1.In the process of DNAzyme detection, the products after cutting single chain of the substrate are closely related to the amount of reaction. However, in the application scene, the cutting effect and products will not be significant. Therefore, we need to introduce the design of signal amplification systems.
Based on this principle, we selected a total of 4 signal amplification systems that can respond to single strand DNA (or SINGLE strand RNA).
Fig. 2.3.1 HTDC mechanism.
The specific reaction process of HTDC is shown in the figure above. When the heavy metal ions were not binded to DNAzyme, the Substrate chain was not cut by the DNAzyme structure, and the fluorescence of FAM was inhibited by BHQ-1 fluorescence quencher. When the heavy metal ions were combined with DNAzyme, the substrate strand was cut open and FAM was not inhibited, enabling fluorescence signals to be detected by the instrument.Meanwhile, in HTDC technology, only the combination of substrate chain, enzyme chain and target can achieve complete DNAzyme function, that is, to achieve the cutting cycle of the substrate chain. If only the substrate strand and enzyme strand bind (S-D complex), as the S-D complex has an unstable structure and cannot react with heavy metal ions, cycle cannot be achieved.
Function of HTDC technology in our project
Fig. 2.3.2 HTDC design. (A) HTDC structure. (B) HTDC sequence.
HTDC originally contains DNAzyme and can generate more signals through cycle, so we need to test its use effect. If it cannot meet the demand, we need to consider abrupt optimization or use it only as a signal amplification system.
To apply target to this experiment, we added the DNAzyme, substrate strand and the target strand together to form a S-D complex which enters a cycle that combines with heavy metal ions and reacts to continuously generate fluorescence signals.
Fig. 2.3.3 target detection limit testing
In order to get the lower limit of target solution, we diluted it into different concentrations: 50nM, 40nM, 30nM, 20nM, 10nM, 5nM and 1nM. Eventually we came to the conclusion that 5nM as the lower limit for target solution. The reason for detecting target lower limit was because that we have to determine what is the lowest concentration of target required to turn on HTDC reaction.
Fig. 2.3.4 HTDC Pb2+ concentration testing
HTDC has the capability to detect numerous heavy metal ions and for this experiment he detection limit we found for Pb2+ is 0.01 μM.
The HTDC system worked in great efficiency and accuracy in detecting heavy metal ions. With the 8-17 DNAzyme already incorporated into the structure, HTDC would serve as a successful signal amplification system for our project.
Fig. 2.3.5 TO-DNA mechanism
TO-DNA is a KIND of DNA with a topological THREE-DIMENSIONAL structure, which is composed of the T7 Promoter and the encoding region. In the initial state, the to-DNA remains off. Because T7 Promoter is folded, the T7 polymerase does not bind to it and transcription does not start. Note: Promoters are DNA sequences that are recognized and bound by RNA polymerase and play a role in initiating transcription.
To start transcription, we add a key Strand (a DNA sequence). When the Key strand binds TO the T7 Promoter, its topological structure will be destroyed and the TO-DNA will be switched on. The T7 polymerase binds TO the T7 Promoter and transcription starts.
Function of TO-DNA in our project
Fig. 2.3.6 TO-DNA design. (A) TO-DNA structure. (B) TO-DNA sequence.
Application of to-dna T7 Promoter technique :(it is highly similar TO the Toehold Switch technique): TO apply the TO-DNA T7 Promoter in this experiment, we add key strand at the end of the Substrate strand. At the same time, we add a sequence that is complementary TO the key strand at the end of the enzyme strand TO prevent the key strand from binding TO the TO-DNA T7 Promoter in advance due TO the lack of sequence that can be paired.
Key strand is added before Substrate strand is cut; When the Substrate strand is cut, the part containing the key strand binds TO the TO-DNA T7 Promoter, so that the coding region is transcribed and fluorescent RNA is formed. The fluorescent RNA reacts with the added compound (DFHBI) to produce a fluorescent signal.
Fig. 2.3.7 key strand detection limit testing.
For our first experiment on TO-DNA, we test the working efficiency under different key strand concentrations and we eventually came to the result of 1nM lower limit.
Fig. 2.3.8 Key strand further testing. (A) Key strand 1*-10 nt structure. (B) Key strand 1* testing.
However, with further testing on TO-DNA, we realized a major problem in the system. The TO-DNA sequence is too long to be paired with the substrate strand and this difference in length would result in an early reaction before the presence of heavy metal ions. (We synthesized complementary sequence to imitate the DNAzyme and TO-DNA combination testings)
In conclusion, we decided to eliminate TO-DNA from our signal amplification system because its reaction might start even before the substrate is cleaved. The key strand will combine with TO-DNA before cleavage.
Fig. 2.3.9 CRISPR-Cas12a mechanism
Cas12a is a Cas protein that, like the traditional Cas9 protein, is guided by GUIDE RNA (gRNA) to cut single - or double-stranded DNA. However, Cas12a also has properties different from Cas9.
First, Cas9 needs to be guided by two RNAs (crRNA and tracrRNA) to cut, while Cas12a needs to be guided by only one RNA (crRNA) to cut. Second, both Cas9 and Cas12a need to recognize PAM (protospacer-motif) sequences to cut double-stranded DNA, but Cas9 corresponds to G-rich PAM sequences, and Cas12a to T-rich PAM sequences. Third, the cutting site of Cas9 is close to the PAM sequence, while the cutting site of Cas12a is far away from the PAM sequence.
Function of Cas12a in our project
Fig. 2.3.10 CRISPR-Cas12a design. (A) CRISPR-Cas12a structure. (B) CRISPR-Cas12a sequence.
