Importance of DNA nanotechnology for DNA methyltransferases in biosensing assays

Abstract

DNA methylation is the process by which specific bases on a DNA sequence acquire methyl groups under the catalytic action of DNA methyltransferases (DNMT). Abnormal changes in the function of DNMT are important markers for cancers and other diseases; therefore, the detection of DNMT and the selection of its inhibitors are critical to biomedical research and clinical practice. DNA molecules can undergo intermolecular assembly to produce functional aggregates because of their inherently stable physical and chemical properties and unique structures. Conventional DNMT detection methods are cumbersome and complicated processes; therefore, it is necessary to develop biosensing technology based on the assembly of DNA nanostructures to achieve rapid analysis, simple operation, and high sensitivity. The design of the relevant program has been employed in life science, anticancer drug screening, and clinical diagnostics. In this review, we explore how DNA assembly, including 2D techniques like hybridization chain reaction (HCR), rolling circle amplification (RCA), catalytic hairpin assembly (CHA), and exponential isothermal amplified strand displacement reaction (EXPAR), as well as 3D structures such as DNA tetrahedra, G-quadruplexes, DNA hydrogels, and DNA origami, enhances DNMT detection. We highlight the benefits of these DNA nanostructure-based biosensing technologies for clinical use and critically examine the challenges of standardizing these methods. We aim to provide reference values for the application of these techniques in DNMT analysis and early cancer diagnosis and treatment, and to alert researchers to challenges in clinical application.

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1. Introduction

DNA methylation is an epigenetic modification in which DNMT catalyzes the binding of a methyl group through covalent bonding of the 5′ carbon atom of the CpG dinucleotide cytosine within the genome, in the DNA nucleic acid sequence, using S-adenosine methionine as a methyl donor.^1–3 DNA methylation is an important epigenetic modification that is inactivated in multiple human biological processes, such as X chromosome genomic imprinting and expression.^4,5 In normal cells, it ensures proper regulation of gene expression and stable gene silencing,^6,7 and DNA methylation is one of the major causes of disease development in cancer. The epigenetic effects of DNA methylation on tumorigenesis occur when hypomethylation leads to the activation of proto-oncogenes or hypermethylation leads to the inactivation of tumor suppressor genes,⁸ which are characterized by the addition of methyl groups to the promoter regions of genes, leading to gene silencing or increasing the likelihood of mutation. Both hypermethylation and hypomethylation are major causes of cancer;⁹ the former is more common and occurs on CpG islands in the promoter regions of genes, whereas the latter is ubiquitous in a wide variety of genomic sequences.¹⁰ DNMT is responsible for the establishment and maintenance of methylation patterns. Aberrant DNA methylation at the 5′-end of cytosine catalyzed by DNMT contributes to various cancers and other diseases by silencing oncogenes.¹¹ Abnormal DNMT activity usually occurs before the appearance of other malignant tumors, and it has been found that DNMT is a potential predictive biomarker and therapeutic target in the diagnosis and treatment of various types of cancer.¹² It is mainly classified into DNMT1, DNMT3A, and DNMT3B, which are typically overexpressed in various cancer tissues and cell lines.¹³ As DNA methylation is reversible, DNMT is often considered an important epigenetic target for drug development.¹⁴

The conventional detection methods for DNMT mainly include radiolabeling,^15,16 methylation-specific PCR,^17,18 gel electrophoresis,^19,20 and high-performance liquid chromatography.^21,22 However, these methods involve the use of radioactive materials, have high sample consumption, low sensitivity, are time-consuming and laborious, and require expensive laboratory equipment and specialized technicians. The development of a target detection method should not be limited by the complexity and inefficiency of the test. Various analytical methods have been developed to overcome these limitations, including fluorescence,^23–25 electrochemical,^26,27 surface-enhanced Raman spectroscopy (SERS),^28,29 electrochemiluminescence,^30–32 chemiluminescence,³³ photoelectrochemical biosensing assays (PEC),^34,35 and colorimetric assays.^36,37 These methods not only enhance the sensitivity of DNMT detection, are easy to operate, and are low cost, but also provide certain advantages in the detection of other molecular biomarkers in the biological field, such as exosomes,^38–40 nucleic acids,^41–43 proteins,^44,45 antibodies,^46,47 cells,^48,49 enzymes,^50,51 and other important biological target assays.

A series of new nanomaterials in the field of biosensing are being developed for the detection of DNMTs. These nanomaterials include gold nanoparticles,^52–54 quantum dots,^55,56 carbon nanotubes,^57,58 and gold nanoclusters,^59,60 all of which have unique physical and chemical properties that can enhance the sensitivity of sensors, making them advantageous for biosensors. Critically, in recent decades, various breakthroughs have been made in DNA structural nanotechnology, employing the properties of nucleic acids, such as the double helix form, Watson–Crick base pairing interactions, and programmable sequences, to build specific nanostructures and nanodevices.^61–63

