Due to the biohazard potential of infectious viruses, such as SARS-CoV-2, and their nucleotide sequences, it is rational to avoid using any of the viruses or their sequences in our research project. Therefore, we want to establish the detection system using the M13 phage geneIII to test the concept. The detection signal can be accelerated through the recombinase polymerase amplification (RPA) technology that specifically and exponentially amplifies the geneIII. Then, the resultant double-stranded DNA forms a specifically nicked site under the targeted nicking system. A special DNA polymerase continues to synthesize nucleotides in 5'-to-3' direction from the nicked site, thus replacing a short single-stranded DNA (ssDNA), which previously hybridizes its complementary strand in the double strand DNA.
The displaced ssDNA enters the rolling circle amplification (RCA) as a primer. The template for the RCA is a complementary sequence that also contains G-quadruplex (G4) motifs. Multiple G4 motifs produce through the RCA bind hemin that acts as an activated peroxidase to oxidize a substrate, 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS). The color's change in the oxidation process can be visualized very quickly, which provides an instant detection of a virus.
Recombinase polymerase amplification (RPA) is an isothermal amplification method based on special enzymes. The RPA system contains three core components, including a recombinase, a single-stranded DNA binding protein and a DNA polymerase . The recombinase binds to a primer to form a nucleoprotein complex that scans double-stranded DNA and facilitate strand exchange at its cognate sites. The resulting D-loop structure is stabilized by the single-stranded DNA binding protein interacting with the displaced template strand. Then, the recombinase disassembles and leaves the primer. The DNA polymerase synthesizes the complementary strand by adding nucleotides to the 3'-end of the primer.
We designed a pair of primers specific to the geneIII of M13 phage. If the tested sample contains the sequence of the geneIII, a specific and exponential replication can be initiated to achieve signal amplification using our detecting system.
In order to create nicks at the specific sites of double-stranded DNA, we explored different strategies and were inspired by the development of gene editing techniques. Three generations of nucleases for targeted gene editing have been developed, including the zinc-finger nucleases (ZFNs), the transcriptional activation-like effector nuclease (TALEN) and the CRISPR/Cas9 system [2-3].
We chose both ZFNs and CRISPR/Cas9 systems to make parallel and comparative designing and studies, to find an optimal targeted nicking system.
In the CRISPR/Cas9 system, Cas9 undergoes conformational changes upon sgRNA binding, and is subsequently directed to its target site. Then, Cas9 introduces a double strand break (DSB) in the DNA sequence of the target site . Cas9 has two nuclease domains: the HNH and RuvC-like domains. The Cas9 HNH nuclease domain cleaves the complementary strand, while the Cas9 RuvC-like domain cleaves the template strand . In our project, we created a HNH nuclease activity-null mutant (H840A), designated as nCas9, which can be used to generate DNA break in the template strand.
The restriction endonuclease BbvCI from Bacillus brevis recognizes a non-palindromic sequence CC^TCAGC/GC^TGAGG. BbvCI consists of two heterogeneous subunits, R1 and R2 . The R1 subunit cleaves the bottom chain (GC^TGAGG, where ^ indicating the cleavage site), while the R2 subunit cuts the top chain (CC^TCAGC). The inactivating mutations of R1 or R2 do not disrupt protein folding and DNA sequence recognition of BbvCI .
In our project, we used the R1+R2- mutant, named as Nb.BbvCI. In addition, we explored the Zinc Finger Tools (URL http://www.zincfingertools.org.) created by Jeffrey G. Mandell et al . Using the experimentally characterized database of zinc finger domains, the expected amino acid sequence of a zinc finger protein bound to the selected target can be generated. The zinc finger protein can meet the needs of specific identification to a certain extent.
Single-stranded DNA Displacing
For the purpose to obtain ssDNA, we added Phi29 DNA polymerase to the system, which is an enzyme widely used in RCA. Phi29 catalyzes 5'-to-3' polymerization and possesses strand displacement activity, which enables it to displace the complementary strand in double-stranded regions during DNA synthesis. In our design, Phi29 continues to add nucleotides from the nick site. During this elongation process, the complementary strand behind the gap at 3'-side can be displaced. The resultant short ssDNA will act as a primer in the RCA reaction.
After extensively exploring literature, we found that the 3'-to-5' ssDNA exonucleolytic activity of Phi29 could adversely affect the results by potentially degrading the displaced ssDNA. Therefore, we decided to test Klenow, the large fragment of DNA polymerase I, in comparison to Phi29, and evaluate the efficiency of the ssDNA production.
Rolling circle amplification (RCA) offers a simple method for DNA amplification, which is an isothermal DNA amplification without a thermal cycling reactor. Thus, it is more suitable to field-test than the methods depending on a thermo cycler .
The displaced ssDNA, complementary to the template sequence, can bridge the nicked site between the 5'- and 3'-termini of the template strand through base pairing. With the help of this displaced ssDNA, the nick at the template can be sealed by adding T4 DNA ligase, which catalyzes phosphodiester bond formation between juxtaposed 5'-phosphate and 3'-hydroxyl termini.
We can skip the primer removal and recombination process, and directly put the ssDNA-template hybrid mix for RCA, which simplifies the procedure and saves possible extra cost of ssDNA-specific exonuclease, such as exonuclease I..
In the RCA reaction, Phi29 continues to synthesize ssDNA from the ssDNA (as a primer) with a high processivity and strong DNA strand displacement activity. The resultant long chain ssDNA is composed of the tandem repeat of G-tract (G ≥ 3) sequences, which can form G-quadruplex needed in the following test. We also used Klenow to replace the role of Phi29 in RCA and evaluated whether it could possibly improve the amplification yield.
G4 is a highly stable DNA secondary structure formed by single-stranded G-rich nucleic acids. This structure can associate with a cofactor, hemin, to produce catalytic activity similar to that of a peroxidase, which is thereby designated as deoxygenase (DNAzyme). We used ABTS as a substrate to analyze the G4 levels produced in the RCA reaction. In the presence of H2O2, the ABTS2- is oxidized by the complex to produce the colored radical anion (ABTS·-). The visible color change can be further quantified in a spectrophotometer at the wavelength of 420nm.
In practical application, RNA is easy to be degraded and its preparation process is relatively difficult. Besides, we discovered that the nick-generating activity of Nb.BbvCI is better than that of nCas9. Therefore, we decided to use ZF-Nb.BbvCI in our detection system. Meanwhile, after comprehensively analyzing the capability of the single chain displacement and RCA using Phi29 and Klenow, we chose the former one to be used in our system.[Results] Furthermore, we generated two fusion proteins by connecting the R1 and R2 domains of Nb.BbvCI to the ZF and Phi29, respectively. This allowed us to produce their quaternary protein complex through in vitro assembly, which could generate an all-in-one reaction module with combined nick-creating, displacement and amplification.
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