Target-induced activation of DNAzyme for highly sensitive colorimetric detection of bleomycin via DNA scission

In this work, a label-free and sensitive colorimetric sensing strategy for the detection of bleomycin (BLM) was developed on the basis of BLM-mediated activation of G-quadruplex DNAzyme via DNA strand scission. A G-quadruplex based hairpin probe (G4HP) containing the scission site (5′-GT-3′) of BLM at the loop region and guanine (G)-rich sequences at its 5′-end was employed in this protocol. In the presence of BLM, it may cleave the 5′-GT-3′ site of the hairpin probe with Fe(ii) as a cofactor, releasing the G-tetrads DNA fragment, which may further bind hemin to form a catalytic G-quadruplex-hemin DNAzyme. The resultant G-quadruplex DNAzyme has notable peroxidase-like activity, which effectively catalyzes the oxidation of 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) by H₂O₂ to produce the blue-green-colored free-radical cation (ABTS^·+). Therefore, the detection of BLM can be achieved by observing the color transition with the naked eye or measuring the absorbance at a wavelength of 420 nm using a UV-Vis spectrophotometer. Attributing to the specific BLM-induced DNA strand scission and the effective locking of G-tetrads in the stem of the G4HP, the colorimetric sensing strategy exhibits high sensitivity and selectivity for detection of BLM in human serum samples, which might hold great promise for BLM assay in biomedical and clinical research.

A label-free and sensitive colorimetric strategy for bleomycin detection was developed based on target-induced activation of DNAzyme via DNA scission.     

Carbon dot mediated G quadruplex nano-network formation for enhanced DNAzyme activity and easy catalyst reclamation

The significant application potential of the DNAzyme activity of G-quadruplex (G4)–hemin complexes has prompted considerable research efforts to amplify their peroxidase mimicking activity to match that of their enzymatic counterparts. However, concurrent improvements in the catalytic cycle and catalyst recovery remain elusive. Herein, we report the creation of a network array of G-quadruplex (G4)–hemin complexes crosslinked by carbon quantum dots (CDs) that not only significantly improves the G-quadruplex–hemin DNAzyme activity, stability, and catalytic cycle, but also points towards easy catalyst regeneration via a semi-heterogeneous catalysis approach. 5′-phosphate terminated G-rich single-stranded DNA molecules proficient in generating intermolecular and intramolecular G-quadruplexes were covalently conjugated to anthrarufin derived CDs through phosphoramidite chemistry. The network array was achieved through K⁺ mediated intermolecular G-quadruplex formation that readily complexes with hemin to give the catalytic core. The presence of CDs in close vicinity ensures a favorable microenvironment that helps in amplifying the DNAzyme activity in both the intermolecular CD–G-quadruplex network assembly and the intramolecular CD–G quadruplex conjugate, while the former is necessary for easy catalyst regeneration. The CD photophysics enable the monitoring of the DNAzyme recovery and reaction progress.

Enhanced DNAzyme activity of G-quadruplex–hemin complex in carbon dot crosslinked nanonetwork with access to easy catalyst regeneration.     

A one-step fluorescent biosensing strategy for highly sensitive detection of HIV-related DNA based on strand displacement amplification and DNAzymes

Sensitive and specific detection of HIV-related DNA is of great importance for early accurate diagnosis and therapy of HIV-infected patients. Here, we developed a one-step and rapid fluorescence strategy for HIV-related DNA detection based on strand displacement amplification and a Mg²⁺-dependent DNAzyme reaction. In the presence of target HIV DNA, it can hybridize with template DNA and activate strand displacement amplification to generate numerous DNAzyme sequences. With the introduction of Mg²⁺, DNAzyme can be activated to circularly cleave the substrate DNA, which leads to the separation of fluorophore reporters from the quenchers, resulting in the recovery of the fluorescence. Under the optimal experimental conditions, the established biosensing method can detect target DNA down to 61 fM with a linear range from 100 fM to 1 nM, and discriminate target DNA from mismatched DNA perfectly. In addition, the developed biosensing strategy was successfully applied to assay target DNA spiked into human serum samples. With the advantages of fast, easy operation and high-performance, this biosensing strategy might be an alternative tool for clinical diagnosis of HIV infection.

