Engineering m6A demethylation-activated DNAzyme for visually and sensitively sensing fat mass and obesity-associated protein

Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N 6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3′-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2′,3′-cyclic phosphate terminus at the 3′-end serving as an intrinsically blocked 3′ end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3′-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Dephosphorylation Switch DNAzyme-RCA Circuit: A Robust Strategy for the Homogeneous and Reliable Detection of FTO Demethylase

Shen,

Huang,

Zhang

et al. 2023

“…Deoxyribozymes (DNAzymes) are a class of functional nucleic acids obtained by in vitro selection, , and they can catalyze a variety of biological reactions (e.g., RNA ligation, RNA cleavage, oxidation–reduction reactions). − In comparison with protein- and RNA-based enzymes, DNAzymes have the characteristics of low synthesis cost, high stability, fast reaction rate, and high programmability, , and they are widely used to detect nucleic acids, proteins, metal ions, small molecules and bacteria . Rolling circle amplification (RCA) is an efficient isothermal amplification approach that employs a circular DNA template to synthesize a long and repetitive single stranded DNA products. , Recently, Jia et al revealed that m 6 A could significantly hinder the single-base elongation activity of Bst DNA polymerases and the ligation efficiency of ligases .…”

Section: Introductionmentioning

confidence: 99%

“…25 Therefore, sensitive and simple methods for monitoring FTO are highly demanded. Deoxyribozymes (DNAzymes) are a class of functional nucleic acids obtained by in vitro selection, 27,28 and they can catalyze a variety of biological reactions (e.g., RNA ligation, RNA cleavage, oxidation−reduction reactions). 29−31 In comparison with protein-and RNA-based enzymes, DNAzymes have the characteristics of low synthesis cost, high stability, fast reaction rate, and high programmability, 32,33 and they are widely used to detect nucleic acids, 34 proteins, 35 metal ions, 36 small molecules 37 and bacteria.…”

Section: ■ Introductionmentioning

confidence: 99%

Construction of Multiple DNAzymes Driven by Single Base Elongation and Ligation for Single-Molecule Monitoring of FTO in Cancer Tissues

Zhao,

Liu,

Zhang

et al. 2023

Fat mass and obesity-associated proteins (FTO) play an essential role in the reversible regulation of N 6 -methyladenosine (m 6 A) epigenetic modification, and the overexpression of FTO is closely associated with the occurrence of diverse human diseases (e.g., obesity and cancers). Herein, we demonstrate the construction of multiple DNAzymes driven by single base elongation and ligation for the single-molecule monitoring of FTO in cancer tissues. When target FTO is present, the m 6 A-RNA is specifically demethylated and subsequently acts as a primer to combine with the padlock probe, initiating single-base elongation and ligation reaction to generate a closed template probe. Upon the addition of phi29 DNA polymerase, a rolling circle amplification (RCA) reaction is initiated to produce large numbers of Mg 2+ -dependent DNAzyme repeats. Subsequently, the DNAzymes cyclically digest the signal probes, liberating numerous Cy5 molecules that can be precisely counted by single-molecule imaging. Taking advantage of the sequence specificity of the polymerase/ligase-mediated gapfilling and ligation as well as the high amplification efficiency of RCA, this biosensor shows excellent specificity and high sensitivity with a detection limit of 5.96 × 10 −16 M. It can be applied to screen FTO inhibitors and quantify FTO activity at the single-cell level. Moreover, the proposed strategy can accurately distinguish the FTO expression level in tissues of healthy individuals and breast cancer patients, providing a new platform for drug discovery, m 6 A modification-related research, and clinical diagnostics.

“…19 Conventional sensing platforms for the m 6 A demethylase activity detection mainly rely on radioisotope-labeled gel electrophoresis analysis, 20 immunoassay, 4 and high-performance liquid chromatography (HPLC), 21 they involve the radiolabeled substrates, large sample consumption, and time-consuming procedures. Alternatively, fluorescent and electrochemical biosensors based on stimulusresponsive DNAzyme 22,23 and specific restriction enzymes (i.e., DpnII endonuclease 24 and Escherichia coli MazF toxin 25,26 ) have been established to convert the demethylase signal into an amplifiable nucleic acid signal. Methylationsensitive DNAzyme-based and DpnII-based strategies exhibit high sensitivity, but they are not suitable for the assessment of the m 6 A demethylase activity against RNA substrates.…”

mentioning

confidence: 99%

Construction of a Quantum-Dot-Based FRET Nanosensor through Direct Encoding of Streptavidin-Binding RNA Aptamers for N6-Methyladenosine Demethylase Detection

Zhang,

Zhao,

et al. 2023

N6-Methyladenosine (m 6 A) demethylases can catalyze the removal of the methyl modification on m 6 A, and it is closely associated with the occurrence, proliferation, differentiation, and metastasis of malignancies. The m 6 A demethylases (e.g., fat mass and obesity-associated protein (FTO)) may act as a cancer biomarker and are crucial for anticancer drug screening and early clinical diagnosis. Herein, we demonstrate the construction of a quantum-dot-based Forster resonance energy-transfer (FRET) nanosensor through direct encoding of streptavidin-binding RNA aptamers (SA aptamers) for m 6 A demethylase detection. This nanosensor employs multiple Cy5-molecule-labeled SA aptamers as the building materials to construct the 605QD-RNA-Cy5 nanoassembly, and it exploits the hinder effect of m 6 A upon elongation and ligation reactions to distinguish m 6 A-containing RNA probes from demethylated RNA probes. When m 6 A demethylase is present, the m 6 A-containing RNA probes are demethylated to generate the demethylated RNA probes, initiating strand extension and ligation reactions to yield a complete transcription template for SA aptamers. Subsequently, a T7-assisted cascade transcription amplification reaction is activated to transcribe abundant SA aptamers with the incorporation of multiple Cy5 fluorophores. The Cy5-incorporated SA aptamers can self-assembly onto the streptavidin-coated 605QD surface to obtain the 605QD-SA aptamer-Cy5 nanoassemblies, resulting in the generation of distinct FRET signals. This nanosensor exhibits ultrahigh sensitivity and excellent specificity, and it can detect endogenous FTO at the single-cell level. Furthermore, this nanosensor can precisely measure enzyme kinetic parameters, screen m 6 A demethylase inhibitors, and differentiate the FTO expression between breast cancer patients and healthy individual tissues, offering a versatile platform for clinical diagnostic and drug discovery.