids and nicked plasmids.
The figure shows that nCas9 possesses the activity of generating nicked DNA.
BbvCI is a restriction endonuclease with two different subunits R1 and R2. After mutating the R2 subunit, we used the R1+R2- mutant, named as Nb.BbvCI, which can only cleaves the bottom chain (see [Design] for more). Initially, we used the commercial Nb.BbvCI to verify our design.
Nb.BbvCI-nickase activity verification
Through communicating with other teams, we decided to use electrophoretic mobility shift assay (EMSA) to verify the activity of ssDNA replacement. Due to the limitation of experimental time, we used commercial Nb.BbvCI for verification. Based on the template DNA sequence, we synthesized a FAM-labeled probe with 23-nucleotide in length, and an unlabeled complementary oligonucleotide, named as c-probe.
Figure 8. EMSA to verify the nickase activity of the Nb.BbvCI. |1. Without Nb.BbvCI. 2. With Nb.BbvCI. 3. FAM-probe alone. 4. Annealed FAM-probe/c-probe.
nCas9 vs Nb.BbvCI
As a result, we verified the nickase activity of both nCas9 and Nb.BbvCI. To further determine which of them should be used in our detection system, we carried out experiments to verify the targeted cleavage ability of the two nickases and compare their cleavage efficiencies.
Figure 9. EMSA to compare the nickase activity of nCas9 and Nb.BbvCI. | 1-4. verification of the nCas9 nickase activity. 5-8. verification of Nb.BbvCI nickase activity.
Based on the EMSA results, the single-strand nickase activity of Nb.BbvCI was higher than that of nCas9 (Figure 9). Therefore, we decided to use the fusion protein Nb.BbvCI in our system to generate nicked dsDNA.
We constructed a Phi29 expression vector, expressed it in E. coli and purified the recombinant Phi29 protein. Phi29 is an efficient DNA polymerase and can continuously synthesize DNA to generate up to 70 kb fragments.
Purification of Phi29
Figure 10. SDS-PAGE analysis of the purified recombinant Phi29 protein. | 1 and 2. Eluent of His×6-tagged Phi29 from Ni-NTA agarose beads.
We successfully expressed and purified the Phi29 protein (Figure 10).
Phi29-single stranded DNA replacement verification
Figure 11. EMSA to verify the Phi29 activity in replacing ssDNA from nicked dsDNA. | 1. Addition of both Nb.BbvCI and Phi29. 2. Reaction without Nb.BbvCI. 3. Reaction without Phi29. 4. FAM-probe alone. 5. Annealed FAM-probe/c-probe.
The ssDNA replacement activity of Phi29 could successfully replace the ssDNA with a newly synthesized strand from the nicked position of a dsDNA (Figure 11).
Klenow and Klenow.mut
DNA polymerase I is from E. coli, and the complete enzyme contains two fragments with molecular weights of 76 and 34 kDa. The large fragment, named as Klenow, retains the 5'-to-3' DNA synthesis activity and 3'-to-5' exonuclease activity, but lacks the 5'-to-3' exonuclease activity of the complete enzyme. Since we worried about that the 3'-to-5' exonuclease activity of Klenow could degrade the replaced ssDNA, we used site-directed mutagenesis to generate a Klenow mutant D424A, designated as Klenow.mut, with its exonuclease activity being eliminated.
Purification of Klenow and Klenow.mut
Figure 12. SDS-PAGE analysis of the purified recombinant Klenow and Klenow.mut. | a. 1 and 2. Eluent of His×6-tagged Klenow from Ni-NTA agarose beads. b. 1 and 2. Eluent of His×6-tagged Klenow.mut from Ni-NTA agarose beads.
We successfully induced and purified the Klenow and Klenow.mut proteins (Figure 12).
Klenow-Single strand replacement verification
We tested the ssDNA replacing activity of Klenow by EMSA.
Figure 13. EMSA to verify the ssDNA replacing activity of the Klenow. | 1. With both Nb.BbvCI and Klenow. 2. Without Nb.BbvCI. 3. Without Klenow. 4. FAM-probe alone. 5. Annealed FAM-probe/c-probe.
In the presence of both Nb.BbvCI and Klenow, the binding of the probe with its complementary strand showed a more intensified band than that of the reaction with Nb.BbvCI alone (compare lane 1 versus lane 3 in Figure 13), suggesting that the reaction with both Nb.BbvCI and Klenow produced ssDNA. In the absence of Nb.BbvCI, the hybrid band of the probe and its complementary strand was absent, proving the nickase activity of Nb.BbvCI. Based on these data, we concluded that Klenow possessed ssDNA replacing activity. However, in the reaction using Phi29, we somehow observed relatively weak band of the hybridized probe with its complementary strand in the system with both Nb.BbvCI and Phi29, compared to that with only the Nb.BbvCI (Figure 11). We are still investigating the underlying reason for this unexpected phenomenon.