DNA, a fundamental part of the human body, is an important nucleic acid genetic material that plays an important role in encoding genetic information.⁶⁴ In 1953, Watson and Crick proposed double-stranded DNA as a double-helix molecular structure with complementary base pairing, which opened the way for subsequent researchers not only to explore the mysteries of DNA itself but also to use the stability of the molecular structure of DNA assemblies as an opportunity to develop DNA nanotechnology, a field where nucleic acids are used as abiotic materials instead of as carriers of genetic information as they would be in living cells, and which is therefore defined as “nucleic acid nanotechnology”. Due to breakthroughs in DNA nanotechnology, it is now possible to create DNA nanostructures “from the bottom up” and integrate them into a variety of shapes and structures. Appropriate nucleic acid design will improve the hybridization efficiency, specificity, and sensitivity of biosensors. The analysis and improvement of the biostability of DNA nanostructures is one of the key areas where advances are needed in the application of DNA nanotechnology.⁶⁵ DNA is biocompatible, biodegradable, and non-cytotoxic because DNA molecules can be readily recognized and accepted by the bodies of living organisms. Under appropriate conditions, DNA nanostructures exhibit high stability and maintain their structure and properties without being damaged. Their high stability ensures the reliability and durability of the DNA nanostructures for various biomedical applications (Table 1). Thus, the rapid development of DNA nanotechnology has led to a wide range of applications of DNA nanomaterials in biomedical fields, including sensing,⁶⁶ diagnostics, therapeutics, and imaging, as functional carriers for drug delivery and nucleic acid detection.⁶⁷ In diagnostics, DNA nanomaterials provide a platform for detecting highly specific biomarkers. As drug carriers, they enable precise drug delivery and release.⁶⁸ Additionally, DNA nanomaterials have been used in gene therapy and high-contrast imaging.⁶⁹ These are the applications of new methods and tools for early disease diagnosis and treatment. The future of DNA nanomaterials in biomedicine remains promising as the technology continues to advance.⁷⁰ In this review, we discuss DNA nanotechnology in biosensor platforms, in which DNA is formed in two-dimensional planar structures, such as HCR, RCA, CHA, and EXPAR, and three-dimensional structures, such as DNA tetrahedra, G-quadruplexes, and DNA hydrogels, for the efficient detection of DNMTs.

Table 1 Overview of DNA nanostructures-based biosensors commonly used in the detection of DNA methyltransferases

DNA nanostructure	Sensing mode	Target	Linear range (U mL^−1)	LOD (U mL^−1)	Ref.
2D DNA planar structure	HCR	Dam MTase	0–1.0	0.011	74
		Dam MTase	0–0.20	2.39	75
		M.SssI MTase	0.002 to 200	2 × 10^−4	76
		DamMTase	0.005 to 100	0.0028	77
		Dam MTase	0.05–100	0.02	78
	CHA	Dam and M.SssI MTase	0.0004–0.4 and 0.0001–0.1	0.001 and 0.0001	82
		M.SssI MTase	0–0.004	1.2 × 10^−4	83
	RCA	Dam and M.SssI MTase	0.001–0.2 and 0.0002–0.1	0.001 and 0.0004	87
		Dam MTase	0.02 to 4	0.0067	88
		Dam MTase	2.5–70	1.8	89
		DnmTl and UDG	0.01–10 and 0.005–10	0.009 and 0.003	90
	EXPAR	M.SssI MTase	0.125–8	0.034	95
		Dam MTase	0.02–10	0.014	96
3D DNA spatial structure	DNA tetrahedron	Dam MTase	0.0025–10	0.001	110
		Dam MTase	0.002–100	0.00036	111
		Dam MTase	0.1–90	0.045	112
	G-quadruplex	Dam MTase	1–7.5	0.3	117
		Dam MTase	0.05–10	0.0085	118
		DNMTl	1.0–30.0	0.09	119
Other 3D spatial structure	DNA hydrogel	Dam MTase	0.001–5.0	0.001 and 0.0004	120

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The aim of this review is to present the applications of 2D and 3D DNA nanostructures in various biosensors, as shown in Scheme 1, where the advantages of these detection methods are briefly described. We hope that this article provides a comprehensive overview of the current state of this field. This review summarizes the latest research on DNMT detection and its significance for future clinical applications of several typical DNA nanotechnologies with unique properties in the field of biosensing in recent years, as well as the challenges faced by DNA nanomaterials in the biomedical field, and provides some insights into their future directions.

Scheme 1 Schematic illustration of several synthetic forms of 2D and 3D DNA nanostructures based on DNA nanotechnology.

2. Two-dimensional DNA planar nanostructures

2D DNA planar structures based on DNA nanotechnology are all 2D products formed by stable and flexible binding between DNA strands under certain conditions, including hybridization chain reaction (HCR), rolling circle amplification (RCA), catalytic hairpin assembly (CHA) and exponential isothermal amplified strand displacement reaction (EXPAR).

2.1. Hybridized strand reaction (HCR)

Specific nucleic acid targets can trigger well-designed, programmable self-assembly reactions. Hybridization chain reaction (HCR) created by Dirks and Pierce in 2004 can be self-assembled through a self-assembly strategy.⁷¹ HCR is a signal-enhancing technique that relies on the autonomous self-assembly of two substable hairpin structures (short DNA fragments) through specific interaction for the generation of long nicked DNA double helices,⁷² the reaction involves only two hairpins of equal concentration of a certain base length, which coexist stably in solution.⁷³ HCR is a simple and efficient enzyme-free isothermal amplification reaction, and the design and characterization of a highly specific and efficient HCR will be useful for biosensor applications.

An isothermal autocatalytic hybridization reaction (AHR) circuit for sensitive detection of DNA methyltransferase and inhibitors assay was developed by Li and others,⁷⁴ by combining the catalytic hairpin assembly (CHA) converter with the hybridization chain reaction (HCR) amplifier, and the schematic diagram of the reaction is shown in Fig. 1(A). The triggering substance promotes the HCR to amplify the fluorescent signals very well. In this study, through the autocatalytic cyclic reaction between HCR and CHA, the final n-fold AHR exponentially increases the signal, and a very small amount of the target can produce exponentially amplified fluorescence (Fig. 1(B)). This protocol can provide a sensitive, stable and diversified assay for DNMT, and the limit of detection (LOD) reached 0.011 U mL⁻¹, which is a good solution for the detection of DNMT in real samples as well as the evaluation of the enzyme's dependence on the cell cycle. It can be seen that this design has higher stability and sensitivity than the traditional assay, and it may have a broad application prospect for better utilization in biomedical analysis and clinical diagnosis. In their work,⁷⁵ Shang et al. combined the Mg²⁺–DNA enzyme module with the HCR amplification cycle reaction to design a promoter repeatedly triggered HCR (IR-HCR) strategy for highly sensitive detection of methyltransferase. As shown in Fig. 1(C), the fluorescence “trigger start” analysis of M.SssI MTase is based on autonomous IR-HCR. The IR-HCR circuit consists of HCR reactants (composed of hairpin H1–H4) and a locked DNAzyme substrate (S–L). In addition, Fig. 1(D) illustrates the detection of M.SssI MTase in the mechanism of IR-HCR. Compared with traditional HCR, the recycling mechanism of Mg²⁺–DNAzyme mediated HCR promoter can significantly improve detection efficiency of M.SssI MTase,and can even reach a level that increases the high signal by 100 times. Perhaps this promoter replication strategy can be easily extended to other amplification fields, thereby significantly enhancing its widespread application in clinical diagnosis and prognosis.