A one-step fluorescent biosensing strategy for highly sensitive detection of HIV-related DNA based on strand displacement amplification and Mg²⁺-dependent DNAzyme reaction.     

DNA circuits driven by conformational changes in DNAzyme recognition arms

DNA computing plays an important role in nanotechnology due to the unique programmability and parallelism of DNA molecules. As an important tool to realize DNA computation, various logic computing devices have great application potential. The application of DNAzyme makes the achievements in the field of logical computing more diverse. In order to improve the efficiency of the logical units run by DNAzyme, we proposed a strategy to regulate the DNA circuit by the conformational change of the E6-type DNAzyme recognition arms driven by Mg²⁺. This strategy changes the single mode of DNAzyme signal transmission, extends the functions of E6-type DNAzyme, and saves the time of signal transmission in the molecular scale. To verify the feasibility of this strategy, first, we constructed DNA logic gates (YES, OR, and AND). Second, we cascade different logic gates (YES–YES, YES–AND) to prove the scalability. Finally, a self-catalytic DNA circuit is established. Through the experimental results, we verified that this DNAzyme regulation strategy relatively reduces the cost of logic circuits to some extent and significantly increases the reaction rate, and can also be used to indicate the range of Mg²⁺ concentrations. This research strategy provides new thinking for logical computing and explores new directions for detection and biosensors.

DNAzyme recognition arms conformational changes drive DNA nanoscale logic circuits without the need for cleavage.     

Visual detection of Fusarium proliferatum based on asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme

A one-step and instrument-free visual method was established based on asymmetric recombinase polymerase amplification coupled with hemin/G-quadruplex DNAzyme for the detection of Fusarium proliferatum.

Asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme-based visual detection of F. proliferatum is demonstrated.

