BACKGROUND: DNA walker-based strategies have gained significant attention in nucleic acid analysis. However, they face challenges related to balancing design complexity, sequence dependence, and amplification efficiency. Furthermore, most existing DNA walkers rely on walking and lock probes, requiring optimization of various parameters like DNA probe sequence, walking-to-lock probe ratio, lock probe length, etc. to achieve optimal performance. This optimization process is time-consuming and adds complexity to experiments. To enhance the performance and reliability of DNA walker nanomachines, there is a need for a simpler, highly sensitive, and selective alternative strategy.
RESULTS: A sensitive and rapid miRNA analysis strategy named hairpin-shaped DNA aligner and nicking endonuclease-fueled DNA walker (HDA-NE DNA walker) was developed. The HDA-NE DNA walker was constructed by modifying hairpin-shaped DNA aligner (HDA) probe and substrate report (SR) probe on the surface of AuNPs. Under normal conditions, HDA and SR remained stable. However, in the presence of miR-373, HDA underwent a conformational transition to an activated structure to continuously cleave the SR probe on the AuNPs with the assistance of Nt.AlwI nicking endonuclease, resulting in sensitive miRNA detection with a detection limit as low as 0.23 pM. Additionally, the proposed HDA-NE DNA walker exhibited high selectivity in distinguishing miRNAs with single base differences and can effectively analyze miR-373 levels in both normal and breast cancer patient serums.
CONCLUSIONS: The proposed HDA-NE DNA walker system was activated by a conformational change of HDA probe only in the presence of the target miRNA, eliminating the need for a lock probe and without sequence dependence for SR probe. This strategy demonstrated a rapid reaction rate of only 30 min, minimal background noise, and a high signal-to-noise ratio (S/B) compared to capture/lock-based DNA walker. The method is expected to become a powerful tool and play an important role in disease diagnosis and precision therapy.

The in vitro detection of circulating tumor cells (CTCs) has been proven as a vital method for early diagnosis and evaluation of cancer metastasis, since the existence and number fluctuation of CTCs have shown close correlation with clinical outcomes. However, it remains difficult and technically challenging to realize accurate CTCs detection, due to the rarity of CTCs in the blood samples with complex components. Herein, we reported a CTCs in vitro detection strategy, utilizing a loop amplification strategy based on DNA tetrahedron and nicking endonuclease reaction, as well as the anti-background interference based on lanthanide metal luminescence strategy. In this work, a detection system (ATDN-MLLPs) composed of an aptamer-functionalized tetrahedral DNA nanostructure (ATDN) and magnetic lanthanide luminescent particles (MLLPs) was developed. ATDN targeted the tumor cells via aptamer-antigen recognition and extended three hybridizable target DNA segments from the apex of a DNA tetrahedron to pair with probe DNA on MLLPs. Then, the nicking endonuclease (Nt.BbvCI) recognized the formed double-strand DNA and nicked the probe DNA to release the target DNA for recycling, and the released TbNps served as a high signal-to-noise ratio fluorescence signal source for CTCs detection. With a detection limit of 5 cells/mL, CTCs were selectively screened throughout a linear response range of low orders of magnitude. In addition, the ATDN-MLLPs system was attempted to detect possible existence of CTCs in biological samples in vitro.

Restriction-modification (R-M) systems form a large superfamily constituting bacterial innate immunity mechanism. The restriction endonucleases (REases) are very diverse in subunit structure, DNA recognition, co-factor requirement, and mechanism of action. Among the different catalytic motifs, HNH active sites containing REases are the second largest group distinguished by the presence of the ββα-metal finger fold. KpnI is the first member of the HNH-family REases whose homologs are present in many bacteria of Enterobacteriaceae having varied degrees of sequence similarity between them. Considering that the homologs with a high similarity may have retained KpnI-like properties, while those with a low similarity could be different, we have characterized a distant KpnI homolog present in a pathogenic Klebsiella pneumoniae NTUH K2044. A comparison of the properties of KpnI and KpnK revealed that despite their similarity and the HNH motif, these two enzymes have different properties viz oligomerization, cleavage pattern, metal ion requirement, recognition sequence, and sequence specificity. Unlike KpnI, KpnK is a monomer in solution, nicks double-stranded DNA, recognizes degenerate sequence, and catalyses the degradation of DNA into smaller products after the initial cleavage at preferred sites. Due to several distinctive properties, it can be classified as a variant of the Type IIS enzyme having nicking endonuclease activity. KEY POINTS: • KpnK is a distant homolog of KpnI and belongs to the ββα-metal finger superfamily. • Both KpnI and KpnK have widespread occurrence in K. pneumoniae strains. • KpnK is a Type IIS restriction endonuclease with a single-strand nicking property.