Fig. 1 (A) Schematic illustration of the AHR circuit that was composed of the HCR amplifier and the CHA converter, schematic illustration of the HCR amplifier, schematic illustration of the CHA converter. (B) Schematic illustration of the AHR and nAHR circuits. Reproduced from ref. 74 with permission of American Chemical Society, copyright 2022. (C) Scheme of the IR-HCR amplification system. Reproduced from ref. 75 with permission of American Chemical Society, copyright 2021. (D) Schematic illustration of the amplified M.SssI detection based on the coupling of IR-HCR circuit with the auxiliary HpaII endonuclease. Reproduced from ref. 75 with permission of American Chemical Society, copyright 2021. (E) Schematic illustration of a strategy for the detection of M.SssI MTase activity. Reproduced from ref. 76 with permission of American Chemical Society, copyright 2020. (F) Schematic Illustration of the Cyclometalated iridium(III) Complex-Based PEC Platform for Dam MTase Detection Based on CHA and HCR Amplification Strategy. Reproduced from ref. 77 with permission of American Chemical Society, copyright 2021. (G) Schematic illustration of the ratiometric ECL signal system. Reproduced from ref. 78 with permission of Elsevier, copyright 2019.

Luo and his team proposed a dual amplification sensing strategy⁷⁶ (Fig. 1(E)), in which the plasma-enhanced Raman intensity of engineered nanohole arrays is assisted by signal amplification through a HCR for ultra-sensitive detection of M.SssI MTase activity and screening of inhibitors. This assay has ultra-high sensitivity with a wide linear range (0.002–200 U mL⁻¹) and a low detection limit of low (2 × 10⁻⁴ U mL⁻¹), which is superior to the reported SERS-based assays. In addition, it can selectively detect M.SssI in human serum samples and evaluate the efficiency of M.SssI inhibitors, which is a good reference for the detection of clinical samples. Cai et al.⁷⁷ reported for the first time an efficient [(C6)₂Ir(dppz)]⁺PF₆⁻ (C6 = coumarin 6, dppz = dipyridazine) sensitized nickel oxide photocathode and its application in photoelectrochemical (PEC) bioanalysis (Fig. 1(F)). The platform integrates the prepared photocathode with an enzyme-free cascade amplification strategy of catalytic hairpin assembly CHA and HCR for DNA methyltransferase (MTase) detection. The cathode PEC platform of this protocol has several merits of extremely high sensitivity, selectivity, and stable analytical performance, resulting in a detection limit of Dam MTase of 0.0028 U mL⁻¹. Currently, Photocathode-based PEC bioanalysis is still in its infancy, and no Ir(III) complexed sensitized nickel monoxide system has been reported yet. This work demonstrates the feasibility and promising prospects of iridium(III) complexed nickel monoxide photocathodes for PEC bioanalysis. Li⁷⁸ and others in their study designed a fouling-resistant electrochemiluminescence (ECL) ratiometric biosensor (Fig. 1(G)) based on a dual-signaling strategy for accurate, selective and sensitive detection of DNA methyltransferase (MTase). The hairpin DNA in this scheme contains symmetric 5′-CATC-3′ sequence and is attached to the ITO electrode. Under the action of Dam MTase enzyme, the ds-DNA of the hairpin is recognized to be sheared, and the sheared DNA of the protruding sequence reacts successively with the subsequent addition of H2 and H3 to carry out HCR, and then it will form a long double-stranded DNA, and when a PTC-NH₂ molecule is inserted into the dsDNA groove When PTC-NH₂ molecules are inserted into the dsDNA grooves, the ECL signal is significantly amplified and also shows a log-linear relationship with the concentration of Dam MTase. This method enables the detection of MTase activity with high sensitivity and selectivity. In addition, this platform enables the detection of the enzyme activity in complex biological media (e.g., FBS samples and human serum), which greatly reduces the non-specific adsorption effect, thus further demonstrating the feasibility of this ECL biosensor for early clinical diagnosis.

2.2. Catalytic hairpin assembly (CHA)

Catalytic hairpin assembly (CHA) was firstly proposed in 2008,⁷⁹ CHA is a typical enzyme-free, highly efficient, isothermal amplification method. Typical CHA reaction utilizes two complementary DNA strands as stable hairpin structures in homogeneous solution, and spontaneous hybridization between the two hairpins is kinetically hindered. However, in the presence of a target strand, one of the hairpins can be opened by a toehold-mediated strand displacement reaction, which in turn enables hybridization between the two hairpins. During this process, the input chain is removed from the annealed hairpin complex, allowing it to initiate the next round of hairpin opening and assembly. CHA circuits provide fast and efficient signal amplification with minimal background and rapid turnover, which can be achieved exponentially within a short period of time and at constant temperature. So CHA circuits are suitable for a variety of analytical applications, including the detection and quantification of therapeutically relevant nucleic acids in vitro and in living cells. In addition, CHA has a promising future as it can be combined with nanomaterials and other molecular biotechnologies to better realize the field of biosensors for substance detection.^80,81

Zhang et al. in their study designed⁸² a simple and novel nanopore sensor, which was combined with a cascade signal amplification technique (Fig. 2(A)-a and b) for the ultrasensitive and rapid detection of DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI), hairpin DNA composed of methylation reaction sequences. In the presence of Dam methyltransferase, the corresponding recognition site of the hairpin DNA is methylated and recognized and sheared by DpnI endonuclease to form a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA–HCR), generating a product that can be absorbed by the Zr⁴⁺-coated nanopore, resulting in a change in the ionic current rectification signal. Conventional methods for detecting DNA methyltransferase generally involve radioactive labeling, expensive equipment and laborious operation, whereas this method is performed in a homogeneous solution under isothermal condition, eliminating the complex process of DNA probe modification on the surface of the nanopore, which also has a wide range of detection, high selectivity, and a detection limit lower than that of most of the existing methyltransferase sensors. It can be better applied in early diagnosis and drug screening, and perhaps this ultra-sensitive methyltransferase detection method will be promising in the field of cancer diagnosis.