Fusarium proliferatum causes rot disease, which is difficult to control worldwide. The vascular systems of F. proliferatum-infected crops are destroyed. It causes rot of the stems, stalks, roots, flowers, and ears of maize^1–3 and decreases its yield and quality remarkably. In addition, the mycotoxins of fumonisin B1, fumonisin B2, beauvericin, enniatins, fusaproliferin, and moniliformin are produced by F. proliferatum during infection processes.^4–6 When these grains are used as food or feedstuffs, the health of consumers or livestock is exposed to danger.⁷ There are many reports on the illnesses of livestock and humans caused by Fusarium mycotoxins.^8–10 Methods based on qPCR have been developed for F. proliferatum detection.¹¹ Although they show high accuracy, these methods require expensive instruments and skilled operators; they are also limited by many factors, such as electric power, high cost, and long testing times, which results in inability to apply these methods outside the lab. Therefore, sensitive and simple detection of F. proliferatum is still needed in field testing of crops and their byproducts.Bio-sensing has become important in recent decades because it provides alternative methods to solve some of the above problems.^12–14 Recombinase polymerase amplification (RPA)¹⁵ is a conventional and isothermal method to obtain double-stranded DNA (dsDNA). There are many reports of rapid detection based on RPA,¹⁶ such as monitoring of viruses, plasmodium, mycoplasma, fungi, and other causative agents. End-point detection is usually performed using lateral flow strips.^17–19 The greatest strengths of the lateral flow strip method are that it is rapid and straightforward. However, its cost is quite high because of the primer labeling and preparing of the strips. In addition, the strip is disposable, which is not suitable for high throughput detection. The product of RPA can also be observed with the aid of SYBR Green I in UV light.²⁰ However, there is no selectivity of binding between SYBR Green I and dsDNA, which results in false positive results of primer dimers and nonspecific amplification of other dsDNA.Methods of visual detection are greatly valued due to the intuitiveness of the results.²¹ Gold nanoparticles are widely used for colorimetric detection of various targets.²² However, preparation of gold nanoparticles with specific sizes is laborious and difficult. Proteins, genomic DNA, salts, et al. are present in the crude liquids of biological samples used in point-of-care detection, which may give rise to nonspecific aggregation of gold nanoparticles. Hemin/G-quadruplex-based visual methods are used to detect targets of nucleotides, proteins, and other signal molecules.²³ In the presence of hemin/G-quadruplex DNAzyme, H₂O₂ and ABTS²⁻ react, and a specific green color is observed; this method is very simple, cost-effective, and straightforward compared with lateral flow strips or gold nanoparticles.Asymmetric RPA is a type of amplification that involves different concentration ratios of primer-F/primer-R. First, the dsDNA product is obtained with the primer pair. As the reaction continues, one primer in a small amount is exhausted, and single-stranded DNA (ssDNA) is produced by the other primer using the newly synthesized dsDNA as a template. In this paper, an one-step and instrument-free strategy for visual monitoring of F. proliferatum is established based on asymmetric recombinase polymerase amplification coupled with hemin/G-quadruplex DNAzyme.The strategy is shown in Scheme 1; it includes extraction of genomic DNA of F. proliferatum DSM62267 (F120), asymmetric RPA, and visual detection. First, genomic DNA was released from F120 cells with NaOH solution. It was used directly as a template for asymmetric RPA with primer-F and primer-R in a certain ratio. The trans-complementary sequence of the G-quadruplex was designed at the 5′-terminal of primer-F. The RPA product was obtained in the presence of a TwistDx/TwistAmp® Liquid Basic kit. The product was a mixture of dsDNA and ssDNA with the G-quadruplex sequence at their 3′-ends. In the presence of NH₄⁺ and hemin, a DNAzyme of hemin/G-quadruplex was constructed at the end of the ssDNA. When ABTS²⁻ and H₂O₂ were added, the resulting solution turned green. However, no color changes occurred in the final solution if no F120 genomic DNA was present, no target DNA was extracted, or no ssDNA was amplified by asymmetric RPA.Open in a separate windowScheme 1Schematic of the asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme-based visual detection of F. proliferatum.A genomic DNA mixture of maize and F120 was extracted from maize flour using NaOH solution. The feasibility of the asymmetric RPA reaction²⁴ was evaluated using this genomic DNA as a template. The ratios of primer-F and primer-R were set as 1 : 1, 1 : 10, 1 : 20, 1 : 50, 1 : 75, and 1 : 100. The products were analyzed by 2.5% agarose gel electrophoresis and stained with ethidium bromide. As shown in Fig. 1A, the bands of ssDNA were all in front of the bands of dsDNA, compared with the band of the 1 : 1 ratio of the primer pair (1). Faint bands of ssDNA were observed when the ratio of primer-F and primer-R was 1 : 75 (5) or 1 : 100 (6). When the ratio was 1 : 20 (3), the band of ssDNA was brighter than those of 1 : 10 (2) and 1 : 50 (4). The yellow line represents the leading edge of the ssDNA bands. At the same time, the bands of dsDNA tapered off with the appearance of ssDNA and the down-ratio of primer-F/primer-R. These data show that genomic DNA of F120 could be extracted by NaOH solution, and the unpurified template of F120 was available for asymmetric RPA. In addition, more ssDNA was produced by the primer pair ratio of 1 : 20 than the other ratios; thus, this ratio was used in the following tests.Open in a separate windowFig. 