Highly convenient and highly specific-and-sensitive PCR using Se-atom modified dNTPs

Hu, Bei; Wang, Ying; Li, Na; Zhang, Shun; Luo, Guangcheng; Huang, Zhen

doi:10.1039/d0cc06172g

Cited by 8 publications

(4 citation statements)

References 25 publications

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“…In order to investigate the mechanism of the de novo DNA synthesis, we have explored the polymerization with the modified dNTPs (Figure ). − We have discovered that the de novo synthesis with polymerases (Pol I, Bst, and Taq functioning under 37, 63, and 72 °C, respectively) can be inhibited by the modified dNTPs (such as dNTPαSe; Figure B). Furthermore, we have found that although the polymerase generates byproduct DNAs more and faster in the presence of the nickase than its absence, dNTPαSe can inhibit the byproduct polymerization as well (Figure D).…”

Section: Resultsmentioning

confidence: 99%

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

et al. 2022

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The template-dependent DNA synthesis, with DNA polymerases, templates, and primers, is essential for disease detection, molecular biology, and biotechnology. However, DNA polymerases can also initiate de novo DNA synthesis without templates and primers, forming byproduct DNAs with random sequences. Herein, we report the mechanisms of the de novo DNA synthesis in the absence or presence of nickase by discovering the reduced bindings between the polymerases and modified dNTPs and between the nickases and the modified DNAs and finding the reduced polymerase synthesis and nickase cleavage. Furthermore, via sequencing, we have identified the mechanism of the de novo synthesis in the nickase-based isothermal amplifications, generating the random DNAs as the major byproducts. Fortunately, we have discovered a novel strategy to inhibit the undesired synthesis with the single-atom-modified nucleotides and achieved the accurate and sensitive detection of clinic samples in the isothermal amplifications. In general, we have revealed the suppression mechanisms on the de novo synthesis and demonstrated that this selenium-atom strategy can allow more accurate and sensitive detection of pathogens via the isothermal amplifications.

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Section: Resultsmentioning

confidence: 99%

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

et al. 2022

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“…Several studies have proposed one-pot detection method that could complete virus detection in one single tube ( Chen et al, 2020a ; Ding et al, 2020 ; Pang et al, 2020 ). However, the detection sensitivity is compromised somehow due to the generation of contaminated nucleic acids, which creates urgent needs for developing contamination-free detection such as high specificity gene amplification technology ( Hu et al, 2021 ). The most feasible strategy for integrating CRISPR-Cas systems with another technology or system is to find a specific nucleic acid molecule as a linker.…”

Section: Challenges and Perspectivesmentioning

confidence: 99%

CRISPR-Cas based virus detection: Recent advances and perspectives

Yin¹,

Man²,

et al. 2021

Biosensors and Bioelectronics

158

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Viral infections are one of the most intimidating threats to human beings. One convincing example is the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2. Rapid, sensitive, specific and field-deployable identification of causal viruses is critical for disease surveillance, control and treatment. The shortcomings of current methods create an impending need for developing novel biosensing platforms. CRISPR-Cas systems, especially CRISPR-Cas12a and CRISPR-Cas13a, characterized by their sensitivity, specificity, high base resolution and programmability upon nucleic acid recognition, have been repurposed for molecular diagnostics, surging a new path forward in biosensing. They, as the core of some robust diagnostic tools, are revolutionizing the way that virus can be detected. This review focuses on recent advances in virus detection with CRISPR-Cas systems especially CRISPR-Cas12a/Cas13a. We started with a short introduction to CRISPR-Cas systems and the properties of Cas12a and Cas13a effectors, and continued with reviewing the current advances of virus detection utilizing CRISPR-Cas systems. The significance and advantages of such methods were then discussed. Finally, the challenges and perspectives were proposed. We tried to provide readers with a concise profile of emerging and fast-expanding CRISPR-Cas based biosensing technology, and highlighted its potential applications in a range of scenarios with regard to virus detection.