Fig. 2 (A)-(a) Scheme of the CHA–HCR cascade amplification system. (A)-(b) Illustration of the sensing strategy based on Zr⁴⁺-coated nanopore for the initiator sequence (T). Reproduced from ref. 82 with permission of American Chemical Society, copyright 2022. (B) Schematic representation of the isothermal nonenzymatic cascaded CHA–HCR system. Reproduced from ref. 83 with permission of American Chemical Society, copyright 2018.

Traditional amplification methods only achieve linear amplification and cannot meet the growing demand for detecting trace targets. Li et al. constructed a multi-stage cascaded amplifier to enhance signal amplification effect, which is very ideal. They designed a powerful enzyme free sensing platform for the detection of MTase⁸³ (Fig. 2(B)). The upstream CHA cycle generates sequential DNA products that are used to activate the downstream HCR cycle, which generates significantly amplified fluorescence signals for the detection of trace amounts of the target, and the method is capable of detecting MTase and its inhibitors in serum and E. coli cells in an ultrasensitive way, and the scheme of the CHA–HCR circuit assay does not require complex preparation of nanomaterials, sophisticated manipulation and fragile enzyme involvement, but ultimately the effective design and application of this scheme holds some promise for from cancer therapy and biomedical research but more validation in terms of clinical samples is needed.

2.3. Rolling circle amplification (RCA) reaction

Rolling circle amplification (RCA) reaction is a highly efficient enzymatic isothermal reaction that produces long tandem single-stranded DNA or RNA products in the presence of short DNA or RNA primers using a ring probe as a template. RCA is a common research tool in molecular biology, material science and medicine.⁸⁴ Since it was discovered at the end of the 20th century, the applications of RCA have increased with the development of science and technology,⁸⁵ and RCA has a number of distinct advantages over other amplification strategies. First, because padlock probes require strict complementarity in the ligation process, which requires a high degree of specificity in the RCA reaction, and second, by introducing multiple primers, exponential amplification can be easily achieved, thereby increasing sensitivity, and RCA is biocompatible, so RCA has received widespread attention in the field of molecular biology as a highly efficient and potentially highly sensitive tool for the detection of biomarkers.⁸⁶

Zhang et al. designed a dual amplification strategy of HRCA and DNA walker⁸⁷ (Fig. 3(A)). In the absence of M.SssI methyltransferase, the corresponding recognition site of the hairpin HM is cleaved by HpaII endonuclease, generating DNA fragments (T-DNA) and inducing the DNA walker-HRCA reaction. SYBR Green I can be embedded in dsDNA to produce a high fluorescence signal. The biosensor has a very high sensitivity, and the detection limits of M.SssI and Dam methyltransferase even reached 0.0004 U mL⁻¹ and 0.001 U mL⁻¹, and the sensing system has a good sensitivity for the detection of endogenous Dam methyltransferase in the actual samples, such as E. coli cell lysates, which is undoubtedly of great significance for the clinical detection of other biomarkers. And also Wen et al. studied the⁸⁸ (Fig. 3(B)) DNA methyltransferase (MTase) detection method for the rolling circle amplification reaction combined with strand displacement amplification (SDA) is also carried out dual-amplification fluorescence strategy, the detection limit of this method can be as low as 0.0067 U mL⁻¹, and the recognition of other types of MTases enzyme has good selectivity, so the establishment of this program has a good generality, can be extended to detect other types of DNA MTases. The method has superior analytical properties and perhaps its application in the field of biosensors is a favorable tool for DNA methyltransferase. Chen et al. designed a fluorescent biosensor for label-free detection of Dam MTase by using methylation-sensitive cleavage primer-triggered hyperbranched rolling circle amplification (HRCA)⁸⁹ (Fig. 3(C)). Since the product of HRCA is a number of double-stranded DNAs (dsDNAs) containing different lengths, the same SYBR Green I can be embedded in the dsDNAs to generate strong fluorescent signals, which can only be detected with weak fluorescence intensity in the absence of a target. In the absence of a target, only a weak fluorescence intensity can be detected to distinguish the presence or absence of the target, and this protocol is designed to be simple, sensitive, and has good selectivity. Fan and his group proposed an exponential amplification strategy mediated by a trifunctional dsDNA probe for the detection of highly sensitive DNA (cytosine-5) methyltransferase 1 (Dnmt1) and uracil-DNA glycosylase (UDG)⁹⁰ (Fig. 3(D)). Primer sequences released during the reaction initiated an exponential rolling circle amplification (ERCA) reaction that produced an extremely strong fluorescent signal. The low cost and simple synthesis design of E-RCA, the high specificity of the reaction, and the high signal-to-noise ratio have greatly improved the sensitivity of the assay, which can well realize the detection of UDG and Dnmt1 at the same time. This strategy has also been successfully applied to the detection of Dnmt1 and UDG activities in samples at the single-cell level. However, additional validation of the clinical application of Dnmt1 and UDG in other real biological samples is also a potentially promising approach.