1(A) 2.5% agarose gel electrophoresis analysis of asymmetric RPA with ratios of primer-F/primer-R of 1 : 1 (1), 1 : 10 (2), 1 : 20 (3), 1 : 50 (4), 1 : 75 (5), and 1 : 100 (6). Line 7 is the blank control, and genomic DNA of healthy maize flour was used as the template. The yellow line represents the leading edge of the ssDNA bands. (B) Colorimetric detection of the solution (pH 7.9) containing 0.6 μM hemin, 150 mM NH₄Cl, 2 mM H₂O₂, and 2 mM ABTS²⁻ within the wavelength range of 390 to 490 nm, (a) blank control, (b) in the absence and (c) in the presence of genomic DNA of F. proliferatum at the primer-F/primer-R ratio of 1 : 20. The inset in B is an image of the resulting colors with the corresponding samples.To demonstrate the feasibility of the asymmetric RPA-hemin/G-quadruplex assay, the absorbance of the resulting solutions was measured by UV-Vis absorption (390 to 490 nm) of NanoDrop 2000. As shown in Fig. 1B, no absorption signal was observed in solutions containing hemin, H₂O₂ and ABTS²⁻ (a) or the assay without genomic F120 DNA near 420 nm (b). An obvious increase of absorbance was observed in the presence of the target DNA (c) at 420 nm. A specific green color appeared in tube c (inserted in the top right corner of Fig. 1B). This result was in keeping with that of the UV-Vis absorption, demonstrating that the asymmetric RPA coupled with hemin/G-quadruplex DNAzyme method is viable. The visual biosensor could monitor F. proliferatum in maize flour.The intensity of UV-Vis absorption generated by the asymmetric RPA-hemin/G-quadruplex assay depended on the temperature gradient and time of the RPA reaction, as well as the different monovalent cations in the final solution (Fig. S1–S3^†). To assess the analytical performance of the asymmetric RPA-hemin/G-quadruplex assay, various amounts of F120 genomic DNA were monitored under the optimal conditions. The UV-Vis absorption increased with the amount of F120 DNA from 0 to 50 ng (Fig. 2A). A larger amount of F120 DNA was used as the template, and more ssDNA was produced by the asymmetric RPA reaction. More hemin/G-quadruplex DNAzyme was constructed, causing an increase of the UV-Vis absorption of the resulting solution. A good linear dependence was observed between the absorbance and the amount of genomic DNA of F120 in a range from 0.01 ng to 50 ng at 420 nm (Fig. 2B and S4^†). The regression equation was A = 0.1042 lg m + 1.331, with a correlation coefficient of 0.989, in which A and m represent the absorbance and quantity of F120 genomic DNA. The detection limit was calculated to be 0.01 ng by three times the standard deviation of the blank. The type of reaction vessel, reaction temperature, and sensitivity of the proposed strategy were compared with RPA-related colorimetric detection methods (ESI Table 1^†).Open in a separate windowFig. 2(A) Absorption curves of solutions containing various amounts of genomic DNA of F120 in the wavelength range of 390 to 490 nm. The arrow from a to h represents DNA amounts of 0 ng (a), 0.01 ng (b), 0.1 ng (c), 0.5 ng (d), 1 ng (e), 5 ng (f), 10 ng (g), and 50 ng (h). The inset shows an image of the visual detection of the corresponding quantities of genomic F120 DNA. (B) The linear correlation between the absorbance and the negative logarithm of genomic F120 DNA quantity at 420 nm.The selectivity of the proposed assay is critical because numerous DNA sequences belonging to the host and other living bodies are present in biological samples. The primer pair used in the strategy was based on the intergenic sequence of the ribosomal RNA gene cluster, which is specific to species of F. proliferatum. Three fungi, F. equiseti RD13 (F216), F. culmorum 3.37 dus Bomm (F109), and F. avenaceum borm (F112), are also species of Fusarium. Ralstonia solancearum and Puccinia sorghi are common causes of disease in corn. The three Fusarium species, R. solancearum, and P. sorghi were used as controls to check the selectivity of the proposed strategy. As shown in Fig. 3, the absorbances of the final solutions of F. equiseti RD13 (2), F. culmorum 3.37 dus Bomm (3), F. avenaceum borm (4), R. solancearum (5), and P. sorghi (6) were as low as that of the blank control (1). However, the absorbance from genomic F120 DNA (7) was obviously high, showing the good selectivity of our strategy.Open in a separate windowFig. 3Selectivity of the proposed method. (1) Blank control, (2) F. equiseti RD13, (3) F. culmorum 3.37 dus Bomm, (4) F. avenaceum borm, (5) R. solancearum, (6) P. sorghi, and (7) F. proliferatum DSM62267.To demonstrate the feasibility of the proposed assay for real sample detection, 4 positive samples and 10 field-collected samples were successively tested by the RPA/G-quadruplex visual assay and PCR. The results show that 2 of the 8 field-collected samples were infected with F. proliferatum; also, the two methods were consistent, indicating that our RPA/G-quadruplex visual strategy is plausible and can be used in field tests (

