DNA amplification circuits are an innovative technology that has emerged in recent years in the fields of molecular biology and biocomputing, with the core idea of efficiently amplifying weak molecular signals by exploiting the self-replicating and amplifying properties of biomolecules. The technology creates a controlled amplification strand reaction in vitro through a series of designed DNA sequences and enzymatic reactions. Its advantage lies in its ability to complete isothermal nucleic acid amplification reactions without the need to design primers and thermal cyclers, and it is characterized by easy operation, rapid reaction and high sensitivity, which can provide faster and more accurate results, and at the same time, it is suitable for a wide range of applications in the biomedical field. Therefore, we would like to develop a polymerase-based self-enhanced signal amplification system for disease diagnosis.

Two types of cancer, ovarian cancer and lung adenocarcinoma, usually have no obvious symptoms in the early stages. According to 2024 cancer statistics, lung cancer, with an incidence rate of 12.4% and a mortality rate of 18.7% in 2022, is the most common cancer and also the leading cause of cancer deaths; ovarian cancer, with an incidence rate of 1.6% and a mortality rate of 2.6%, is a common female cancer. Many patients are already in the advanced stages of the disease when diagnosed, missing the best time for treatment, and the five-year survival rate of lung cancer is often less than 20%. Through early diagnosis, lesions can be detected before the cancer spreads, thus improving the cure rate and long-term survival of patients. Secondly, early diagnosis can dramatically reduce the complexity and cost of treatment, patients do not have to undergo intense chemotherapy or surgery, and their quality of life is significantly improved. In addition, early detection of cancer can also reduce the risk of recurrence and metastasis and prolong the disease-free survival of patients. Overall, early diagnosis not only saves more lives, but also reduces the economic burden on patients and society, with far-reaching implications for public health.

Therefore, we constructed a diagnostic platform based on polymerase self-enhanced signal amplification system and strand replacement reporter system for the early diagnosis of ovarian cancer and non-small cell lung cancer in Engineering Cycle 1.

Target screening: We performed correlation analysis, expression analysis and machine learning validation of microRNA gene expression profiles of ovarian cancer and non-small cell lung cancer in the NCBI database, and screened four targets:

We provided the designed DNA sequence to the gene synthesis company. After obtaining the ssDNA synthesized by the company, we completed the self-assembly of the weight probe and the fluorescent reporter probe by mixing the various molecules together and then slowly cooling them down:

1. strand Displacement Probe
2. DDSD Probe
3. Fluorescent Reporting Probes

We used the PCR instrument to complete the cooling process, and the procedure of temperature reduction was as follows:

At the end of the probe synthesis, we used electrophoresis to verify the construction of the weight probes.

The electrophoresis results showed a successful synthesis of all the weight probes.

Verify the feasibility of the primary amplification system:

This figure shows that the reaction process can only be completed in the presence of Rep, Target, Bst, DDSD, F, RP to finally generate the report probe RP, that is, to realize the first level of signal amplification.

In order to verify that our system is able to produce a quantitative response to the target, we set up a concentration gradient of the target in the solution, and completed the verification by examining the fluorescence curve produced by the reaction:

As shown in the figure our fluorescence curves can be well distinguished under different target concentrations, i.e., the primary amplification system can realize the quantitative response to the target.

During this engineering cycle, we have identified a number of areas for improvement to further refine our diagnostic platform:

1. Limited by the magnification of the current level of amplification, the sensitivity of the current detection, the intensity of the signal is still poor, so we would like to introduce a new level of amplification of the reaction to achieve a more accurate early diagnosis.
2. Currently, the means we use to detect fluorescence signals is to use a qpcr instrument (non-qpcr reaction) to capture fluorescence signals and draw curves, so the accuracy is also limited by the instrument, so we also need to detect fluorescence methods and tools with higher accuracy.
3. Because we want to realize a highly reliable and translatable diagnostic platform, it is not enough to use synthetic targets in the selection of test samples, so we need to further use tissue and blood samples for validation.