1.DNAzyme combines with heavy metal ions to cut substrate Strand.
2.After cutting, a segment of substrate Strand, as target DNA, combines with binary complex (Cas12a+crRNA) to form a ternary complex, showing the activity of trans cutting.
3.Add a large number of single-stranded DNA with fluorescent groups and quenched groups, which are cut by ternary complex to produce fluorescence signal.
4.Cas combines with double chain, but in the technology of DNAzyme, single chain is required. In combination with double chains, we can express fluorescent signal without cutting DNAzyme, so we need single chain to cut DNAzyme.
Fig. 2.3.11 ssDNA testing:(A) ssDNA-11nt testing.(B) ssDNA-13nt testing.(C) ssDNA-15nt testing.
For our first experiment of Cas12a, we chose to select a fitting sequence length to be paired with DNAzyme: screening between 15nt, 13nt and 11nt. Eventually, we came to the conclusion of 15nt because it has highest efficiency and lowest detection limit of 50nM.
Fig. 2.3.12 ssDNA further testing
With further testing on Cas12a, we have proved that dsDNA would not trigger the Cas12a reaction and therefore early reaction is not likely to occur. When incorporated with DNAzyme, we noticed Cas12a's outstanding working efficiency among all the signal amplification systems. As a result, this would be another potential amplification system for the project.
After rounds of testing on Cas12a, we have proven its working efficiency with DNAzyme. The experiments were very successful and we have decided to choose Cas12a as one of our potential signal amplification systems.
Fig. 2.3.13 Toehold Switch mechanism
In the initial state, the Toehold Switch remains off because the Switch RNA contains hairpin structures. Hairpin structure is a structure that forms a closed loop at one end when a single strand of DNA /RNA self-folds and some of its regions complement each other with bases. The sequence in the ring region cannot be paired, so ribosomes cannot bind to RBS (ribosomal binding site), and translation cannot begin.
To start translation, we add trigger RNA. The A 'and B' sites on the trigger RNA are paired with and bound to the A and B sites on the Switch RNA, making the hairpin structure become normal. The Toehold Switch entered the open state, the ribosome was combined with RBS, and the translation began.
At the same time, we will use the Cell Free System to make up for the defects of traditional Cell experiments (such as restricted experimental environment, complicated experimental process and restricted experimental products), and provide materials (such as various enzymes) for the translation process.
Note: The principle of cell-free systems: Destroy bacteria (e. coli), retain the inclusions, and place them in the Toehold Switch as translation materials.
Function of Toehold-Switch in our project:
Fig. 2.3.14 Toehold Switch design. (A) Toehold Switch structure. (B) Toehold Switch sequence
In order to apply Toehold Switch to this experiment, we added Trigger DNA at the end of substrate strand (Trigger RNA was not used in this experiment, because of its high cost and instability).
1.Heavy metal ions will bond to the middle part for reaction. In order not to affect this reaction, we chose to add trigger DNA at the end.
2.Heavy metal ions bind to enzyme strand for reaction. In order not to affect this reaction, we choose to add trigger DNA in substrate strand.
3.At the same time, we add a sequence that is complementary to trigger DNA at the end of enzyme strand to prevent trigger DNA from binding to Toehold Switch in advance due to the lack of sequence that can be matched.
4.Before cutting Substrate strand, trigger DNA was added. After cutting the Substrate strand, parts containing trigger DNA are combined with the Toehold Switch. We replaced the original sequence of Toehold Switch with lacZ sequence to express it and produce lacZ enzyme. The lacZ enzyme reacts with the added compound (X-Gal) to produce a fluorescence signal.
Fig. 2.3.15 Trigger DNA testing
For the first experiment, we tested the working efficiency of Trigger DNA in the toehold switch system. However the Trigger DNA showed no rising trend except for the positive control and therefore the result didn't reach our expectations. We believe it might not be suitable for Toehold Switch to combine with the purchased cell-free system.
Fig. Trigger DNA further testing
After the previous experiment on Trigger DNA, we redesigned the DNA sequence and performed the same experiment again. Unfortunately, there was still no rising trend on the graph and we believed that the
Eventually, we did not choose Toehold Switch for our signal amplification system. The testing results are unreliable and the fluorescence signal was not shown at all. However, we do believe that our experiment results would set as a background knowledge on future experiments.
Signal Amplification System Conclusion
Out of the four signal amplification systems that we chose to incorporate with the function of DNAzyme, HTDC and CRISPR-Cas12a proved to be efficient and successful. HTDC incorporated with 8-17 DNAzyme in its structure has a lower limit of 5nM for detection and repeated experiments have also proved its high accuracy in identifying heavy metal ions. CRISPR-Cas12a has a detecting lower limit of 50nM and with its outstanding working efficiency. Cas12a is the major signal amplification system that our team eventually decided to focus on. However, regarding TO-DNA and Toehold-Switch. These two systems were incapable of detecting heavy metal ions when combined with DNAzyme. Due to the TO-DNA incompatibility with the DNAzyme sequence, the system could not provide an accurate detection of heavy metal ions. The key strand will combine with TO-DNA before cleavage. Through repeated experiments with the Toehold Switch system, the results showed no detection of heavy metal ions and we believed that the system design itself experienced issues when combined with the DNAzyme. As a result, we eventually decided to put our attention towards HTDC and CRISPR-Cas12a systems for signal amplification. But we do hope that experiment data for TO-DNA and Toehold-Switch would help with future researches.
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