Recently, Professor Wang Pengfei and his team at Shanghai Jiao Tong University designed a new DNAzyme sensor that does not require preamplification and named it SPOT.
图 | 王鹏飞（来源：王鹏飞）
For a variety of clinically significant nucleic acid markers represented by miRNA and viral RNA, this sensor has the ability to detect and realize rapid and convenient clinical molecular diagnostic applications.
Sensitive and specific detection of serum miRNAs can be used for molecular diagnostics of a wide range of cancers, including breast, gastric, and prostate cancers.
In addition, SPOT provides sensitive and accurate detection of SARS-CoV-2 RNA from clinical swabs. At the same time, SPOT can be combined with lateral flow flow technology to achieve point-of-care testing (POCT) detection of nucleic acid targets.
(来源:Applied Chemistry International Edition)
Because SPOT is built entirely from synthetic DNA molecules, it avoids the need for expensive protein enzymes and is cost-effective.
Experiments have shown that the cost of materials for a single SPOT assay is as low as approximately $0.02, which is significantly lower than existing detection systems.
This sensitive and specific analytical performance, one-step protocol, low cost, and POCT capabilities make SPOT a better assay than existing DNase-based or CRISPR-based assays for nucleic acid targets.
In the future, in the field of laboratory medicine, the team hopes to use the SPOT system to detect circulating nucleic acids in vivo to achieve sensitive and specific molecular diagnosis of multiple types of diseases, so as to provide new possibilities for early diagnosis and precision treatment of diseases.
Further, they also hope that the combination of aptamers can be used to detect small molecules and proteins, expanding the clinical diagnostic applications of the SPOT system in many aspects.
Previously, traditional DNAzyme did not have programmable targeting for in vivo delivery of therapeutics. The new DNAzyme system designed by the team can quickly design and implement programmable target strands and cleave them to achieve therapeutic targets.
Overall, this innovation is expected to provide more precise and effective means for disease treatment, and bring important impetus to the development of gene therapy and precision medicine.
(来源:Applied Chemistry International Edition)
So, what is the need for the development of this result, and what does it make up for in the existing measurement tools?
According to reports, nucleic acid molecules present in biological fluids (blood, urine, sweat, etc.) are a class of key biomarkers for molecular diagnosis of major diseases, including cancer and viral infections.
However, nucleic acid markers have the characteristics of low abundance, high dynamics, high heterogeneity, and large background interference, and their clinical detection faces challenges such as undetectable, inaccurate, incomplete, undetectable, and undetectable.
Therefore, there is an urgent need to develop ultra-sensitive, highly specific, high-throughput, convenient and economical nucleic acid biomarker detection and analysis methods.
DNAzymes are a class of in vitro-screened synthetic DNA molecules with enzyme-like catalytic activity, such as cleavage of nucleic acid phosphodiester bonds.
A typical cleavaged nucleic acid DNAzyme consists of a catalytic core with catalytic capabilities and two arms for substrate recognition through sequence complementarity.
As a result, many DNAzymes are now widely used for metal ion detection in vitro and in vivo because their catalytic activity is highly dependent on metal ions.
However, DNAzyme with small molecule, protein, or nucleic acid detection capabilities is rarely reported, making these targets difficult to detect.
Inspired by natural enzymes and nucleases, heterogeneous DNAzyme biosensors are designed by implementing heterogeneous modules (e.g., aptamer, toehold) to mediate their catalytic activity through regulators such as small molecules, proteins, nucleic acids, or bacteria.
Traditional heterogeneous DNAzyme biosensors are often designed as multi-component molecular complexes that inhibit and release DNAzyme through a toehold inhibitory strand.
and direct detection of nucleic acid targets by splitting and recovering the catalytic core after target binding, or by target-induced stabilization of DNAzyme-substrate-target complexes.
However, these biosensing systems typically exhibit picomolar to nanomolar sensitivity for direct detection of directed nucleic acids, and therefore cannot detect miRNA or viral RNA markers in clinical samples.
While many miRNAs are considered to be potential biomarkers for a variety of cancers, few have been shown to be clinically useful due to the lack of sensitivity and specificity for cancer diagnosis.
