Apoptosis is a typical programmed death mode with complex molecular regulation mechanisms. Developing advanced strategies to monitor apoptosis progression is conducive to disease treatment related with apoptosis. Herein, we developed a regulator-carrying dual-responsive integrated AuNP composite fluorescence probe for in situ real time monitoring apoptosis progression. The nanoprobe is constructed by modifying specially designed double-stranded DNA (dsDNA) and caspase 3-specific cleavable peptides (pep) to the surface of AuNP. After uptake by cells, the nanoprobe recognizes miRNA 21 and triggers fluorescence recovery, enabling silencing and imaging of the upstream signaling molecule miRNA 21. Once miRNA 21 is silenced, the downstream signaling molecule caspase 3 is activated and cleaves the substrate peptides, and fluorescence is restored for in situ imaging of caspase 3. The apoptosis induced by silencing miRNA 21 has been successfully implemented in HeLa and A549 cells. The expression level of miRNA 21 and corresponding changes of caspase 3 have also been effectively monitored. These results suggested this nanoprobe will be a potential tool for apoptosis-related biomedical research and clinical application.

Sensitive and accurate detection of flap endonuclease 1 (FEN1) is essential to understand its roles in DNA replication and repair and explore its functions in tumor diagnosis and prognosis. Here, we designed a magnetic separation-assisted cascade hybridization chain reaction (HCR) amplification strategy for enzyme-free and sensitive detection of FEN1. Firstly, the forked dsDNAs contained the recognition site (5 ' overhanging DNA flap) of FEN1 were modified on magbeads surface to form magnetic recognition probes. By the recognition and catalytic cleavage of FEN1, the 5 ' flaps were removed into solution and then purified by magnetic separation. Then, the obtained flaps served as triggers to activate the linear-assembly of hairpin probes H1 and H2 (primary HCR), forming nicked double helices with split fragments. Finally, the fragments acted as new triggers to activate the branched-assembly of hairpin probes H3 and H4 (secondary HCR), causing fluorophore/quencher labeled on H3 far away and fluorescence recovery. Benefiting from the purification of triggers by magnetic separation and the signal amplification effect of cascade HCR, this strategy achieved a low detection limit of 3.6 x 10(-5) U mu L-1 for FEN1, and enabled the assay of it in actual samples. The results demonstrate that our strategy will provide a potent approach for sensitive detection of FEN1 in early tumor diagnosis and prognosis.

Acquiring information on telomerase activity at multiple levels contributes to a better understanding of its role in various physiological and pathological processes. Herein, a primer extension activating 3D DNAzyme walker is developed for in situ imaging and sensitive detection of telomerase activity. This walker is constructed via co-modifying specially designed hairpin structured walking strands and track strands on a gold nanoparticle (AuNP). The walking strand contains a pre-blocked DNAzyme sequence and a telomerase primer hybridized to its root. The track strand embeds at an RNA cleavage site and is labeled with the FAM group. After this walker is taken up by cells, the telomerase primer is extended under the action of endogenous telomerase to liberate DNAzyme. The liberated DNAzyme cuts track strands in the presence of the cofactor Mn2+ to drive the walker's processive operation, resulting in an enhanced fluorescence recovery of the AuNP-quenched FAM fluorophore. In situ imaging of telomerase activity in three different cell lines (MCF-7 cells, HeLa cells and HL-7702 cells) was well implemented. The discrimination of cancer cells from normal cells and the screening of telomerase inhibitors have been achieved. The sensitive detection of telomerase activity in HeLa cell lysate has also been realized with a detection limit of 10 cells. This walker performed a new approach for monitoring telomerase activity from different levels, providing a potential tool for clinical diagnosis, prognostic evaluation and drug screening.