Fig. 3 (A) Scheme of Ultrasensitive fluorescence detection of multiple DNA methyltransferases based on DNA walkers and hyperbranched rolling circle amplification. Reproduced from ref. 87 with permission of Elsevier, copyright 2023. (B) Mechanism of the dual-amplification method for detecting Dam MTase activity. Reproduced from ref. 88 with permission of Royal Society of Chemistry, copyright 2022. (C) Principle of HRCA-based fluorescent biosensor for Dam MTase activity detection. Reproduced from ref. 89 with permission of Elsevier, copyright 2020. (D) Schematic illustration of the tri-functional probe mediated cascade amplification strategy for highly sensitive detection of Dnmt1 and UDG activities. Reproduced from ref. 90 with permission of Elsevier, copyright 2020.

2.4. Exponential isothermal amplification reaction (EXPAR)

In addition to the various reactions commonly used to form two-dimensional DNA planar structures mentioned above, other strand-bound reaction signal amplification techniques such as isothermal amplification, including linear amplification, cascade amplification and exponential amplification according to the reaction kinetics, exponential isothermal amplification and strand displacement amplification, etc., have been frequently used in the detection of DNA methyltransferase in recent years. Isothermal exponential amplification reaction with high amplification efficiency,^91,92 among the cleaving enzyme-based methods, the isothermal exponential amplification reaction (EXPAR) has attracted greater attention since it was proposed by Galas and coworkers in 2003. Isothermal exponential amplification techniques are mainly divided into two categories: enzyme-based isothermal exponential amplification and enzyme-free isothermal exponential amplification. Exponential amplification technology has the advantages of high sensitivity and selectivity, high signal-to-noise ratio, low cost, and fast response time,⁹³ and this technology has been used for the detection of various types of analytes, including nucleic acids, proteins, biomolecules, ions, and even cells, so the exponential amplification electrochemical biosensors have received a lot of attention. However, there is still more room for the development of this technology, and it will be more promising if this solution can be applied to the detection of enzymes (e.g., polymerase) in cells in the fields of biosensing, biomedicine, and biotechnology. Briefly, EXPAR amplifies DNA in four main steps. Firstly, EXPAR begins amplification when the target binds to the trigger sequence of the template, forming a partial double strand. Subsequently, DNA polymerase extends it to form an extended double-stranded DNA containing the cleavage enzyme recognition site, and then the cleavage enzyme cleaves the upper strand, and DNA polymerase displaces the cleaved trigger sequence by strand displacement, generating additional cleavage enzyme-recognizable sequences, a process that repeats itself in an exponential manner.⁹⁴

The detection of methyltransferase (MTase) is important for the diagnosis of methylation-related diseases and drug screening. An et al. proposed an hpa II-assisted linear amplificationenhanced exponential amplification strategy on a fluorescent biosensor for sensitive and label-free detection of M.SssI MTase activity. The strategy⁹⁵ showed higher amplification efficiency with a detection limit of 0.034 U mL⁻¹, good selectivity and simple design, and the reaction process avoided labeling, isolation steps and cumbersome synthesis, which is expected to be a good platform for the detection of M.SssI MTase. Utilizing the in vivo DNA lesion repair mechanism, Zhang et al. developed a new fluorescent method for the specific and sensitive detection of DNA methyltransferase (DNA MTase) on the basis of targeted cascade isothermal amplification of DNA lesion repair.⁹⁶ Because the uracil repair-mediated exponential isothermal amplification reaction (EXPAR) has high amplification efficiency, the endonuclease cleaves DNA methyltransferase (DNA MTase) efficiently, the single uracil repair-mediated non-specific amplification inhibition produces only a low background signal, and the amplification reaction is carried out under homogeneous isothermal conditions without thermal cycling, washing, and isolation steps, the method has a high sensitivity. In addition, by designing appropriate reactants, this method can also be used for the detection of other DNA methyltransferases, and the design of this protocol shows great potential for biomedical research and clinical diagnostics. Cui and his research group⁹⁷ proposed a novel DNA-based biosensor named probe-amplifier-reporter trifunctional integrated platform (PARTIP), which consists of a single DNA substrate, and this single-stranded DNA plays the roles of probe, amplifier, and reporter at the same time. Multiple amplification processes are spontaneously initiated in the presence of the target to achieve highly sensitive detection of the target. It is worth noting that although multiple amplification processes are included, the entire process can be completed in a simple “one-pot” mode because all reactions are synchronized. The ultra-simple “one-pot” assay mode ensures the potential of PARTIP for general applications, and overall, this strategy provides a versatile assay tool for clinical diagnostics, pharmacological analysis and therapeutic analysis of diseases (Fig. 4).

Fig. 4 (A) Schematic illustration of HpaII-EXPAR-based assay for DNA methyltransferase activity. A The generation of P1 duplex, B the detection principle of M.SssI MTase. Reproduced from ref. 95 with permission of Springer-Verlag GmbH Germany, copyright 2023. (B) Schematic illustration of the Dam MTase assay based on DNA lesion repair-directed cascade isothermal amplification. Reproduced from ref. 96 with permission of Royal Society of Chemistry, copyright 2019. (C) Mechanism of PARTIP for the detection of PNK activity. Reproduced from ref. 97 with permission of Royal Society of Chemistry, copyright 2019.

3. DNA three-dimensional spatial nanostructures

With the rapid development of DNA nanotechnology, DNA nanotechnology has shown great potential for application in the fields of biosensing, bioimaging, drug delivery, cell biology and materials manufacturing,⁹⁸ compare Typical DNA nanostructures include regular shapes (e.g., Y-scaffold), DNA tetrahedron, polyhedron, prisms, DNA dendrimers, amorphous structure (e.g., DNA hydrogel), and DNA origami structure, which are more complex to synthesize. All of these structures are precisely designed to be assembled into spatial structures of specific size and shape due to the programmability of DNA, and DNA is an ideal sensing molecule not only because of its specificity, but also because it is very robust and can function at a wide range of biologically relevant temperature and condition.^99,100 DNA nanostructure sensors based on DNA molecular recognition properties are biocompatible and highly specific, scientists have come to realize the crucial role of DNA as an analytical tool in biomedical, analytical and material science application.¹⁰¹ DNA methyltransferase detection based on three-dimensional DNA nanostructure has also proliferated in recent years, and the commonly used ones are mainly the DNA tetrahedral structure, the G-quadruplex structure, and the DNA hydrogel and DNA origami structure.