Phi29 vs Klenow and Klenow.mut
We compared the exonuclease activity of Klenow, Klenow.mut and Phi29.
Figure 14. DNA agarose gel of the FAM-labeled probe after incubated with Klenow, Klenow.mut and Phi29. | 1. Klenow + FAM-probe. 2. Klenow.mut + FAM-probe. 3. Phi29 + FAM-probe. 4. FAM-probe alone. 0.5 µg of each enzyme and 15 pmol of the probe were used.
Based on the figure 14, the Klenow enzyme apparently degraded the probe in different degrees with relatively small and diffused bands, likely due to its exonuclease activity. However, the degree of degradation in the presence of Klenow.mut was markedly reduced, suggesting that the abrogation of its 3'-to-5' exonuclease activity could stabilize ssDNA (Figure 14). Meanwhile, we also detected that the exonuclease activity of Phi29 was significantly higher than that of both Klenow and Klenow.mut. Based on these results, we concluded that the Klenow.mut would be the best choice among the three enzymes in mediating the ssDNA replacement.
As indicated above, we also hoped the DNA polymerase in the ssDNA replacement reaction to be used in the subsequent RCA reaction. Therefore, in the following investigation, we verified the activity of these enzymes in the RCA reaction and evaluated the overall performance when combining the two reactions into a single step.
The RCA is another important application of the polymerase in our design. We would like to identify a DNA polymerase with the strongest RCA-driving activity with a ssDNA template containing G-quadruplex (G4) motifs initiated by our desired primers. In our initial design, we planned to first cyclize the linear template prior to the RCA reaction. This could be achieved by adding a specially designed primer that could anneal or bind to both end of the linear G4-containing template. With the addition of T4 DNA ligase, the 5'-phosphate group and 3'-hydroxyl group of the two ends could ligated to form a phosphodiester bond leading to the formation of a cyclized ssDNA to be used a template in the RCA. However, prior to the subsequent RCA reaction, an additional step would be needed to purify the circular template by removing the primers. Unfortunately, following this experimental design, we observed inextricable high background signal and potential false positive detection. Therefore, we optimized the experimental strategy by directly using a single primer to drive both template cyclization and RCA reaction, which effectively reduced the possibility of false positive results. Based on this design, we verify the RCA ability of Phi29, Klenow and Klenow.mut.
Figure 15. The agarose gel electrophoresis of the RCA results using Phi29, Klenow and Klenow.mut. | Lane 1-4: Klenow, Klenow.mut, Phi29 and negative control. The RCA reaction was conducted for 90 min.
In the RCA experiment, Phi29 showed a robust activity in driving RCA with intensive DNA products present in the loading well or beneath it (Figure 15). However, no detectable product was observed in the RCA reaction using either Klenow or Klenow.mut.
In our detection system, the ssDNA from the replacement step is used as a primer for RCA reaction. Therefore, we combined the two steps by individually adding the replacement products of Phi29 and Klenow enzymes to the RCA reaction, and evaluated the amplified products by DNA agarose gel electrophoresis.
Figure 16. Agarose gel electrophoresis to verify the activity of Phi29 and Klenow in driving ssDNA replacement and RCA reaction in combined reaction systems. | a. 1-3. RCA after BbvCI+Klenow-mediated ssDNA replacement. b. 1-3. RCA after BbvCI+phi29 chain replacement. 1, 2 and 3 indicate increasing amounts of the ssDNA products used in the RCA reaction.
As shown in Figure 16, in the combinatorial reaction of the two steps, Phi29, but not Klenow, could produce significant amplification products. Overall, based on our experimental data, we decided to use Phi29 as the DNA polymerase to drive both ssDNA replacement and RCA reaction in our detection system.
To enhance the specificity and programmability of our detection system, we also planned to use a zinc finger (ZF) protein as an anchoring tool. Through designing a ZF to bind a specific sequence and attaching it to a single strand-cleavage enzyme, we could generate a guided nickase to create a nick at a desirable site. This construction integrated the functions of DNA anchorage and nickase cleavage. As indicated above, BbvCI is a restriction endonuclease with two subunits R1 and R2. First, through linking the R1 subunit and the ZF together, we constructed the BbvCI-R1-ZF fusion protein. Second, we also linked the mutated BbvCI-R2 subunit (i.e., Nb.BbvCI-R2) and Phi29 to generate the Nb.BbvCI-R2-phi29 fusion protein. (BBa_K3894024)
Figure 17. SDS-PAGE analysis of purified recombinant BbvCI-R1-ZF. | 1 and 2. Elutent of His×6-tagged BbvCI-R1-ZF from Ni-NTA agarose beads.
Figure 18. SDS-PAGE analysis of purified recombinant Nb.BbvCI-R2-Phi29. | 1 and 2. Elutent of His×6-tagged Nb.BbvCI-R2-Phi29 from Ni-NTA agarose beads.