Wang Pengfei believes that this multi-component design may have a negative impact on its detection capabilities.
Low-abundance targets are more difficult to measure due to reasons such as imperfect stoichiometry, kinetic molecular traps, impaired catalytic activity, and increased risk of signal leakage.
The main research interest of the research group is to develop simple, fast and sensitive new molecular diagnostic methods for diseases.
Through their research, the team found that circulating nucleic acids in body fluids have become important biomarkers for liquid biopsy for a variety of diseases.
Due to the high dynamics, heterogeneity, and low abundance of these nucleic acid biomarkers in biological specimens, their clinical detection is a great challenge.
Traditional polymerase chain reaction (PCR) techniques require rigorous sample handling, expensive instrumentation, and specialized handling.
而新型等温扩增方法，比如重组酶聚合酶扩增（RPA，Recombinase Polymerase Amplification）、环介导等温扩增（LAMP，Loop-mediated isothermal amplification）等则能简化操作过程。
However, while CRISPR in combination with isothermal amplification technology has shown high sensitivity and convenience, it has drawbacks such as non-specific amplification, sequential procedures, and the need for expensive and vulnerable enzymes that limit its clinical application.
Through the consideration of the above factors and the advantages of the research group, they decided on this topic.
(来源:Applied Chemistry International Edition)
Through theoretical simulation and design, they designed the new DNAzyme sensor, SPOT. In order to clarify the mechanism of SPOT and optimize the experimental parameters, the research group studied the specific activation mode and the length of the nucleic acid target that can be applied.
Due to the nature of the DNAzyme sensor, the original DNAzyme sensor was designed with severe signal leakage, and no matter how much the experimental conditions were optimized, it could not achieve stable and highly sensitive detection.
Through a large number of literature research, as well as learning and understanding the design principles of the previous DNAzyme sensor, combined with the discussion within the group, they believe that the design of self-blocking nucleic acid strands may be able to solve the problem of signal leakage.
Later, through a series of experiments, the signal leakage problem has been solved. However, the DNase sensor cannot be activated.
They then further optimized the parameters and shortened the length of the binding arm to build the DNAzyme sensor.
From this, they constructed a single-stranded, self-locking single-molecule system and named this sensing system SPOT (a sensitive loop-activated DNAzyme biosensor for nucleic acid detection).
At the same time, they further combined SPOT with test strips, laying the foundation for SPOT to implement POCT detection.
Fully optimized and SPOT assays provide robust detection sensitivity of 15 fM for miR-21, 1.9 aM for viral RNA, and detection specificity for nucleic acid targets (variant differentiation) in a single-tube, one-step, no preamplification, and isothermal assays.
最终，相关论文以《一种用于核酸敏感检测的可编程 DNAzyme》（A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids）为题发在 Angewandte Chemie International Edition[1]。
Shi Chenzhi is the first author, and Wang Pengfei is the corresponding author.
图 | 相关论文(来源:Angewandte Chemie International Edition)
However, only a small number of clinical samples are currently being tested on SPOT. In the future, the research group will conduct clinical studies on more patients in order to fully evaluate the reliability of the SPOT assay.
At the same time, due to the preamplifier-less approach used in this study, the SPOT was not sensitive enough to produce a clearly distinguishable reading on a test strip that was visible to the naked eye.
Therefore, the team intends to further increase the sensitivity of SPOT or combine it with a portable plasma/fluorescence-based visualization device so that SPOT's POCT capabilities can be used in real-world applications.
In the future, at the level of clinical application, they hope to establish a multi-center, large-scale clinical cohort to demonstrate the disease diagnostic efficacy of the SPOT system on a larger scale.
In terms of technical improvements, the team plans to expand the use of SPOT. In addition to nucleic acid detection, they also hope to achieve the detection of small molecule and protein markers by introducing aptamers.
Resources:
1.Shi, C., Yang, D., Ma, X., Pan, L., Shao, Y., Arya, G., ... & Wang, P. (2024). A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids. Angewandte Chemie International Edition, e202320179.
Operation/Typesetting: He Chenlong