Label-free fluorescent aptasensor for chloramphenicol based on hybridization chain reaction amplification and G-quadruplex/N-methyl mesoporphyrin IX complexation

The use of the broad-spectrum antibiotic chloramphenicol (CAP) in food is strictly regulated or banned in many countries. Herein, for the sensitive, rapid, and specific detection of CAP in milk, a label-free fluorescence strategy was established based on guanine (G)-quadruplex/N-methyl mesoporphyrin IX (NMM) complex formation and hybridization chain reaction (HCR) amplification. In this system, CAP can specifically bind to an aptamer (Apt) to release an Apt-C sequence from double-stranded DNA (Apt·Apt-C). Apt-C, can further hybridize with a functional hairpin DNA probe to release a primer sequence. The released primer sequence causes HCR and the formation of a nicked double-helix polymer, which contains G-quadruplex DNA. The recognition of G-quadruplex DNA by the NMM fluorochrome results in fluorescence enhancement. Consequently, CAP can be quantitatively detected by measuring the fluorescence intensity at 612 nm. The reliability of the aptasensor method was confirmed by comparison with an enzyme-linked immunosorbent assay. The proposed aptasensor was found to have a limit of detection of 0.8 pg mL⁻¹ for CAP. Moreover, when the aptasensor was applied to the detection of CAP in milk samples, the average recoveries were 99.8–108.3% with relative standard deviations of 4.5–5.2%. Thus, this CAP detection method, which is rapid with high sensitivity and selectivity, has considerable potential for a wide range of food analysis applications.

For the sensitive and specific detection of CAP in milk, a label-free fluorescence strategy was established based on guanine (G)-quadruplex/N-methyl mesoporphyrin IX (NMM) complex formation and hybridization chain reaction (HCR) amplification.     

Proximity hybridization triggered strand displacement and DNAzyme assisted strand recycling for ATP fluorescence detection in vitro and imaging in living cells

We developed a novel strategy for ATP detection in vitro and imaging in living cells based on integrating proximity hybridization-induced strand displacement and metal ion-dependent DNAzyme recycling amplification. Four DNA oligonucleotides were used in the sensing system including two aptamer probes, enzymatic sequences and FAM-linked substrate strands. Upon the addition of ATP, the proximity binding of two aptamers to ATP led to the release of the enzymatic sequences, which hybridized with the FAM-linked substrate strand on the graphene oxide (GO) surface to form the ion-dependent DNAzyme. Subsequent catalytic cleavage of the DNAzyme by the corresponding metal ions results in recycling of the enzymatic sequences and cyclic cleavage of the substrate strand, liberating many short FAM-linked oligonuleotide fragments separated from the GO surface, which results in fluorescence enhancement due to the weak affinity of the short FAM-linked oligonuleotide fragment to GO. The amount of produced short FAM-linked oligonuleotide fragments is positively related to the concentration of ATP. This means that one target binding could result in cleaving multiplex fluorophore labelled substrate strands, which provided effective signal amplification. The vivo studies suggested that the nanoprobe was efficiently delivered into living cells and worked for specific, high-contrast imaging of target ATP. More importantly, this target-responsive nanoscissor model is an important approach for intracellular amplified detection and imaging of various analytes by selecting appropriate affinity ligands.

A novel strategy for ATP imaging was developed based on proximity binding-induced strand displacement and metal ion-dependent DNAzyme recycling.     

