To date, deoxyuracil (dU) has not been detected in humans at a single base resolution level. This becomes one of the blind spots and bottlenecks in DNA sequence detection, seriously hindering the recognition of dU function and the understanding of the genetic code of DNA.
On January 17, chen Yihan, academician of the Chinese Academy of Sciences and professor of Tongji University, Ma Honghui, a researcher at Tongji University, and Hu Jinchuan, a researcher at Fudan University, jointly published an article in the Journal of the American Chemical Society. With the help of a special enzyme molecule, the researchers invented a DNA detection technology with good sensitivity, strong specificity and high resolution, and for the first time used enzymatic method to accurately detect the dU in DNA at the level of single base resolution, which achieved a fundamental breakthrough in the detection technology of dU bases in DNA.
Break through the sequencing bottleneck
As we all know, DNA is the genetic code of an organism. They are generally thought to include 4 bases, including adenine (A), thymine (T), guanine (G), and cytosine (C). Later studies found that there was an additional base dU in the DNA. Together, these bases make up the basic elements of DNA.
"From prokaryotes to eukaryotes, from single-celled organisms to humans, in addition to A, T, G, and C, their DNA contains varying proportions of dU. But until now, humans have struggled to detect dU from single-base resolution levels, which hampers cognition of dU function and understanding of the GENETIC code of DNA. Chen Yihan told China Science Daily.
In HIV DNA, there is more than one dU for every 20 bases; in the DNA of malaria parasites, dU accounts for about one in 100,000 bases. dU can be produced by C base deamination and can be "impersonated" into the genome by T bases.
Due to the lack of sensitive and specific single-base resolution dU sequencing technology, people cannot achieve the precise localization of dU in DNA like other bases (A, T, G, and C).
"That is, current dU detection techniques can confirm the presence of dU bases in several bases, but it is not possible to determine which specific bases the dU bases are located between." Jiang Liudan, the first author of the paper and a doctoral student at Tongji University, told China Science Daily, "What is the biological significance of dU bases? What is the significance of dU bases in the occurrence and development of diseases? To answer these questions, breakthroughs in dU base detection and localization at the single-base resolution level must be achieved. Without precisely locating bases at the single-base resolution level, it is impossible to infer the amino acid and protein sequences that the bases encode at the DNA sequence level. ”
Accurate "fishing" turns enemies into friends
dU has "two-sidedness" – sometimes a friend of human health, and sometimes an enemy of human health.
"Many reports have found that when the body is confronted with different antigens, immune cells need dU as an intermediate to produce a variety of antibodies to help defend against pathogens such as the new crown virus." Ma Honghui, co-corresponding author of the paper, said, "When dU appears in patients with tumors or cardiovascular diseases, it may lead to the instability of the patient's genome and accelerate the development of the disease." ”
Obviously, accurate detection of the distribution of dU in DNA will help assess the physiological function of individual humans and the prognosis of disease. However, finding the exact location of the dU in DNA is like finding a needle in a haystack.
After years of exploration, researchers eventually invented dU sequencing technology with superior single-base resolution.
Ma Honghui said that the sequencing technology must first find a suitable "hook" - a new glycosidase called UdgX, a source of mycobacterium difficile. This glycosidase is able to excise the dU of DNA, forming a gap and at the same time forming a covalent bond with the corresponding ribose, eventually capturing it.
After the glycosidase as a "hook" is "fished" into the DNA fragment containing dU, the dU location needs to be further determined. Next, dna high-fidelity polymerase properties are used. The enzyme is like a train on a DNA track, and when it hits a dU gap marked by this glycosidase, it will passively "stop" in place.
The researchers then combined high-throughput sequencing techniques to amplify the "stop" signal and ultimately pinpoint the location of the dU on DNA and even the genome at the level of a single base.
Enzymatic detection has obvious advantages
To facilitate the spread and popularity of this sequencing technique, the researchers named it Ucaps-seq. Since then, a dU detection technology at the level of single base resolution based on a new type of glycosidase has been born.
Based on this technology, people can accurately detect the dU in DNA just as they detect A, T, G and C in DNA.
"Ucaps-seq sequencing technology is the first enzymatic method in the world to detect dU bases in DNA." Chen Yihan said, "The existing dU sequencing technology is chemical, and the enzymatic sequencing technology is obviously superior to the chemical sequencing technology. ”
Enzymatic sequencing techniques are sensitive, specific and high resolution. In addition, the technology also has the characteristics of high efficiency and low cost in practical applications, rarely false positives, and rarely affected by interference factors. For example, existing dU chemical sequencing techniques require the use of an enzyme to excise the dU to make it a pyrimidine-free site, which is difficult to distinguish from the presence of dna itself without pyrimidine sites, inevitably leading to false positives.
In order to further verify the effectiveness of Ucaps-seq sequencing technology, the researchers first verified the principle of the sequencing technology on the synthesized DNA probe model, and then verified the single-base resolution efficacy of Ucaps-seq sequencing technology in mutagenic cancer cells and B cells, and finally evaluated the gene editing off-target, and found that the sequencing technology has a strong recognition ability for gene editing off-target.
"The birth of Ucaps-seq technology, especially the application of its detection kits, will be as convenient and efficient as other base detections." Chen Yihan said, "This will greatly promote nucleic acid sequence detection, genetic code deciphering and human cognition of nucleic acids." (Reporters Zhang Shuanghu and Huang Xin)
Related paper information:
https://doi.org/10.1021/jacs.1c11269
Source: China Science Daily
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