We successfully expressed and purified the recombinant BbvCI-R1- ZF and Nb.BbvCI-R2-Phi29 proteins in E. coli (Figures 17 and 18). They will be assembled in vitro to test their combinatorial activity.
G4 chromogenic detection
In the last step of detection, the G-rich DNA sequence produced by the RCA reaction forms an atypical secondary structure, G-quadruplex (G4), using four G-tract strands stabilized by the Hoogsteen hydrogen bond. The G4 DNA can combine with hemin to form a deoxyribozyme with peroxidase-like activity, which catalyzes its substrate 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) to produce a green color, and thus can be used as a biosensor. This is also a key step to realize the visualization of our testing platform. Next, we will show the results of the G-quadruplex sequence amplification and the chromogenic reaction.
Figure 19. Chromogenic reaction of a G-quadruplex sequence. | 1, 2 and 3. G-quadruplex sequence template with final concentrations of 0.2, 0.5 and 1.0 µM. 4. Negative control of reaction without G-quadruplex.
By exploring literature, we learned that many G-rich sequences could form G-quadruplex structures. Based on relevant publications, we chose a validated G-quadruplex sequence with substantial experimental evidence. The results showed that the G-quadruplex in different concentrations and mixed with hemin could catalyze its substrate ABTS to produce products with green color that was discernible from the negative control. The color intensity showed a concentration dependence with the strongest color change at 1.0 µM of G-quadruplex among the three samples (Figure 19). Our data verified the chromogenic effects of the selected G-quadruplex sequence.
Figure 20. Results of G-quadruplex electrophoresis by Phi29-mediated RCA.
Figure 21. Chromogenic reaction of Phi29-mediated RCA products. | 1. Positive control with a synthesized G-quadruplex sequence. 2. Products of Phi29-mediated RCA. 3. Reaction without primers. 4. Reaction without template. 5. Reaction without Phi29 polymerase. 6. Reaction without T4 DNA ligase.
Figure 22. Absorbance at 420 nm of the chromogenic reaction of the Phi29-mediiated RCA products.
We used purified Phi29 polymerase to carry out RCA for the amplification the G-quadruplex template. Based on the DNA agarose electrophoresis, the RCA products could be steadily detected in the experimental group but not in the controls (Figure 20). Meanwhile, the color change could also be observed in the experimental group versus the controls (Figure 21), and their absorbance at 420 nm was also quantified (Figure 22). From the results of these three experiments, we conclude that Phi29 can efficiently amplify the G-quadruplex sequence through the RCA reaction and the amplified product can generate chromogenic reaction.
In order to put our detection device into actual application, we explored the relationship between absorbance and the primer amount according to the detectable color range, and finally determined the required sample size through the modeling. Therefore, we designed a gradient of primer concentrations for RCA and chromogenic reaction, and obtained the following results.
Figure 23. DNA agarose electrophoresis to detect the products of the RCA reaction using a gradient of primer concentrations. | 1. Negative control without any primer. 2-7. 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 µM of primers.
Figure 24. Absorbance at 420 nm of the chromogenic reaction by the RCA products using a gradient of primer concentrations.
The above experiments prove that primer concentration can reach a saturated level of about 0.6 µM in mediating the RCA reaction to promote the subsequent chromogenic reaction (Figure 24), which provides supportive data to our modeling. In the future research to improve our detection system, we will further define the color threshold, optimize the chromogenic reaction system, and increase the detection sensitivity.
In the last part, we wanted to verify the overall detection performance of our assembled and integrated protein complex. In this experiment, we mimicked the actual use environment of the device as much as possible in the following procedure. We first assembled the BbvCI-R1-ZF (BBa_K3894025) and Nb.BbvCI-R2-Phi29 (BBa_K3894024) complex in vitro by incubating their purified recombinant proteins. Second, the assembled protein complex was used to treat the dsDNA containing a BbvCI site to create a nick on it, and then generated a replaced ssDNA in a system containing dNTPs with 1-hour incubation. After that, the produced ssDNA was used as a primer in the subsequent RCA reaction for 90 minutes, followed by the addition of hemin, ABTS and H2O2 for the chromogenic reaction.
Figure 25. DNA agarose electrophoresis of the amplified G-quadruplex-containing sequence by the protein complex of our integrated system.
Figure 26. Chromogenic reaction of the RCA products in by the protein complex of the integrated system. | PC: positive control; NC: negative control; EG: experimental group using the integrated detection system.
As shown in the Figures 25 and 26, our integrated detection system could carry out the sequential reactions to steadily detect the template DNA in the sample and produce chromogenic changes. Therefore, we have successfully verified the overall function of the detection platform that can complete the entire process from sampling to result reporting within 3 hours, with the reactions at 30℃ and the device in an ambient temperature.
Our future efforts will be focused on optimizing the detection system, reducing the detection time, and improving the detection sensitivity.