Adenosine triphosphate (ATP), as the primary energy source and significant biomarker, is associated with the occurrence and progression of various diseases. Therefore, detecting ATP sensitively is essential for ATP-related biochemical study, disease diagnosis and treatment. Herein, sensitive electrochemical aptasensing of ATP was proposed based on MOF-derived porous Co3O4 coupled with AuNPs and nucleic acids as a novel electrocatalysis signal probe. Firstly, Au@Co3O4 nanocomposite was synthesized via pyrolysis Co-zeolitic imidazolate framework and then functionalized with AuNPs by in situ chemical reduction, which showed excellent electrocatalytic activity towards hydroquinone (HQ) as well as excellent stability. Then, DNA capture probe (CP) was connected to Au@Co3O4 nanocomposite via Au-S bond to form Au@Co3O4-CP signal probe for signal transduction. Meanwhile, well-designed aptamer probe (AP) containing ATP aptamer and its complementary sequence was immobilized on AuNPs modified electrode surface via Au-S bond. In the presence of ATP, the aptamer in AP interacted with ATP, resulting in the dissociation of AP. Consequently, Au@Co3O4-CP signal probe could be captured on electrode surface via hybridization, leading to a significant amplified electrocatalytic signal towards HQ. Finally, the quantitative determination of ATP was achieved by determining the current signal generated on electrode surface. The proposed electrochemical aptasensor demonstrated excellent sensitivity and selectivity that could detect ATP as low as 0.3 nM. Moreover, the aptasensor showed excellent performance for detecting ATP in complex serum matrix. These results indicate that MOF-derived nanocomposites have promising application in construction of novel electrocatalysis signal probe as well as versatile bioassays.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase found to be overactive in most leukemia cells and is recognized as a biomarker of leukemia. Here, we develop a strand-elongation initiated DNAzyme walker to detect TdT activity. The walker is constructed by modifying specially designed track strand (TS) and incomplete walking strand (WS) onto the surface of a mesoporous silica nanoparticle (MSN) loaded with large amounts of rhodamine 6 G (Rh6 G) in its pores. The TS is a hairpin DNA with an RNA cleavage site, serving as substrate for DNAzyme and encapsulating the Rh6 G. The incomplete WS contains one side of binding arm and partial catalytic core of DNAzyme. Under the action of TdT, the incomplete WS is elongated with polyA, completing the lacking part of catalytic core and the other side of binding arm of DNAzyme. Then the completed DNAzyme cleave the TSs, unblocking and releasing the Rh6 G to indicate the TdT activity. This walker is completed and initiated only after the action of TdT, ensuring its strict specificity for TdT activity. Besides, each WS cleave multiple TSs, and each cleavage leads massive Rh6 G to be released. Such double-amplified signal accumulation endows the walker with good sensitivity. This walker specifically detected TdT activity down to 0.093 U mL-1, and successfully detected TdT activity in human serum. The proposed walker demonstrates a novel method for specific and sensitive detection of TdT activity, providing an effective tool for TdT-related biological research and leukemia theranostics.

Uracil DNA glycosylase (UDG) is a key base excision repair (BER) enzyme and its abnormal expression is nearly relevant to several diseases including cancer. The sensitive detection of UDG activity is beneficial for biomedical studies and clinic diagnosis. In this work, we proposed a dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of UDG activity. The specially designed dumbbell probe contained two uracil bases, two recognition sites for nicking enzyme and a split sequence of DNAzyme. Unsealed dumbbell probes were first connected into sealed dumbbell probes by T4 DNA ligase, and then the unsealed probes were hydrolyzed by exonuclease to ensure the purity of probes. Under the influence of UDG, two uracil bases were removed to produce two apyrimidinic (AP) sites, which were subsequently cleaved by Endo.IV. The probes after cleavage acted as primers and templates for double nicking sites strand displacement amplification (SDA) to produce a mass of two products. The products of SDA continued to act as primers and templates for rolling circle amplification (RCA) to produce repeats containing complete DNAzyme sequences. The DNAzyme repeatedly cleaved multiple molecular beacons (MB), resulting in remarkable fluorescence enhancement. Benefiting from the triple cascade signal amplification, the sensitivity was improved and the detection limit was 7.2 × 10-5 U mL-1. The method could well distinguish UDG from other interfering enzymes and detect UDG activity in real biological samples, showing good specificity. In addition, this method could be used for screening inhibitors. The above results suggested that the method provided a promising analytical means for UDG related biomedical research and clinic diagnosis.

