Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging

Dong, Kejun; Shu, Wan; Zhang, Jiarui; Cheng, Shuangshuang; Zhang, Jun; Zhao, Rong; Hua, Teng; Zhang, Wei; Wang, Hongbo

doi:10.1016/j.bios.2023.115118

Cited by 13 publications

(3 citation statements)

References 33 publications

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“…The clustered regularly interspaced short palindromic repeat (CRISPR) system has been recently adopted as a flexible and robust tool for genome editing, − bioengineering, , and diagnostics. − In particular, the CRISPR-Cas12a and CRISPR-Cas13a systems have shown potential for molecular diagnostics owing to their trans-cleavage activity. − In CRISPR-based diagnostics, the Cas-gRNA complex can recognize target nucleic acids and initiate enzymatic activities for nonspecifically degrading the surrounding single-stranded nucleic acids. By using single-stranded nucleic acid labeled with a fluorophore/quencher, the collateral cleavage activity can generate enhanced fluorescent signals in response to the target nucleic acid.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection

Lee,

Min,

Lee

et al. 2024

Anal. Chem.

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The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cisand trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.

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Section: Introductionmentioning

confidence: 99%

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection

Lee,

Min,

Lee

et al. 2024

Anal. Chem.

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“…15 More recent examples of glycosylase assays incorporate DNA amplification techniques 16,17 and signal amplification techniques using DNAzymes 18,19 and CRISPR-Cas trans-cleavage. 20 Although sensitive, DNA amplification techniques are complicated and time-consuming, involving multiple technical steps and incubation periods. DNAamplification techniques can detect intracellular glycosylases after their introduction into the cell by lipofectamine transfection or MnO 2 nanosheets, 21−23 but the engulfed DNA is prone to nuclease digestion, which risks false positive results or attenuates detection altogether.…”

mentioning

confidence: 99%

“…These methods used simple DNA substrates labeled with fluorophores, radioisotopes, , and chemiluminescent molecules . More recent examples of glycosylase assays incorporate DNA amplification techniques , and signal amplification techniques using DNAzymes , and CRISPR-Cas trans-cleavage . Although sensitive, DNA amplification techniques are complicated and time-consuming, involving multiple technical steps and incubation periods.…”

mentioning

confidence: 99%

Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker

Tao,

Zhang,

Weinfeld

et al. 2024

ACS Meas. Sci. Au

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DNA glycosylase dysregulation is implicated in carcinogenesis and therapeutic resistance of cancers. Thus, various DNA-based detection platforms have been developed by leveraging the base excision activity of DNA glycosylases. However, the efficacy of DNA-based methods is hampered due to nonspecific degradation by nucleases commonly present in cancer cells and during preparations of cell lysates. In this report, we describe a fluorescence-based assay using a specific and nuclease-resistant three-dimensional DNAzyme walker to investigate the activity of DNA glycosylases from cancer cell lysates. We focus on DNA glycosylases that excise uracil from deoxyuridine (dU) lesions, namely, uracil DNA glycosylase (UDG) and single-stranded monofunctional uracil DNA glycosylase (SMUG1). The limits of detection for detecting UDG and SMUG1 in the buffer were 3.2 and 3.0 pM, respectively. The DNAzyme walker detected uracil excision activity in diluted cancer cell lysate from as few as 48 A549 cells. The results of the UDG inhibitor experiments demonstrate that UDG is the predominant uracil-excising glycosylase in A549 cells. Approximately 500 nM of UDG is present in each A549 cell on average. No fluorescence was generated in the samples lacking DNAzyme activation, indicating that there was no nonspecific nuclease interference. The ability of the DNAzyme walker to respond to glycosylase activity illustrates the potential use of DNAzyme walker technology to monitor and study biochemical processes involving glycosylases.

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Enzyme‐Powered, Label‐Free DNA Walker for Uracil‐DNA Glycosylase Detection at Single‐Cell Level

Li,

Wang,

Zong

et al. 2024

Chemistry An Asian Journal

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Uracil‐DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme‐powered, label‐free DNA walker was devised for UDG activity detection. Initially, a label‐free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G‐quadruplex sequence, enabling the generation of a label‐free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme‐powered walking process and generating significant dissociative G‐quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label‐free fluorescence response was achieved. The proposed DNA walker presented a direct and label‐free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single‐cell level was accomplished. Consequently, the developed DNA walker serves as a label‐free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.

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Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging

Cited by 13 publications

References 33 publications

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection

Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker

Enzyme‐Powered, Label‐Free DNA Walker for Uracil‐DNA Glycosylase Detection at Single‐Cell Level

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