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Adenosine is closely related to the development of cancer, and it can be regarded as a biomarker for cancer diagnosis and therapy. Here, a T7 exonuclease (T7 Exo)-assisted and target-triggered cascade dual recycling signal amplification strategy was developed for the sensitive and specific detection of adenosine. In this strategy, a capture strand (Cap)-inhibit strand (Inh) duplex and a DNA hairpin with intact G-quadruplex sequence are designed. In the presence of adenosine, Cap binds with adenosine specifically to form Cap-adenosine complex with recessed 5′-hydroxyl termini, causing the release of Inh. Under the action of T7 Exo, the adenosine is released from Cap-adenosine complex. Then the released adenosine interacts with the next Cap-Inh duplex to promote the release of a new Inh and the recycle I is completed. After that, the released Inh hybridizes with hairpin to form Inh-hairpin duplex with blunt 5′ -hydroxyl termini. Under the same action of T7 Exo, the Inh gets released and a G-quadruplex sequence is obtained. Subsequently, the released Inh continues opening hairpin and the recycle II is accomplished. Finally, plenty of G-quadruplex sequences are generated and then interact with N-methyl-mesoporphyrin IX (NMM) to obtain enhanced fluorescence signal. The limit of detection (LOD) of the proposed strategy is estimated to be 9.8 × 10−7 mol L−1, and the linear range of the strategy is from 5.0 × 10−6 mol L−1 to 7.0 × 10−4 mol L−1. Besides, the proposed strategy is capable of distinguishing adenosine from its analogues. This strategy holds promise in adenosine related biomedical research, clinical diagnosis and disease treatment.

DNA methyltransferase (MTase) aberrant expression has a close relationship to tumorigenesis. DNA MTase activity detection is of great importance to its biomedical research and theranostics study. Here, multiple sealed primers-mediated rolling circle amplification (RCA) strategy is developed for sensitively and specifically detecting DNA MTase activity. The DNA probe has a folded, double-loop structure that seals multiple primers. First, in the presence of DNA MTase, the DNA probe is methylated, which then gets cleaved by the restriction endonuclease and breaks into multiple DNA oligonucleotide fragments. Second, each DNA oligonucleotide fragment acts as an independent primer for triggering RCA reaction respectively, producing long DNA strands that contain several interval G-quadruplexes. Finally, copious of G-quadruplexes are obtained, which bind N-methylmesoporphyrin IX (NMM) to generate significantly enhanced fluorescence. When DNA MTase is absent or inactive, the DNA probe is stable and cannot release the primers for RCA reaction. In the proposed strategy, the action of DNA MTase on one DNA probe is converted to the multiple amplifications triggered by multiple released primers. The detection limit for Dam MTase is down to 0.0085 U/mL, and the target MTase can be well discriminated from its MTases analogues. The method is utilized in screening of Dam MTase inhibitors and analyzing of spiked Dam MTase in biological samples. The results suggest that the strategy may provide a promising tool for DNA MTase activity detection in biomedical research and cancer theranostics.

In the regulatory network, miRNAs play a regulatory role in a cooperative or antagonistic manner. Simultaneous accurate detection and imaging of multiplexed miRNAs in living cells are of great significance for miRNA-associated biological research and disease diagnosis and treatment. Herein, a MnO

Human telomerase is an endogenous ribonucleoprotein that is over-expressed in most types of malignant cancer cells. Sensitive and specific detection of telomerase activity is crucial for better understanding its role in cancer cells and further exploring its function in cancer diagnosis. Here, we develop convertible DNA ends-based silver nanoprobes for sensitive and specific colorimetric detection telomerase activity. Silver nanoprobes are constructed by modifying telomerase binding substrates (TS) that are pre-hybridized with complementary sequences onto silver nanoparticles (AgNPs), via the coordination between consecutive cytosines in TS strand and AgNPs. This forms blunt-end terminated, double-stranded DNA on the surface of AgNPs. Under the action of telomerase, TS on the silver nanoprobes are elongated with telomeric repeats, converting DNA stiff blunt ends to flexible single-stranded dangling ends. The dangling ends enhance the stability of nanoprobes and relieve their salt-induced aggregation, and the solution shows a yellow color. When telomerase is inactive, the blunt end-terminated nanoprobes cannot resist salt-induced aggregation, resulting in a gray color of solution. Based on telomerase-regulated DNA “blunt-dangling” ends conversion-induced AgNPs' dispersity and color change, colorimetric detection of the endogenous telomerase with AgNPs is realized. The detection limit is equivalent to 1 cell/μL of telomerase activity, and extracts from cancer cells and normal cells are visually distinguished through color difference. The proposed strategy will offer a new approach for reliable, convenient quantification of telomerase activity in biochemical research and clinical diagnosis.