DNA walkers are a type of self-assembled nanodevices based on the specificity, predictability and programmability of DNA hybridization. The walking components can move autonomously and gradually along the predetermined track under specific external stimuli. These features of autonomous movement and repetitive stepping endow them with excellent signal amplification capability, making the DNA walker show a good application prospect in chemical measurement. This article systematically reviewed the research progress of the DNA walker in the fields of biosensing and living cell imaging.

While proteasome inhibitors such as bortezomib showed satisfactory clinical benefits in the initial treatment of multiple myeloma (MM), drug resistance and relapse are unavoidable. Recent studies suggested inhibition of histone deacetylases (HDACs) restored sensitivity of bortezomib-resistant MM. Hence, we designed dual inhibitors targeting both HDACs and proteasomes to address the resistance of bortezomib. The most potent inhibitors, ZY-2 and ZY-13 showed excellent inhibition against proteasome and good selectivity against HDACs. In particular, ZY-2 not only exhibited good antiproliferative activities on the MM cell lines RPMI-8226, U266, and KM3 (IC50 values of 6.66, 4.31, and 10.1 nM, respectively) but also showed more potent antiproliferative activities against the bortezomib-resistant MM cell line KM3/BTZ compared with bortezomib (IC50 values of 8.98 vs. 226 nM, P < 0.01) and even better than the combination of the HDAC inhibitor MS-275 and bortezomib (1:1) (IC50 values of 8.98 vs. 98.0 nM, P < 0.01).

Herein, a bipedal-unequivalent three-dimensional DNA walker is designed and applied to biosensing. It involves three components: (i) DNA three-way junction switch (DTWJS), in which two longer strands containing different sequences are partially complementary serving as bipedal-unequivalent walking strand (B-UWS) and the shortest one serves as blocking strand; (ii) fluorophore-labelled hairpins H-1 and H2, co-modified onto gold nanoparticles (AuNPs), serving as track strands; (iii) hairpins H3 and H4, contain sequences complementary to H1 and H2, respectively, serving as fuel strands. By target binding to blocking strand, B-UWS is liberated to hybridize with H1 and H2 in turn. The unfolded H1 and H2 get hybridized with H3 and H4, respectively, accompanying by B-UWS sequential release and fluorescence signal accumulation. Then B-UWS continues to hybridize with another track strands to walk. Compared with unipedal and bipedal-equivalent DNA walkers, this bipedal-unequivalent DNA walker has twice the sustainable operation capability through kinetic and affinity studies. Using let-7a as a model target, it shows a detection limit of 68 pM and satisfactory reproducibility in biological fluids. This DNA walker provides a new sustainable operation mode and will be a potential analytical tool in clinical diagnosis.

A bimolecular i-motif mediated FRET strategy was developed based on the proximity-induced folding of two identical cytosine-rich DNA strands. This strategy affords a FRET signal that is highly matched to the dimerization event, and enabled accurate and dynamic in situ imaging of Met homodimerization on a living tumor cell surface.

Overexpression of adenosine 5'-triphosphate-binding cassette transporters is one of the primary causes of drug resistance in cancer. Downregulating the expression of these transporters by inhibiting the mRNA translation process is an effective approach to cope up with this situation. Herein, multifunctional molecular beacons (MBs)-modified gold nanoparticle (AuNP) as a nanocarrier (MBs-AuNP) is developed for synergistic inhibition and in situ imaging of drug-resistant-related mRNAs in living cells. MBs-AuNP is composed of (i) triple specially designed molecular beacons modified on the surface of AuNP, for binding drug-resistant-related mRNAs, loading doxorubicin (Dox), and reporting the fluorescence signal, and (ii) AuNP, for loading MBs, introducing them into cells, and quenching their fluorescence. After uptake by cells, MBs-AuNP will hybridize with three different drug-resistant-related mRNAs (MDR1 mRNA, MRP1 mRNA, and BCRP mRNA), respectively, which could inhibit their translation to decrease efflux protein expression and lead to AuNP-quenched fluorescence recovery for in situ imaging. Real-time quantitative-polymerase chain reaction and western blot results showed that drug-resistant-related mRNAs and efflux proteins expression both decreased. Dox-loaded MBs-AuNP exhibited higher suppression efficacy compared to that of free Dox against HepG2/ADR (0.35 vs 1.06 mu M of IC50) and MCF-7/ADR (2.78 vs >5 mu M of IC50). Direct observation of intracellular hybridization events and differentiation of drug-resistant cancer cells or non-drug-resistant cancer cells could be accomplished through fluorescence imaging analysis. This nanocarrier is capable of downregulating the expression of multiple efflux proteins by gene silencing, allows in situ monitoring of silencing events, and thus provides a powerful strategy to cope up with drug resistance at the gene level.