The CRISPR system is a gene editing technology that centers on the enzyme Cas and its guide RNA (crRNA). However, in addition to gene editing, the CRISPR system has also been developed to detect specific nucleic acid sequences. This detection technology relies on the trans-cleaving activity of the Cas protein: when activated, the Cas protein's trans-cleaving activity cleaves probes containing fluorescent labels in the system, leading to the generation and amplification of fluorescent signals.

The CRISPR assay is particularly noteworthy for its speed and sensitivity, which make it stand out from other assays. Its significant advantage is that it is easy to operate and does not rely on complex instrumentation, a feature that makes it particularly suitable for rapid screening and diagnosis in field environments, revolutionizing the field of immediate medical testing, food safety monitoring, and environmental pathogen detection.

Therefore, we introduced the CRISPR system into the polymerase-based self-enhanced signal amplification system and designed a secondary signal amplification system, aiming to achieve higher sensitivity detection. As shown in the figure, this system utilizes a specific miRNA that binds to the rep strand and then generates a wt strand through two strand displacement reactions, which in turn activates Casase together with the crRNA. the non-specific DNA endonuclease paracrine activity of Casase further cleaves the reporter probe with fluorescent and quenching groups, leading to the restoration and enhancement of fluorescence signals, thus realizing the amplification of the signals.

In order to ensure that the diagnostic system is effective, we need to adopt a rigorous preparation process: washing, silanization and sealing steps ensure that the slide surface is clean and suitable for fixation and observation of the sample. The silanization step is particularly critical, as it adsorbs fluorescent signals by forming a layer of reactive silane, which is essential for the observation of sheared fluorescent markers. This adsorption enhances signal capture and visualization. The sealing film then protects the sample from the external environment, ensuring stability and observability of the sample.

After the preparation of the film, we placed the sample under a single-molecule fluorescence super-resolution microscope for observation and analysis. The core principle of single-molecule fluorescence microscopy, which recognizes fluorescent dots in the sample solution on the slide, is to utilize the photoswitching properties of fluorescent molecules to achieve super-resolution imaging. Specifically, fluorescent groups can briefly fluoresce strongly (bright state) when excited by laser light at a specific wavelength, and then rapidly switch back to a stable dark state. By controlling the transition of fluorescent molecules between the fluorescent and non-fluorescent states, temporal sparse excitation is achieved so that only a small number of fluorescent molecules emit light at the same moment. These glowing fluorescent molecules can be spatially resolved, and by accurately localizing the position of each molecule, this localization information is accumulated to ultimately construct a super-resolution image that exceeds the limiting resolution of conventional diffraction.

For the acquired super-resolution images, we first imported multi-frame .tiff images using the tifffile library and converted them to NumPy arrays for storage. Next, bright spot detection is performed, which includes normalizing the pixel values, calculating the mean and standard deviation of the image, setting a threshold, and identifying the local brightest region as a bright spot using a non-maximal value suppression algorithm. After the detection is complete, we label these bright spots in the image and count them.

To further improve the accuracy of the detection, we train the experimental and control group images by adjusting the confidence intervals. This process involves cyclically comparing the difference in the number of bright spots under different thresholds to determine the optimal threshold for accurate single-molecule detection and analysis.

microRNAs (miRNAs), as a class of small-molecule non-coding RNAs, are expressed at abnormal levels in a wide range of diseases, especially cancer. They can be used as biomarkers of diseases to help physicians for early diagnosis and treatment monitoring. Therefore, in the wet-lab phase of this project, we extracted miRNAs from ovarian and lung cancer cell samples (A549, H157) as well as samples from cancer patients' tissues and blood for early diagnosis of cancer, respectively. The specific operations are as follows:

Sample preparation

Tissue samples: remove from liquid nitrogen and grind into powder then add appropriate amount of lysate MZ for homogenization;

Cell samples: can be lysed directly by adding lysate MZ, or collected by centrifugation and then added, oscillated or pipetted to mix, avoid washing in the process to prevent miRNA degradation;

Blood samples: separate the serum or plasma first, then add the lysis solution to oscillate and mix.