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“…[4][5][6][7] Recently, Huang and coworkers found that dNTPαSe substrates can significantly improve specificity and enhance sensitivity of polymerase chain reaction (PCR) via inhibiting nonspecific polymerization, which could greatly facilitate PCR amplification, therapeutic discovery, and X-ray structural studies. [8,9] In addition, NTPαXs are also valuable in medicinal chemistry. Shaw and Fischer have shown that α-P-BH 3 -modified triphosphates of antiviral nucleosides and adenosine analogues are potent HIV-1 transcriptase inhibitors [10] and P2Y 1 receptor agonists, [11] respectively.…”

Section: Introductionmentioning

confidence: 99%

“…In comparison to β‐ and γ‐ P ‐modified NTPs, the most distinct feature of NTPαXs is that they could be recognized by polymerases to incorporate S, Se, and BH 3 ‐modified phosphate linkages into DNA and RNA, which has made NTPαXs versatile tools in molecular biology [4–7] . Recently, Huang and coworkers found that dNTPαSe substrates can significantly improve specificity and enhance sensitivity of polymerase chain reaction (PCR) via inhibiting nonspecific polymerization, which could greatly facilitate PCR amplification, therapeutic discovery, and X‐ray structural studies [8,9] . In addition, NTPαXs are also valuable in medicinal chemistry.…”

Section: Introductionmentioning

confidence: 99%

Efficient Synthesis of α‐P‐Modified Nucleoside Triphosphates and Dinucleoside Tetraphosphates via Linear P^VP^VP^III‐Nucleoside Intermediates

et al. 2023

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As an important class of nucleoside triphosphate analogues, α‐P‐modified nucleoside triphosphates (NTPαXs) have long been recognized as versatile tools in molecular biology and medicinal chemistry. However, the available synthetic methods based on modified nucleoside monophosphate and cyclic PVPVPIII‐nucleoside intermediates are only low to moderate‐yielding. Inspired by recent progress on PV‐PIII coupling reactions, we attempted to access NTPαXs via the putative linear PVPVPIII‐nucleoside intermediates. The current research found that a specific type of nucleoside phosphoramidites, fluorenylmethyl (Fm) nucleosidyl‐phosphoromorpholidites, are highly reactive in coupling with nBu4NH3P2O7 even without acidic activators. In situ oxidation of linear PVPVPIII‐nucleosides with specific sulfurizing, boranylating, and selenylating reagents followed by deprotection of α‐P and nucleoside moieties afforded NTPαXs (X=S, Se, and BH3) in excellent isolated yields. By exploiting dicyanoimidazole (DCI)’s promoting effect on PV‐PIII coupling, the current linear PVPVPIII‐nucleoside‐based method was further developed into an efficient route to α‐P‐modified dinucleoside tetraphosphates.

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Highly convenient and highly specific-and-sensitive PCR using Se-atom modified dNTPs

Abstract: Primer design and condition optimization for PCR are tedious and labour-intensive.

Cited by 8 publications

References 25 publications

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

CRISPR-Cas based virus detection: Recent advances and perspectives

Efficient Synthesis of α‐P‐Modified Nucleoside Triphosphates and Dinucleoside Tetraphosphates via Linear P^VP^VP^III‐Nucleoside Intermediates

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Highly convenient and highly specific-and-sensitive PCR using Se-atom modified dNTPs

Abstract: Primer design and condition optimization for PCR are tedious and labour-intensive.

Cited by 8 publications

References 25 publications

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

CRISPR-Cas based virus detection: Recent advances and perspectives

Efficient Synthesis of α‐P‐Modified Nucleoside Triphosphates and Dinucleoside Tetraphosphates via Linear PVPVPIII‐Nucleoside Intermediates

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Product

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About

Efficient Synthesis of α‐P‐Modified Nucleoside Triphosphates and Dinucleoside Tetraphosphates via Linear P^VP^VP^III‐Nucleoside Intermediates