3.1. DNA tetrahedron

DNA tetrahedron is a classical three-dimensional framework first proposed by Turberfield and colleagues in 2004. The synthesis of DNA tetrahedron requires only an annealing process.¹⁰² Four carefully designed oligonucleotides containing 55 bases are mixed in equimolar concentration in a salt-containing buffer. Orthotetrahedra with six edges and four vertices were synthesized by increasing the temperature to 95 °C for a few minutes and then rapidly cooling to room temperature, and the DNA tetrahedron is synthesized in high quantity. DNA tetrahedron is an ideal carrier for biosensor application with multiple function, mainly focusing on the four vertices and six double-helical edges. DNA tetrahedral nanostructure is characterized by a simple preparation process, a high yield, a clear structure, a uniform size, a good biocompatibility, programmable, rich in functional modification sites, and strong mechanical rigidity.

DNA nanotechnology¹⁰³ specializes in exploiting the molecular property (e.g., self-assembling property) of DNA to construct new types of nanoscale structure that can be manipulated, and the assembly of DNA nanostructure has the advantages of programmability,¹⁰⁴ ease of modification and ease of synthesis.^105,106 In these technologies, DNA is not used as genetic material, but is used as a template for guiding other molecules in the direction or position of their movement, or for fabricating DNA nanospatial structures that are themselves functional, and so on. The unique properties of DNA strands have potential applications in genetic diagnosis and gene therapy.¹⁰⁷

DNA tetrahedron has been used for in vitro application in the last decades and is excellent tool for molecular biology. DNA tetrahedron can also be used in living cells and in vivo studies such as drug delivery,¹⁰² and we believe that living-cell studies can advance the understanding of cellular metabolism, elucidate disease mechanism, and provide methods for drug discovery, diagnosis, and therapeutic application.^108,109

For DNA methyltransferase assay, Cao and colleagues¹¹⁰ designed a DNA tetrahedral nanostructure (DTN)-initiated hybridization chain reaction (HCR) to assemble large-scale nanozymes for ratiometric fluorescence assay of DNA adenine methyltransferase (Dam). In this study, DNA tetrahedral structure with an alkyne-modified DNA probe (Alk-DTN) is assembled on magnetic beads (MB) and used as a scaffold for click chemistry. The efficiency of the surface-based DNA click reaction was improved and the catalytic activity of the gold nanoparticle nanoenzyme was maintained due to the inherent mechanically rigid structure, good orientation and size tuning of the DNA tetrahedron. Due to the multiplexed amplification technique, the biosensor had a detection limit for Dam MTase as low as 0.001 U mL⁻¹. The protocol was effective in detecting real samples and in screening for methylation inhibitors, providing a promising platform for bioanalysis. The constructed biosensing system has excellent analytical performance, ultra-low detection limit and high selectivity, and has been successfully applied in complex matrices and maybe has the potential to be a promising tool for early clinical diagnosis and drug development.

The author himself has also used DNA tetrahedron to detect DNA methyltransferase,¹¹¹ the protocol design is simple, the detection is fast and the sensitivity of the detection is extremely high. The whole experiment first synthesized the tetrahedron, and then the group designed the dumbbell single chain reaction to form a stable symmetrical double-ring dumbbell structure, and the double-stranded site 5′-GATC-3′ formed by the dumbbell stem was methylated under the catalytic recognition of the Dam methyltransferase, and then carried out the DpnI-specific enzymatic reaction, and the dumbbell was sheared with the DpnI-specific enzymatic reaction when the Dam methyltransferase exists and catalyzes the dumbbell to make the dumbbell methylated. When Dam methyltransferase was present and catalyzed the methylation of the dumbbells, the dumbbells were sheared and reacted with the hairpins at each tip of the tetrahedron to produce a strong fluorescence signal. The stable 3D tetrahedral structure can greatly reduce the interference background signal, and at the same time, the dumbbells can be combined with their four apical fluorescent hairpins after shearing to improve the recognition efficiency of the tetrahedral hairpins, so that the fluorescence can be greatly stimulated, and the fluorescence signal increases with the concentration of Dam methyltransferase to achieve a certain range of linear detection and ultra-low detection limit, and the 5-fluorouracil methyltransferase was found to be the most efficient and effective method for the detection of 5-fluorouracil methyltransferase in the serological experiments. At the same time, serological experiments and 5-fluorouracil methyltransferase inhibitor experiment has also verified the superiority of this method and provided a better reference value for the early diagnosis and treatment of cancer and other diseases, as well as drug research. Zhou et al. utilized the high rigidity and versatility of DNA tetrahedron nanostructure to design a novel multifunctional nanostructure as an “Off–On” fluorescent probe for detection of target methyltransferase.¹¹² This sensing system is initially in the “off” state due to the close proximity of the fluorophore and the quencher. After the substrate is recognized by the target methyltransferase, the DNA tetrahedron can be methylated, producing methylated DNA sites. These sites can be recognized and cleaved by the restriction endonuclease DpnI, which causes the DNA tetrahedron to disintegrate and the fluorescent signal to recover the fluorescent signal, producing an “on” state. The proposed DNA tetrahedron-based sensing method can detect Dam methyltransferase in the range of 0.1–90 U mL⁻¹ and its detection limit is lower than previously reported. The multifunctional DNA tetrahedral nanostructure has many outstanding advantages, such as easy to synthesize, structurally stable and rigid, and rich in modification sites, etc. DNA tetrahedral nanostructure is highly efficient in biosensing, and this scheme also has good specificity and reproducibility in detecting Dam methyltransferase in real samples. Therefore, this work is promising for the detection of DNA methyltransferase in clinical diagnostics, we anticipate that this DNA nanostructure-based strategy will be useful for bioassays, clinical diagnostics and drug discovery (Fig. 5).

