An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

Magnetic separation-assisted cluster-amplified versatile fluorescent aptasensors for the sensitive detection of target biomolecules.

A sensitive and versatile platform for detecting diverse target biomolecules was developed by combining a magnetic separation module and a fluorescence amplification module in a plug-and-play manner. The magnetic separation module was constructed using magnetic beads (MBs), whose surfaces were modified with aptamer-blocked captor DNAs. The fluorescence amplification module was constructed by loading the fluorescent dye rhodamine 6G (Rh6G) into the pores of mesoporous silica nanoparticles (MSNs). The MSN surfaces were modified with prey DNAs, of which the MSN-near ends hybridized with complementary DNAs (sealing DNAs) to form duplexes to seal the pores, and the free ends were designed to be single-stranded that were complementary to the captor DNAs. Upon binding of targets to their aptamers, the captor DNAs were unblocked and thus were able to hybridize with the prey DNAs, to capture Rh6G-laden MSNs, forming MB-MSN clusters. The clusters were isolated by magnetic separation and heated to dissociate the DNA duplexes, to unseal the MSN pores and release the inner Rh6G; thus a target was converted into a cluster of Rh6G dyes. By simply changing the target aptamers and related DNA connectors, this strategy detected ATP, thrombin, and platelet-derived growth factor BB with detection limits of 2.1 nM, 4.1 pM, and 2.4 pM, respectively. A wide range of targets, high amplification efficiency and universal functional modules endow the aptasensors with good potential as versatile platforms for detecting target molecules in vitro and in medical research.

A magnetic DNAzyme walker for both in-situ imaging and sensitive detection of MUC1 on living cells.

Mucin 1 (MUC1) is a transmembrane glycoprotein commonly expressed in epithelial cells with stable levels and polarized distribution. Their expression levels and spatial distribution abnormally altered during oncogenesis and play tumor-promoting roles synergistically. We herein propose a magnetic DNAzyme walker (MDW) for both in-situ imaging and sensitive detection of MUC1. This MDW was constructed by modifying specially designed track strands (TSs) and walking strands (WSs) on a streptavidin magnetic bead (SA-MB). The TSs contained cleavage sites for DNAzymes and were labeled with Cy3 at free ends. The WSs contained DNAzyme sequences and were firstly blocked by hybridizing with Cy5-labeled aptamers of MUC1. The DNAzymes were unlocked upon aptamers binding to MUC1 on cells. MDWs were then transferred to a buffer suitable for DNAzyme action, where the unlocked DNAzymes cleaved multiple TSs, releasing amplified Cy3-fragments, which were separated from the uncleaved ones by magnetic separation. In-situ imaging of MUC1 were achieved by the fluorescence of Cy5 on aptamers bound to MUC1. Sensitive detection of MUC1 were achieved by the amplified fluorescence of released Cy3. In-situ imaging and walker operation for detection were triggered by the same targets at the same time, ensuring the signals are real-time correlative. Moreover, MDWs' operation was separated from cells, reducing interference between imaging and detection. The proposed MDW offers a potential approach for comprehensive analysis of MUC1 in early diagnosis and progression assessment of tumor.

Primer-template conversion-based cascade signal amplification strategy for sensitive and accurate detection of polynucleotide kinase activity.

Here, a primer-template conversion-based cascade signal amplification strategy is described for the sensitive detection of polynucleotide kinase (PNK) activity. This strategy integrated rolling circle amplification (RCA) and multiple-repeated-strand displacement amplification (MRSDA) with G-quadruplex based fluorescence lighting-up assay. A delicate dumbbell-shaped DNA probe with 5'-hydroxyl terminus was designed, in which G-quadruplex and half recognition site of nicking enzyme Nb.BbvCI were encoded in two loops respectively. Under the action of PNK, the 5' terminus on dumbbell probe was firstly phosphorylated, and then the dumbbell was cyclized with the catalyzation of T4 ligase to become the RCA template. The RCA process produced multiple copies of the prolonged primer. After that, under the assistance of nicking enzyme Nb.BbvCI, a primer-template conversion occurred, which converted the primer and template of RCA into the template and primer of the subsequent MRSDA, respectively. The MRSDA generated multiple repeated ssDNA sequences which possessed G-quadruplexes for outputting signal by lighting-up fluorescence of thioflavin T (ThT). The cascade signal amplification of RCA and MRSDA provided high detection sensitivity, and the target-dependence of template in cascade signal amplification led to a low background. The method showed excellent detection limit of 0.2 × 10-6 U µL-1 in buffer and 5 cells in cell lysate sample. Moreover, this method displayed favorable selectivity when interfering proteins were present. The developed strategy has good practical potential for PNK activity detection in clinical diagnosis and medical research.

A functional DNA-modified dual-response gold nanoprobe for simultaneously imaging the acidic microenvironment and membrane proteins of tumor cells.

Tumor progression is a complicated process influenced by multiple factors, in which the acidic tumor microenvironment (TME) and altered tumor-associated membrane proteins (TA-MPs) are closely involved. Monitoring the status of these factors is of significance for tumor progression research. Here, we develop a novel probe for simultaneously imaging the acidic TME and TA-MPs in situ. In this probe, i-motif-forming sequences (strand I) are conjugated to a gold nanoparticle (AuNP) via gold-sulfur bonds for acid-response. Extended aptamers (strand A) for protein recognition are labeled with Cy3 and Cy5 respectively at two ends. The extended part of strand A hybridizes with strand I to quench Cy3 by the proximal AuNP, and the protein recognition part hybridizes with a strand labeled with BHQ2 (strand Q) to quench Cy5. When the integrated probe encounters an acidic TME, the strand I fold into i-motif quadruplexes and release the AQ duplexes from the AuNP, enabling Cy3 to be lit to indicate the acidic TME. The aptamers in AQ duplexes bind to target proteins, removing the hybridization between strand A and Q thus leading to the fluorescence recovery of Cy5 for in-situ imaging of the proteins. Fluorescence measurement and confocal microscopy imaging showed that the probe could sensitively respond to the alteration in acidity from pH 7.4 into pH 6.5, which is coincide with the acidity gap of extracellular microenvironment between normal and tumor cells. Besides, it enabled the in-situ imaging of MUC1 proteins on living cell surface, revealing their expression level and distribution. This probe demonstrates a new approach for simultaneously imaging the acidic TME and TA-MPs, providing a useful tool for multifactor research of tumor progression.

MnO2 nanosheet-mediated target-binding-induced FRET strategy for multiplexed microRNAs detection and imaging in living cells.

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.

A bimolecular i-motif mediated FRET strategy for imaging protein homodimerization on a living tumor cell surface.

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.