To enhance efficacy of chemotherapy and achieve real-time imaging of cancer cells, it is crucial to develop nanocarriers with targeted drug delivery capacity and fluorescence property for cancer theranostics. Herein, a dual-targeting DNA tetrahedron nanocarrier (MUC1-Td-AS1411) was constructed for breast cancer cell imaging and targeted drug delivery. This nanocarrier consisted of three components: (i) DNA tetrahedron core for multivalent conjugation of function ligands and loading doxorubicin (Dox); (ii) activatable MUC1 aptamer probe (MUC1-probe), formed by the hybridization of MUC1 aptamer sequence with fluorophore extended from one vertex and complementary sequence with quencher, for targeting and imaging MUC1 protein on cytomembrane; (iii) AS1411 aptamer, which was hybridized to the overhang on three vertexes via prolonged sequence, for binding to nucleolin. Firstly, MUC1-probe of this nanocarrier targeted MUC1 protein of MUC1-positive cells, causing a conformational reorganization of MUC1 aptamer, releasing complementary sequence with quencher and leading to fluorescence recovery. Subsequently, after internalizing into cells, AS1411 aptamer moiety of nanocarrier bound to nucleolin selectively, then the whole nanocarrier targeted nucleus and released Dox into nucleus. MUC1-positive cells and MUC1-negative cells could be differentiated by means of fluorescence imaging. Versus free Dox, Dox-loaded MUC1-Td-AS1411 showed lower cytotoxicity to MUC1-negative HL-7702 cells (P < 0.01), approximately equal lethality to sensitive MCF-7 cells (P > 0.05) whereas more effective to doxorubicin-resistant MCF-7 cells (P < 0.01). Therefore, this nanocarrier could be used as a promising candidate for cancer theranostics.

Drug resistance arising from overexpressed efflux transporters increases the efflux of drugs and accordingly restricts the efficacy of chemotherapy. Advances in nanocarriers have provided potential strategies to cope with drug resistance. Herein, endogenous stimuli-responsive nucleus-targeted nanocarrier is developed for intracellular multidrug resistance protein 1 (MRP1) mRNA imaging and drug delivery. This nanocarrier (AuNP-mRS-DSs) is composed of three parts: (i) gold nanoparticle (AuNP), for loading DNA and quenching fluorescence; (ii) mRNA recognition sequence (mRS) modified on the surface of gold nanoparticle by gold-thiol bond, for the specific recognition of MRP1 mRNA; (iii) detachable subunit (DS), hybridized with Cy5-labeled DNA linker and nucleolin recognition motif and grafted onto mRS via the DNA linker for loading doxorubicin (Dox), binding to nucleolin, and reporting signal. First, nucleolin recognition motif of this nanocarrier targets nucleolin, which is overexpressed on cancer cells surface; subsequently, the whole nanocarrier enters the cell via nucleolin-mediated internalization. Subsequently, mRS will specifically recognize overexpressed MRP1 mRNA, leading to the release of trapped DS and followed by AuNP-quenched Cy5 fluorescence recovery. Finally, by translocation of nucleolin from cytoplasm to nucleus, the DS targets nucleus to delivery Dox. By intracellular fluorescence imaging, the differentiation of drug-resistant and nondrug-resistant cells could be achieved. Compared with free Dox (IC50 > 8.00 μM), Dox-loaded AuNP-mRS-DSs (IC50 = 2.20 μM) performed superior suppression efficacy toward drug-resistant cancer cells. Such a nanocarrier provides an effective strategy to synergistically sense and circumvent drug resistance, which may be exploited as a candidate for personalized medicine.