Fig. 5 (A) Schematic illustration of the (A) Alk-DTN-MB probe design and preparation and (B) the conceptual method for DNA adenine methyltransferase (DAM) detection. Reproduced from ref. 110 with permission of Royal Society of Chemistry, copyright 2023. (B) Scheme showing the ultra-sensitive detection of Dam MTase using DTFS assisted by SDRDs. Part A: DNA methylation reaction and Dpn I cleavage reaction of the dumbbell-shaped probe in the experiment. Part B: synthesis of the DTFS. Part C: hybridization of the sheared SDRDs with the tetrahedral hairpins. Reproduced from ref. 111 with permission of Elsevier, copyright 2021. (C) Construction of “off–on” fluorescence sensing system with DNA tetrahedron: (a) schematic representation of (1) assembly of DNA tetrahedron, (2) methylation by Dam MTase with the help of SAM, and (3) Cleavage of methylated sites by DpnI to collapse the DNA tetrahedron for recovering the fluorescence signal; (b) complementary sequences in four oligos (S-a, S-b, S-c, and S-d) for construction of tetrahedron. Reproduced from ref. 112 with permission of American Chemical Society, copyright 2017.

3.2. G-quadruplex

The widespread use of G-quadruplex has been discussed by many researchers since the 1980s,¹¹³ and the structure of G-quadruplex nucleic acid is different from that of the typical double-helix structure. The G-quadruplex is a stable coplanar tetragonal nucleic acid secondary structure formed by the pairing of four guanine bases via Hoogsteen hydrogen bonds.¹¹⁴ Four guanines interact to form the guanine quaternary structure, which is the structural unit and building block of the G-quadruplex structure. G-quadruplex structure come in a variety of conformation and can be formed by the stacking of two, three, or more G-quadruplexes. DNA G-quadruplex is specialized three-dimensional DNA nanostructure formed by DNA sequences enriched with specific G. In these G-rich DNA sequences, four guanine bases form a G-quadruplex by Hoogsteen base pairing, and then two or more G-quadruplexes are stacked on top of each other. A G-quadruplex structure is formed, and the intervening sequences are extruded into a single-stranded loop.¹¹⁵ G-quadruplex has become increasingly popular in the supramolecular arena, and the fascinating structure has been used in molecular diagnostic over the past decade. Sen pioneered the discovery that DNA G-quadruplex can enhance hemin peroxidation, and since then G-quadruplex has been commonly used in the detection of DNA, RNA, and other molecules, including proteins, small molecules, and metals.¹¹⁶ Subsequent studies have shown that the DNA G-quadruplex structure is formed not only within a single strand (intramolecular) but also between multiple strands (intermolecular).

The application of G-quadruplex in the detection of DNA methyltransferase is also fruitful, such as Yuan et al. studied a colorimetric biosensor (PER-FHGD nanodevice), which detects DNA methyltransferase activity by integrating primer exchange reaction (PER) amplification and functionalized hemin/G-quadruplex DNAzyme (FHGD),¹¹⁷ and the scheme has excellent sensitivity and selectivity. By replacing the natural hemin cofactor with a functionalized cofactor mimic, FHGD exhibits significantly higher catalytic efficiency, thereby improving the assay performance of the FHGD-based system. This aspect was also well validated for Dam MTase activity in serum and E. coli cell extracts, while by simply changing the recognition sequence of the substrate, the protocol can be well applied to point-of-care (POC) assays.

In a study by Xu and co-workers,¹¹⁸ they developed a multiple sealed primers-mediated rolling circle amplification (RCA) strategy for sensitive and specific detection of DNA methyltransferase activity. The DNA probe is a double ring structure with a folded loop that can be sealed with multiple primers, and in the presence of DNA MTase, the DNA probe is methylated and then cleaved into multiple DNA oligonucleotide fragments by a restriction endonuclease. The DNA oligonucleotide fragments serve as independent primers for triggering the RCA reaction, producing long DNA strands containing several spacer G-quadruplexes. Finally, a large number of G-quadruplexes are obtained, which bind N-methyl mesoporphyrin IX (NMM) and produce significantly enhanced fluorescence. When DNA MTase was absent or inactivated, the DNA probe remained stable and could not release primers for the RCA reaction. The results suggest that this strategy could be a promising tool for DNA MTase activity detection. While Zhang et al. in their study designed a more simple and direct ECL analysis coupled hybridization chain reaction with G-quadruplex/hemin DNAzyme biosensing strategy for the detection of DNA methyltransferase,¹¹⁶ so it can be seen that the G-quadruplex was used in the detection of DNA methyltransferase also play an important role (Fig. 6).

Fig. 6 (A) The schematic illustration of the PER-FHGD nanodevice for DNA MTase activity detection. (A) Working principle of FHGD and HGD. (B) Step of Dam methylation and DpnI digestion. (C) Step of cascade PER amplification. (D) Step of FHGD-catalyzed colorimetric reaction. Reproduced from ref. 117 with permission of Elsevier, copyright 2023. (B) Schematic illustration of the multiple sealed primers-mediated rolling circle amplification strategy for sensitive and specific detection of DNA methyltransferase activity. Reproduced from ref. 118 with permission of Elsevier, copyright 2019. (C) Schematic diagram of the ECL assay for detecting the activity of DNMT1. Reproduced from ref. 119 with permission of Royal Society of Chemistry, copyright 2017.