An ultrasensitive electrochemical biosensor for detecting p53 gene was fabricated based on heated gold disk electrode coupling with endonuclease Nt.BstNBI-assisted target recycle amplification and alkaline phosphatase (ALP)-based electrocatalytic signal amplification. For biosensor assembling, biotinylated ssDNA capture probes were first immobilized on heated Au disk electrode (HAuDE), then combined with streptavidin-alkaline phosphatase (SA-ALP) by biotin-SA interaction. ALP could catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA). While AA could induce the redox cycling to generate electrocatalytic oxidation current in the presence of ferrocene methanol (FcM). When capture probes hybridized with p53, Nt.BstNBI would recognize and cleave the duplexes and p53 was released for recycling. Meanwhile, the biotin group dropt from the electrode surface and subsequently SA-ALP could not adhere to the electrode. The signal difference before and after cleavage was proportional to the p53 gene concentration. Furthermore, with electrode temperature elevated, the Nt.BstNBI and ALP activities could be increased, greatly improving the sensitivity and efficiency for p53 detection. A detection limit of 9.5 × 10-17 M could be obtained (S/N = 3) with an electrode temperature of 40 °C, ca. four magnitudes lower than that at 25 °C.

Early screening of biomarkers benefits therapy and prognosis of cancers. MiRNAs encapsulated in tumor-derived exosomes are emerging biomarkers for early diagnosis of cancers. Nevertheless, traditional methods suffer certain drawbacks, which hamper their wide applications. In this contribution, we have developed a convenient electrochemical approach for quantification of exosomal miRNA based on the assembly of DNA triangular pyramid frustum (TPF) and strand displacement amplification. Four single-stranded DNA helps the formation of primary DNA triangle with three thiols for gold electrode immobilization at the bottom and three amino groups on overhangs for the capture of silver nanoparticles. On the other hand, target miRNA induced strand displacement reaction produces abundant specific DNA strands, which help the DNA structural transition from triangle to TPF. Amino groups are thus hidden and the declined silver stripping current can be used for the evaluation of target miRNA concentration. This biosensor exhibits excellent analytical performances and successfully achieves analysis of exosomal miRNAs from cells and clinical serum samples.

Elevated C-reactive protein (CRP) levels are linked with bacterial infection, local inflammation in osteoarthritis and the increased risk of developing cardiovascular disease. Here, a sensitive and label-free CRP assay is developed by combining cyclic enzymatic signal amplification and capillary electrophoresis (CE) with UV detection. This assay is constructed of base pairing and target recognition. Thereinto, nicking endonuclease (NEase) can recognize the specific nucleotide sequences in double-stranded DNA (dsDNA), which is formed by a CRP aptamer and its complementary DNA (cDNA). Sequentially, NEase cleaves only cDNA to produce signal DNAs. Therefore, a large number of signal DNAs are generated through continuous enzyme cleavage. In the presence of CRP, the aptamer recognizes and binds to CRP with high affinity and selectivity, which results in a decrease in signal DNAs, and thus the UV absorption value of CE significantly decreases, too. A wide linear range was obtained between 0.0125 and 15 μg mL-1 (0.11-130.5 nM) in 1% human serum with a detection limit of 4 ng mL-1 (35 pM). Additionally, the proposed method is universal and can be applied to analyze other similar substances by altering the matched aptamer.

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is \'nicked\' rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I-DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I\'s binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs.

Molecular machines constructed by three-dimensional (3-D) DNA walker have emerged as a hot topic in applications such as novel biosensors, cargo delivery platforms and intracellular imaging. Herein, we first propose a lame DNA walker that can randomly and autonomously move on microsphere-based 3-D track. The stochastic lame walker has a long leg mainly responsible for persistent movement and a short leg cutting substrates rapidly. Its motion is propelled by a nicking endonuclease cleavage of hybridized DNA tracks. Kinetic and persistent study show that the lame DNA walker enables reaction equilibrium at 30 min, need a cleat domain of at least 14 nucleotides and can persistently move on 3-D tracks with an average rate of 6.467 × 10-11 M s-1. We also demonstrate that the lame walker can be used to detect target DNA in the detection range of 10 pM-5 nM with high specificity by toehold exchange mechanism. This work will further expand the performance of 3-D DNA walkers and substantially contributes to the improved understanding of DNA walking systems.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2\' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.