DNA adsorption on nanoscale zeolitic imidazolate framework-8 enabling rational design of a DNA-based nanoprobe for gene detection and regulation in living cells

DNA functionalized nanomaterials have attracted tremendous attention for bioanalytical applications. Owing to exceptional fluorescence quenching ability, most DNA-based nanoprobes were designed with turn-on signals for target gene detection, while only a few of them could simultaneously achieve gene detection and regulation in one system. In this study, we explored the use of nanoscale zeolitic imidazolate framework-8 (ZIF-8) as a building block to construct a DNA-based nanoprobe. We found ZIF-8 could stably adsorb DNA to resist the dissociation by various biological ligands, enabling potential biological applications. However, ZIF-8 was not a nano-quencher to turn off the fluorophore labeling on the adsorbed DNA. We therefore designed a DNAzyme embedded molecular beacon (DMB) to functionalize ZIF-8. After endocytosis, ZIF-8 was disintegrated to release DMB for target mRNA detection, and the co-released Zn²⁺ acted as an effective cofactor to activate the embedded DNAzyme for mRNA regulation. This study provides a versatile nano-platform to realize multiple functions inside cells by using functional nucleic acids, which holds great promise for theranostic applications.

Boosting DNA-based nanotheranostics for gene detection and regulation by ZIF-8.     

Detection of nucleic acids via G-quadruplex-controlled l-cysteine oxidation and catalyzed hairpin assembly-assisted signal amplification

The development of simple, sensitive and cost-effective methods for specific nucleic acid detection has attracted tremendous attention due to its importance to the early diagnosis of genetic diseases and to biodefense applications. In this work, we demonstrated a fluorescent turn-off mode DNA assay based on l-cysteine-modulated synthesis of CdTe quantum dots (CdTe QDs), horseradish peroxidase-mimicking G-quadruplex–hemin–K⁺ complex controlled oxidation of l-cysteine to cystine, and catalyzed hairpin assembly (CHA)-assisted signal amplification. After the addition of target DNA, the CHA signal amplification reaction was triggered and numerous H1–H2 double-stranded DNA were formed, initiating the release of G-quadruplex sequences in H2 simultaneously. Thus, the degree of inhibition of the synthesis of CdTe QDs is proportional to the concentration of the G-quadruplex sequence in this method. In contrast, when the target DNA was absent, the CHA could not be triggered, and the fluorescence signal was high due to the remaining intact l-cysteine. Under optimal experimental conditions, the homogeneous fluorescence method achieved the detection of HIV DNA with a linear range from 0.1 pM to 1 nM and a detection limit of 0.12 pM. This novel biosensor exhibits excellent specificity in differentiating DNA sequences with a single-base and two-base mismatch. To the best of our knowledge, this a label-free and highly sensitive bioassay utilizing CHA-assisted signal amplification and G-quadruplex control of in situ synthesis of CdTe QDs strategy was not reported in previous. Thus, this proposed strategy is anticipated to find use in basic biochemical research and clinical diagnosis.

A novel homogeneous strategy for detection of DNA via biomimetic synthesis of luminescent QDs coupled with nucleic acid signal amplification.     

Rational design of sequestered DNAzyme beacons to enable flexible control of catalytic activities

DNAzymes as functional units play increasingly important roles for DNA nanotechnology, and fine control of the catalytic activities of DNAzymes is a crucial element in the design and construction of functional and dynamic devices. So far, attempts to control cleavage kinetics can be mainly achieved through varying the concentrations of the specific metal ions. Here we present a facile sequestered DNAzyme beacon strategy based on precisely blocking the catalytic core of the DNAzyme, which can flexibly regulate the DNAzyme cleavage kinetics without changing the concentrations of metal ions. This strategy can be extended to couple with a large number of other RNA-cleaving DNAzymes and was successfully applied in designing a dual stem-loop structure probe for arbitrary sequence biosensing, which provides the possibility of scaling up versatile and dynamic DNA devices that use DNAzymes as functional modules.

We present a sequestered DNAzyme beacon strategy based on precisely blocking the catalytic core for flexible regulation of DNAzyme kinetics.     

Significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium

We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10). Our CD results showed that the peptide bound to the c-Myc G-quadruplex and induced a significant blue shift in the positive peak of 20 nm in KCl alone or with 40wt% PEG200 or 20wt% PEG8000 in comparison to NaCl. Our Native Gel results confirmed that peptide binding destabilized the duplex and stabilized the unimolecular G-quadruplex and not binding to i-motif. UV thermal results confirmed destabilization of bimolecular structure and stabilization of unimolecular G-quadruplex. QW10 showed preferential binding towards c-MYC promoter G4 with binding constant (K_b) values of the order of 0.05 ± 0.2 μM, 0.12 ± 0.1 μM and 0.05 ± 0.3 μM for complexes in K⁺ alone or 40wt% PEG 200 or 20wt% PEG 8000 respectively. QW10 showed preferential cytotoxicity with IC₅₀ values of 11.10 μM and 6.44 μM after 72 and 96 hours'' incubation on Human Breast Carcinoma MDA-MB 231 cells and was found to be non-toxic with Human Embryonic Kidney (HEK-1) cells. Interestingly, we observed reduction of c-Myc gene expression by 2.5 fold due to QW10 binding and stabilizing c-MYC G4. Our study for the first time provides an expanded overview of significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10).     

Formation of G-quadruplex structure in supercoiled DNA under molecularly crowded conditions

G-quadruplex is a secondary structure of nucleic acids that plays crucial roles in many significant biological processes. Potential G-quadruplex-forming sequences exist widely in various regions of the genome such as telomeres and gene promoters. In spite of the fact that G-quadruplex can be readily assembled from a single-stranded segment of DNA, its formation from duplex DNA is very difficult under physiological conditions because Watson–Crick interactions in guanine rich segments need to be weakened first. It is demonstrated in our studies that intrastrand G-quadruplex generated from a perfectly matched guanine-rich duplex in a circular DNA as a result of significant quadruplex stabilization and duplex destabilization created by the combined actions of negative DNA supercoiling and molecular crowding conditions.

It is demonstrated that G-quadruplex generated from G-rich duplex in a circular DNA as a result of quadruplex stabilization and duplex destabilization created by the combined actions of negative DNA supercoiling and molecular crowding condition.     

Highly sensitive colorimetric sensing of copper(ii) ions based on “CLICK-17” DNAzyme-catalyzed azide modified gold nanoparticles and alkyne capped dsDNA cycloaddition

A click chemistry assay based on a newly discovered DNAzyme, CLICK-17, with azide modified gold nanoparticles (azide-AuNPs) and alkyne capped dsDNA (alkyne-linker DNA) was employed for novel and selective detection of Cu²⁺ visually. The strategy involved using CLICK-17 to mediate a catalytic reaction for triazole formation between azide-AuNPs and alkyne-linker DNA under the help of Cu²⁺ (without sodium ascorbate) or Cu⁺, which eventually led to the aggregation of AuNPs. The obvious color change from ruby red to bluish purple was then observed by the naked eye and the absorbance peak shifted from 525 to 570 nm. Interestingly, CLICK-17 and Cu⁺-catalyzed click reaction had the best performance compared to either Cu⁺ alone or CLICK-17 and Cu²⁺-mediated reaction in terms of the reaction time and sensitivity. This system has been demonstrated to allow quantitative measurement of Cu²⁺ with a detection limit as low as 26.8 nM and also has high specificity that can distinguish Cu²⁺ from other metal ions. Further, the method was tested with a real mineral water sample for Cu²⁺ concentration determination. Satisfactory recoveries of 90.8% and 99.8% were achieved.

We report selective and visual detection of Cu²⁺ based on aggregation of azide modified gold nanoparticles induced by CLICK-17 DNAzyme and Cu²⁺ or Cu⁺ catalyzed click reaction between azide-AuNPs and alkyne-dsDNA.     

A DNA small molecular probe with increasing K+ concentration promoted selectivity

DNA small molecular probe study was considered as a promising approach to achieve DNA related disease diagnosis. Most related reports were performed under specific salinity. Herein, 4-imino-3-(pyridin-2-yl)-4H-quinolizine-1-carbonitrile (IPQC) was generated via a facile procedure with high yield (85%). It is found that IPQC could act as a universal probe for most tested ssDNA, dsDNA and G4 DNA in low [K⁺] concentration (less than 20 mM). However, IPQC showed highly selective G4 DNA binding via UV-vis and fluorescence response in increasing [K⁺] (e.g., 150 mM) conditions. The ion atmosphere effects are instructive for DNA probe exploration. This provides guidance for the design, selection and optimization of the probes for target DNA sensing.