The homogenized samples were allowed to stand in order to promote the separation of nucleic acid-protein complexes in preparation for subsequent miRNA extraction. miRNA isolation, purification and decontamination:

The aqueous and organic phases were separated by centrifugation and chloroform shaking to remove protein and lipid impurities;

Subsequently anhydrous ethanol precipitation and purification of miRNA with the help of adsorption column miRspin were performed to further remove residual proteins and salts;

The adsorption column was treated with deproteinizing solution MRD and rinsing solution RW to ensure high purity miRNA;

Finally, the purified miRNA was eluted and collected from the adsorption column, and its concentration and purity were determined using NanoDrop to ensure that the experimental requirements were met.

Rack synthesis

Just like engineering cycle 1, we provide the designed DNA sequences to gene synthesis companies, and after obtaining the synthesized ssDNA, we mix the various molecules in proportion and then slowly cool down to complete the self-assembly of the weight probe and fluorescence reporter probe.

Preparation

In order to provide a standardized reaction environment for the secondary amplification system and to facilitate observation under a single-molecule fluorescence super-resolution microscope after completion of the reaction, we took the following steps for preparation:

Slide cleaning:

The slides were submerged with KOH solution and sonicated to remove some of the impurities on the slide surface, and washed with deionized water to wash away the residual KOH;

The slides were submerged in chromatography grade methanol and sonicated to remove grease and other organic contaminants from the slide surface, and acetone washed to remove residual methanol.

Silanization of slides:

Slides were submerged in a mixture of acetone: APTES = 49:1, sonicated to remove light, then washed with deionized water and blown dry under nitrogen.

Slide sealing:

Deionized water submerged the bottom of the empty gun nose box and placed the slide on its holder.

Cut about 0.5cm of the end of the tip of the gun, set the mouth upward, the outer wide mouth dipped full of A glue: B glue = 1:1 mixture of glue and paste it on the slide, wait for the glue to solidify and then drop the sample.

Verify the feasibility of the secondary amplification system:

This figure shows that non-specific shearing of the reporter probe, i.e., secondary amplification of the signal, can only be accomplished in the presence of wt, cas12A, crRNA, and RP all together.

In order to verify that our system is able to produce a quantitative response to the target, we set up a concentration gradient of the target in the solution, and completed the verification by examining the fluorescence curve produced by the reaction:

We validated our system using 20 clinical tissue samples. The results show that the system is sensitive enough to detect targets at very low concentrations (9.3 pM) and fast enough that the whole process takes only 10 minutes. The mean values of the cancer samples and normal samples are shown in the figure, showing significant differences with p-values of less than one in a thousand. The ROC curve in the figure has an AUC value of 0.96, showing very high diagnostic accuracy. This is a fluorescence plot taken continuously with our single molecule platform.

During this engineering cycle, we have gained valuable insights that will guide the further development of our polymerase self-enhanced binding CRISPR system:

1. Clinical sample validation: Initial testing on clinical samples has shown promise, but more validation is needed. We plan to expand sample size and diversity, such as using blood and other assays to better assess the diagnostic accuracy and reliability of our platform in the real world.
2. Target screening: The TCGA database we are currently using has limited samples, resulting in some of the microRNA expression up-regulation/down-regulation information in the results being different from those reported in other literature, we should subsequently continue to expand the sample set and optimize our model to improve the credibility.
3. Kinetic simulation: We have only validated the reaction process on basic reaction kinetics, and in the future, we can incorporate better tools such as molecular simulation to fully ensure that our reaction proceeds as desired.
4. Signal amplification: In the first level of signal amplification, we used the polymerase self-amplification reaction, in addition to other reactions that can be used to incorporate the CRISPR reaction, such as RCA, RPA, PER and so on.
5. Diagnostic scope: In fact, our diagnostic platform can be used not only for early diagnosis, but also for determining the extent of the disease, such as cancer staging, as long as there is a corresponding well-labeled database, and it can also be used for postoperative testing.

In conclusion, these will guide us to improve and optimize the polymerase self-enhancement coupled with CRISPR system in the future, which will bring us closer to building a powerful and reliable early diagnostic platform.