In the regulatory network, miRNAs play a regulatory role in a cooperative or antagonistic manner. Simultaneous accurate detection and imaging of multiplexed miRNAs in living cells are of great significance for miRNA-associated biological research and disease diagnosis and treatment. Herein, a MnO2 nanosheet-mediated target-binding-induced fluorescence resonance energy transfer (FRET) strategy was developed for detection and imaging of multiplexed miRNAs in living cells. Two pairs of DNA probes (P1-AF 488/P1′-Cy3 and P2-AF 488/P2′-AF 594) contained the complementary sequence to target miRNAs (miRNA-373 and miRNA-96) and labelled with different fluorescence dyes were designed. They were adsorbed onto MnO2 nanosheets by physisorption to form DNA/MnO2 nanocomposite probes. When the DNA/MnO2 nanocomposite probes were taken up by cells, the MnO2 nanosheets were reduced by intracellular glutathione, accompanying the release of DNA probe pairs. Then the DNA probe pairs specifically recognized and combined with miRNA-373 and miRNA-96 to form stable duplexes, respectively, bringing labelled fluorophores into close proximity to occur FRET. Based on this, the simultaneous imaging of miRNA-373 and miRNA-96 in MDA-MB-231 and L02 cells was successfully implemented. The results displayed a higher expression level of target miRNAs in MDA-MB-231 cells compared to L02 cells. The changes in expression levels of miRNA-96 induced by anti-miRNA-96 or mimics in MDA-MB-231 cells could also be monitored. In addition, the ratiometric detections of multiplexed miRNAs were achieved by utilizing the DNA probe pairs. The proposed strategy provides an alternative method for simultaneous accurate detection and imaging of multiplexed miRNAs and has potential application in biomedical applications.

Sensitive and reliable detection of transcription factors (TFs) is crucial for disease diagnosis and drug discovery. Herein, a protein binding protection in combination with DNA masking-based strategy is developed for sensitive and reliable detection of NF-κB p50. First, NF-κB p50 binds specifically to a hairpin probe to form a protein-DNA complex. Then, the formed complex protects the hairpin probe from hybridization with the masking strand and subsequently triggers the cascade signal amplification of strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Excess hairpin probes that do not bind NF-κB p50 are masked through hybridization with masking strands into stable duplexes, prohibiting the non-specific amplification and avoiding the risk of false positive signals. The signal results from the presence of NF-κB p50, ensuring the detection reliability. Through the cascade amplification specifically triggered by the target, the method can detect the purified recombinant NF-κB p50 down to 1.0 × 10–13 M. It is further employed for the NF-κB p50 inhibitor screening and analysis, which shows the potential in anti-NF-κB p50 drug discovery. Moreover, the method is applied in detection NF-κB p50 in cell nuclear extracts with a detection limit of 0.1 ng μL−1. The proposed strategy will provide a promising tool for sensitive and reliable assaying NF-κB p50 activity in biomedical study and disease diagnosis.

Folate receptor (FR) is over-expressed in most human tumors and has been regarded as biomarker and therapeutic target. Specific and sensitive detection of FR is essential for tumor treatment and drug development. Here, a specific, sensitive and rapid FR detection strategy was proposed based on terminal protection-mediated autocatalytic cascade amplification coupled with graphene oxide fluorescence switch. Firstly, the specific binding of FR to the folate terminally-labeled on a primary trigger DNA (PT-DNA) could protect the PT-DNA from exonuclease I degradation, converting FR detection to PT-DNA detection. Subsequently, the PT-DNA hybridized with the overhang of 3'-FAM labeled hairpin probe to initiate exonuclease III-assisted hydrolysis, accompanied with the PT-DNA recycling and autonomous generation of secondary trigger DNA and fluorophore. The secondary trigger DNA could as a PT-DNA analogue for the successive hybridization and hydrolysis process, liberating numerous fluorophores within 40min. Finally, The fluorophores kept away from the surface of graphene oxide, achieving significantly amplified fluorescence signal. The specific interaction between FR and folate guaranteed the FR could be distinguished from other proteins with high selectivity. The high fluorescence quenching efficiency of graphene oxide guaranteed a low background. Due to the highly autocatalytic cascade amplification efficiency and low background, sensitive detection of FR had been achieved with the detection limit of 0.44pM. The recoveries from 92% to 107% were achieved by detecting FR in spiked human serum. These results indicate this strategy holds a great potential for reliable quantification of FR in clinical diagnosis and disease treatment.

Sensitive detection of telomerase activity is critical to cancer diagnosis, screening of anticancer drugs and evaluation of cancer therapy. Herein, a label-free molecular beacons-based DNA machine was developed for sensitive detection of telomerase activity. The DNA machine consisted of T7 exonuclease (T7 Exo), label-free recognition molecular beacon (RMB) and signal molecular beacon (SMB) with projecting 5'-terminuses, which can protect RMB and SMB from being digested by T7 Exo. Firstly, telomerase elongated telomerase substrate (TS) primer, generating a telomerase elongation production (TEP) with tandem repeats (TTAGGG)n. Next, TEP activated the DNA machine by hybridizing with RMB, unfolding RMB with a recessed 5'-terminus, making RMB deprotection from T7 Exo. Then T7 Exo-assisted cycling cleavage was incurred, releasing intact TEP and numerous DNA fragments (trigger DNA), which got recycling I. Subsequently, trigger DNA specifically opened SMB and was recycled by T7 Exo, liberating multiple G-quadruplex (G4) structures, which got recycling II. Finally, TEP and the liberative G4 structures strongly interacted with N-methyl-mesoporphyrin IX (NMM), yielding a significantly enhanced fluorescence together. In this way, per telomerase-mediated elongation event was efficiently converted into the greatly amplified fluorescence signals. Telomerase activity in crude HeLa cells extracts equivalent to 50 cells/mL was successfully measured with a linear range from 50 cells/mL to 2000 cells/mL. Besides, the strategy was also successfully used to assay the inhibition effect of a telomerase-inhibiting drug, demonstrating the strategy holds the potential to screen telomerase inhibitors.