Design
Introduction
The development of molecular neural networks is mainly based on the principle of artificial neural networks, which realize complex information processing by using chemical or biological materials to build elements similar to neurons, and using chemical reactions or biological processes to simulate the connection and information transmission between neurons.
In order to realize the detection and intelligent analysis of multiple nucleic acid targets, we plan to use the in vitro biochemical circuit of neural network structure ——a single layer of perceptron constructed with DNA probes as building blocks, to achieve accurate diagnosis of diseases.
In practice, we have designed a general platform for detecting nucleic acid markers of disease using the polymerase-mediated strand displacement and toehold-mediated reaction as the core principle. This non-invasive platform has the advantage of accurate judgment and simple usage.
While using it, molecular calculation operations are performed through in vitro detection, weighting, summing and fluorescence reporting of multiple nucleic acid targets，so it can transform multidimensional concentration signals into intuitive low-dimensional results to achieve accurate diagnosis of diseases.
Implementation of the molecular computing function
Screening of targets and the determination of weights
Initially, we selected disease-related data from NCBI. Then, Linear Regression, Support Vector Machine (SVM), Logistic Regression, ElasticNet, LASSO and other algorithms were used to analyze the relationship between the amount of gene expression and the detected diseases. Also, ACC, AUC, ROC and F1 equivalents were evaluated comprehensively to select the target with the best effect and the corresponding weight.
Construction of the weight probes
Polymerase-mediated strand displacement reaction
As shown in the figure, A and A*, B and B* , and C and C*complement each other, respectively. This reaction is similar to the polymerase chain reaction. First, the primer and the partially complementary double-stranded sticky ends of DNA bind together according to the principle of complementary base pairing, that is, A and A* bind together.
Then, the temperature was adjusted to the optimal reaction temperature of DNA polymerase, and the DNA strand was synthesized to replace the DNA complementary to the template 5'-C-B-A-3' and displaced the single-stranded 5'-C*-3' of the partially complementary duplex DNA.
The probe of the target with the weight less than 5 binds the weight strand corresponding to the weight number of the target DNA. In the process of DNA polymerase, the weight strand is displaced by the newly synthesized substrand.
Through this process, the signal of the target is quantitatively converted into the weight strand number. For targets with the weight large than 5, in the process of DNA polymerase, the primary weight chain CODE 1 is replaced by the newly synthesized subchain of DNA polymerase and used as the primer for the secondary weight probe after release, then the polymerase-mediated strand displacement reaction occurs again, and the secondary weight chain CODE 2 is replaced and released, realizing the multiplication of first-level weight and second-level weight.
Therefore, the signal of the target is quantitatively converted into the product of two-level weight multiplication. Additionally, on the weight probe, we will assign a blocker shorter than the target to prevent the suspended 3′ end of the primary weight chain CODE 1 from acting as primers for the secondary weight probe to trigger an unwanted DNA polymerization reaction.
The realization of summation
In the process of strand displacement reaction, the weight strand is replaced and released to the solution subject, and the target with the same weight symbol corresponds to the same weight strand, so the weighted target signal is uniformly converted into the weight strand signal, and the signal will be automatically summed.
The strand displacement reaction mediated by Toehold
The Toehold-mediated strand displacement reaction is a reaction process in which a long single-stranded DNA replaces a short duplex DNA into a thermodynamically more stable long duplex DNA structure through Toehold-mediated three-stranded migration.
DNA strand displacement reactions generally undergo three steps of different intermediate migration reactions. The first reaction phase is the initialization process.
Firstly, the Toehold 3 region of the input signal chain A (Input A) is complementary hybridized to the Toehold 3 * region of complex X (Complex X) to form a substable state duplex product;
Then it enters the second reaction phase (branch migration), in which the recognition region 2 of Input A will gradually replace the hybridization region 2 of the output signal B (Output B) to achieve branch migration;
Finally, the reaction enters the third stage (Output signal), also called the strand replacement reaction stage. In this process, the 2 region of Output B is completely replaced by the 2 region of Input A, and a new hybrid double-stranded product Y (Complex Y) and Output B are generated.
Throughout the DNA strand displacement reaction, the sticky terminal region is usually composed of 4 to 6 base sequences. The binding strength of the sticky end can be regulated by changing the base composition or base number of the sticky terminal region, thus moving the reaction in a dynamically controlled direction, finally achieving a six order of magnitude change in the reaction rate constant 1.
In the absence of the target nucleic acid, the reporter probe composed of short chain ssDNA modified by fluorescent molecule 6 FAM (18 nt) and longer chain ssDNA (7 + 18 nt) modified by quenching molecule BHQ 1 forms a stable double-stranded structure, which narrowed the distance between the fluorescent molecule and the quenched molecule, resulting in fluorescence resonance energy transfer (FRET) and fluorescence quenching;
When the target appears in solution, the weight strand produced by the polymerase-mediated strand displacement is hybridized to the double-stranded DNA long strands by the toehold-mediated strand displacement reaction, thus forming a thermodynamically more stable double-stranded structure while competing the DNA short strands down to achieve fluorescence recovery.In the previous step, the free weight strand signal is converted into the fluorescence signal in a 1:1 ratio.
- Wang Huizhen. Tumor exosome detection study based on DNA nanoprobe and Toehold-mediated strand replacement reaction [D]. Hunan University, 2021.DOI:10.27135/d.cnki.ghudu. 2021.004388.↩