Overview
The overall reaction is divided into three parts.denoted as reaction A.B.and C.

Reaction A:

        MiRNA will bind to the complementary region in the probe and form a local DNA-RNA hybrid double strand through base complementary pairing.Subsequently,the DSN enzyme will degrade the DNA in the hybrid double strand and break the probe cicular structure.resulting in the release of miRNA in the blood and the unpaired RNA in the probe.The released MiRNA will carry out a new round of reaction,realizing the linear amplification of the signal.Compared to the DNA isothermal amplification process,the reaction process is fast,and the amount of miRNA added can be approximately amplified into the amount of sgRNA in a short time.

Figure 1.1 Reaction A flow diagram

Reaction B:

        sgRNA forms a ternary complex with Cas12a and assistant DNA.which can trans-cleave the prominent structure of inhibit DNA and release the sgRNA sequences.These sgRNAs combine with Cas12a to be activated to carry out a new round of reaction and achieve exponential amplification of the signal.
The reaction process is shown in Fig.1.2.

Figure 1.1 Reaction A flow diagram

Reaction C:

1.

Colloidal gold particles appear purplish red when they are dispersed in the solution.When colloidal gold particles are cross-linked by single-stranded DNA,the distance between colloidal gold particles is shortened,which changes the color of the solution.(Plasma nanoparticles aggregate to a certain extent,and the distance between particles is reduced,the plasma resonance on their surface will be enhanced,and the LSPR absorption summit will move.changing the color of the solution.(Localized Surface Plasmon Resonance,LSPR)
Nucleic acid sequences (SH-5'-AAAAAAAAAATAGCTTATCAGAAAAAAAAA-3'-SH,9nm)connect to the 20 nm colloidal gold particle:

Figure 1.1 Reaction A flow diagram

2.

Cas12a is activated in the first steps of the reaction and has the trans-cleavage abllity,which can cleave the single strand DNA between colloidal gold particles,so that the colloidal gold particles can evenly disperse in the solution.As the amount of activated cas12a gradually increases,the solution gradually returns to its original color.
Activated cas12a with trans-cleavage activity cleaves ssDNA between the gold particles: ssDNA (above 9nm sequence)Different lengths of DNA fragments or single nucleotides

Figure 1.1 Reaction A flow diagram

Reaction Simulation (Mass-action Kinetics)
        The change of the concentration of each compound with time is described by differential equation'.and the whole regulation route is simulated.According to the mass action law,for the reaction
        At a certain temperature,the reaction rate is proportional to the product of the reactant concentration cA. cB power.that is
k is called the rate constant, and at some temperature k is con

For reaction A：

The simplified isothermal amplification model of DSN is shown in Fig.2.1.

Figure 2.1 Reaction A flow diagram

The reaction equation is as follows:

Remarks:

A:miRNA
B:Cicular probe
C:Intermediate chimeric
D:sgRNA containing small fragments
E:sgRNA'
F:sgRNA
The differential equation of the reaction process is described by using the discrete time method and the Mi equation of the enzymes involved in the reaction2 as follows:
Through the reaction kinetics simulation,the relationships of each component and time of the reaction3 are obtained in Fig.2.2.

Figure 2.2 Relationships of each component and time in reaction

(A:miRNA:B:Cicular probe:C:Intermediate chimeric:D:sgRNA containing small fragments;E:sgRNA': F:sgRNA)
The relationship between the amplified sgRNA and time can be obtained as Fig.23.

Figure 2.3 Change of sgRNA concentration with time

Finally,it is concluded that after the reaction of part A,miRNA can basically expand to 9.95 times sgRNA. https://github.com/xyf1041632550/AFMU-China-Reaction The Reaction_A.py scripts can be found on GitHub.

For reaction B:

Cas cutting amplification model is shown in Fig.2.4.

Figure 2.4 Reaction B flow diagram

        The reaction equations and the differential equations that describe the reaction process using the Millis equation4 of the enzymes involved in the reaction are as follows:
For the rate of reaction 1.reaction2,reaction3 and reaction 4.respectively:
For each component：
        Through the reaction kinetics simulation,the https://static.igem.wiki/teams/4778/wiki of each component and time of the reaction are ob- tained as Fig.2.5.

Figure 2.5 Relationships of each component and time in reaction

After the simulation in reaction B,the sgRNA of the input system can be approximately amplified as 1.2210n7.
The relationship between reaction rate and time of each reactio

Figure 2.6 Relationship between reaction rate of each step and

https://github.com/xyf1041632550/AFMU-China-Reaction The Reaction_B.py scripts can be found on GitHub.
Find out optimal reaction time allocation
Ideally,reaction A is a process in which the substrate is linearly amplified.
        x is the substrate concentration and k is the rate constant of the reaction A.
Reaction B is a process in which the substrate is exponentially amplified.
        m is the number of reactions in reaction B
We need to explore how long reaction A and reaction B can separately reach to the maximum expansion increment in a fixed total reaction time.According to the above reaction,the function of substrate with re- spect to time can be obtained.
        q is the reaction constant,T is the total reaction time,and t is the time required for the reaction to occur once
        Since the reaction B and C concur simultaneously by the same Cas protein. are fixed values.Plug the k value of reaction A(obtained from the previous reaction kinetics simulation equation)in the above function,and set the total reaction time as 10 minutes,so that the optimal reaction time alloca- tion scheme can be obtained.

Results

Figure 2.7 Time allocation of reaction A, B and C

        As shown in Fig.2.7.by simulating the above functions in Python,we found that the amplification effect was best when the ratio of reaction time between reaction A and reaction B was 1.2:8.8.For example.if the total reaction time is 10 minutes,the reaction time of reaction A and reaction B should be 72 seconds and 528 seconds respectively.to achieve the best amplification amount.
https://github.com/xyf1041632550/AFMU-China-Reaction The Optimal_time_allocation.py scripts can be found on GitHub.
Prospect
        Due to the limit of time.our study mainly used MiRNA-34a as the biomarker for NAFLD.a representative disease of metabolic syndrome.In our wet lab.the substrate in reaction A increases completely linearly, but with the continuous extension of reaction time,the k value may become smaller,resulting in non-lin- ear growth.When the substrate is replaced,whether the k value is constantly remains to be verified by fur- ther experiments.Similarly.reaction B grew to the power of 2 in this study,and it remains to be verified whether there is a possibility of 1-molecule Cas protein cleaving multiple substrates.Therefore,when the detection target is changed,the optimal duration of reaction A and B should be reassigned.
Reference
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