3.3. Other three-dimensional spatial structures

Other three-dimensional DNA nanotechnologies involving the detection of DNA methyltransferase have also made great breakthrough over the years, such as DNA hydrogel and DNA origami. DNA is used as the sole component, backbone or cross-linking agent of the hydrogel, and pure DNA hydrogel is formed by chemical action (by chemical bonds as cross-linking points) or physical action (non-covalent action such as hydrogen bonding, van der Waals force, or entanglement between strands of DNA molecules, etc.). Since DNA sequences provide programmability based on predictable Watson–Crick base-pairing interaction and structural transformation capability, it is easy to integrate nucleic acid recognition elements and DNA nanostructure into DNA hydrogel, which can be efficiently used for sensing and other purposes as their large specific surface, high biocompatibility, etc. Gao and his associated researchers developed an enzyme-encapsulated target-responsive DNA tetrahedra-cross-linked DNA hydrogel and combined it with commercial glucose test strips to form a POCT tool for portable, sensitive, selective and quantitative determination of Dam activity.¹²⁰ A simple and POCT tool for the timely detection of DNA adenine methyltransferase (Dam) activity by capturing glucose-producing enzyme for target recognition and signaling using DNA tetrahedral hydrogel. This POCT assay tool is capable of highly sensitive and selective determination of Dam activity, even reached a detection limit of 0.001 U mL⁻¹, which is superior to most previously reported biosensors. Thus, DNA tetrahedron-based hydrogel is expected to be an excellent candidate for the construction of target-responsive DNA hydrogel. Simple and quantitative evaluation of Dam can be achieved, and the design of this protocol may enable timely medical diagnosis in the home.

Christian Heck and colleagues proposed a method based on the idea that non-DNA labeling is a key component in the construction of functional DNA nanostructures, grafting covalent labels onto DNA origami nanostructure in a one-pot reaction with an enzyme.¹¹⁸ The DNA methyltransferase M. TaqI labels the DNA nanostructure with an azide moiety, which is used as a generalized attachment point. Direct labeling with fluorescent dyes was also demonstrated. This procedure produces structure with high fluorescence intensity and narrow fluorescence intensity distribution. Combined with UV cross-linking, it produces temperature-stable, intensely fluorescent beacons. This is merely an indication that DNA methyltransferase can be well integrated with DNA origami, however, application in the detection of DNA methyltransferase using DNA origami techniques is few and far between and may be a direction for our future research (Fig. 7).

Fig. 7 (A) Principle of the developed method for Dam activity assay. Reproduced from ref. 120 with permission of American Chemical Society, copyright 2020. (B) Schematic labeling strategy: (1) while the DNA origami partially folds, M.TaqI labels scaffold and staples with azide groups. (2) The enzyme is digested and the DNA origami is properly folded. (3) The label of interest (here: Cy5) is attached via copper-free click reaction. Reproduced from ref. 121 with permission of Royal Society of Chemistry, copyright 2020.

4. Summary and conclusion

DNA methylation is an important genetic modality in human biology, and DNMTs play an important role in the constant development of methylation. Tumor formation is influenced by both genetic and epigenetic modifications, with DNA methylation being one of the most important modifications in the field of epigenetics. Numerous studies have shown that DNA methylation is inextricably linked to tumor development and likely plays a crucial role in the early stages of tumorigenesis. In this review, we summarize some methods for detecting DNMTs. The traditional DNMT assay has obvious drawbacks, mainly including high sample consumption, low sensitivity, time-consuming and laborious operation process, and the need for expensive laboratory equipment.

In the past several years, significant progress has been made in the detection of DNMT activity, which has been of great use in the diagnosis, prognosis, and treatment of cancers. Colorimetric, fluorescent, and electrochemical methods are both convenient and cost-effective, with reduced sample consumption and a relatively desirable limit of detection, and have been successfully applied to human serum samples and even to cells. In the field of DNA nanotechnology, DNA nanostructures have been widely utilized in biomedical research due to advances in their design, shape, and size. This has led to a broad range of applications of DNA nanomaterials in biomedicine, including sensing, diagnosis, therapy, and imaging, as well as functional carriers that play an important role in areas such as drug delivery and nucleic acid detection. Due to the unique characteristics of DNA nanostructures, such as biocompatibility, stability of synthetic structures, and simplicity of the synthesis process, DNA nanostructures are excellent for the transportation and delivery of biomolecules. The flexibility of DNA molecules enables the transformation and creation of a wide variety of DNA nanostructures used in various fields of biomedicine.

For the detection of DNMTs, we outlined several two-dimensional planar DNA nanotechnology and three-dimensional DNA spatial nanotechnology techniques, including HCR, RCA, CHA, and EXPAR, and three-dimensional structures such as DNA tetrahedrons, G-quadruples, DNA hydrogels, and DNA origami. They demonstrate the significant potential of DNA nanomaterials in the biomedical field. We believe that the target detection system constructed based on DNA nanotechnology can be verified in human serum isolation and even live cell tests in the laboratory. However, a number of challenges remain. Firstly, these DNA nanostructure-based sensing techniques are currently in the experimental laboratory phase. The design of DNA nanostructures is crucial, yet external environmental fluctuations may cause detrimental conformational changes, potentially rendering the biosensor ineffective. Additionally, designing diverse DNA structures, heavily reliant on literature and iterative experimentation, is time-intensive due to the complexity of chain interactions, contributing to system instability and necessitating continuous optimization. A significant hurdle lies in translating these sensors for clinical use and point-of-care testing, an area underscored by extensive research. We believe that refining experimental conditions, such as the efficient design of DNA sequences aided by advanced computing technologies for precise DNA strand configuration, could enhance experimental stability. Choosing a consistent environment for stable DNA interactions will also be crucial for assay accuracy, efficiency, and the improvement of specificity and sensitivity. Concurrently, this experimental design effort supports the development of demethylation drugs like 5-fluorouracil, an epigenetically targeted inhibitor approved by the U.S. FDA for cancer treatment. Overall, with the advancement of technology in the field of biological research, possibilities for future improvements are limited, and the transition from laboratory benches to practical or clinical use is just around the corner. We believe that DNMT biosensors based on DNA nanotechnology may find increasing applications in biomedical research, disease treatment, clinical diagnosis, and drug design in the near future.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by Chongqing Natural Science Foundation (grant no. CSTB2022NSCQ-MSX0892) and Jiulongpo district-level technology foresight and system innovation project (grant no. 2023-03-006-Z).

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