DNA small molecular probe study was considered as a promising approach to achieve DNA related disease diagnosis.     

Selective recognition of human telomeric G-quadruplex with designed peptide via hydrogen bonding followed by base stacking interactions

We described a novel synthetic peptide in which a glutamine residue binds through hydrogen bonding to a guanine-base and a trytophan residue intercalates with K⁺ resulting in stabilization of a human telomeric G-quadruplex with high selectivity over its complementary c-rich strand and a double-stranded DNA and its complementary C-rich strand. This peptide offers great potential for cancer treatment by inhibiting the telomere extension by telomerase.

A new synthetic peptide is presented. A Glu residue binds through H-bonding to a guanine-base and a Trp residue intercalates with K⁺ resulting in stabilization of a human telomeric G-quadruplex with high selectivity over a complementary c-rich strand and double-stranded DNA.     

Monitoring spin coherence of single nitrogen-vacancy centers in nanodiamonds during pH changes in aqueous buffer solutions

We report on the sensing stability of quantum nanosensors in aqueous buffer solutions for the two detection schemes of quantum decoherence spectroscopy and nanoscale thermometry. The electron spin properties of single nitrogen-vacancy (NV) centers in 25 nm-sized nanodiamonds have been characterized by observing individual nanodiamonds during a continuous pH change from 4 to 11. We have determined the stability of the NV quantum sensors during the pH change as the fluctuations of ±12% and ±0.2 MHz for the spin coherence time (T₂) and the resonance frequency (ω₀) of their mean values, which are comparable to the instrument error of the measurement system. We discuss the importance of characterizing the sensing stability during the pH change and how the present observation affects the measurement scheme of nanodiamond-based NV quantum sensing.

We report on the sensing stability of quantum nanosensors in aqueous buffer solutions for the two detection schemes of quantum decoherence spectroscopy and nanoscale thermometry.     

Mesoporous MnFe2O4 magnetic nanoparticles as a peroxidase mimic for the colorimetric detection of urine glucose

Mesoporous MnFe₂O₄ magnetic nanoparticles (mMnFe₂O₄ MNPs) were prepared with a one-step synthesis method and characterized to possess intrinsic peroxidase-like activity, and had obvious advantages over other peroxidase nanozymes in terms of high catalytic affinity, high stability, mono-dispersion, easy preparation, and quick separation. The mMnFe₂O₄ MNPs were used as a colorimetric sensor for indirect sensing of urine glucose based on the sensing principle that H₂O₂ can be produced from glucose oxidation catalyzed by glucose oxidase (GOx), and under the catalysis of the mMnFe₂O₄ MNPs nanozyme, H₂O₂ can oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a blue color in a few minutes. This sensor is simple, cheap, sensitive, and specific to glucose detection with a detection limit of 0.7 μM, suggesting its potential for on-site glucose detection.

Schematic illustration of glucose detection with glucose oxidase (GOx) and mMnFe₂O₄ MNPs-catalyzed system.     

A new G-triplex-based strategy for sensitivity enhancement of the detection of endonuclease activity and inhibition

EcoRI is an important biomacromolecule in live cells and protects bacterial cells against foreign DNA. In this work, we developed a simple and convenient G-triplex (G3) (5′-TGGGAAGGGAGGGAATTCCCT-3′)-based colorimetric assay for the rapid and selective detection of EcoRI activity and inhibition. The sequence specifically responds to EcoRI in the presence of K⁺ and hemin to form a G-triplex/hemin complex. Taking advantage of G-triplex, EcoRI activity was investigated under the optimized conditions. The absorption intensity ratio displayed a linear relationship against the concentration of EcoRI in the range 0 to 100 U mL⁻¹, and the detection limit was 5.7 U mL⁻¹. Furthermore, G3 showed good selectivity, and the ability to be used to screen for EcoRI inhibitors, indicating its potential in detection and analysis applications.

A new G-triplex-based probe was developed for detecting EcoRI activity and inhibition. The probe showed good selectivity towards EcoRI. The assay was colorimetric and can be monitored by the naked eye.