A cascade amplification platform assisted with DNAzyme for activity analysis, kinetic study and effector screening of Fpg in vitro.

As a highly conserved damage repair protein, Fpg can specifically recognize and digest 8-oxoG from a damaged DNA backbone. Meanwhile, DNAzyme, a single-stranded DNA with enzymatic activity, can cleave RNA in the presence of cofactors. In this study, we established a highly sensitive method for Fpg assay using a DNAzyme-mediated signal cascade amplification strategy. Based on the Fpg-dependent fluorescence response of the "turn-on" manner, we could reliably determine Fpg activity down to 0.14 U mL-1 with a linear response from 0.10 to 40 U mL-1 under optimal conditions. In addition, this strategy was successfully applied to analyze the kinetic parameter of Fpg with Km of 0.061 µM. Furthermore, the developed sensing system was used to screen the regulators of Fpg from natural compounds and antibiotics. These results indicated that all of the 14 natural compounds and 6 kinds of antibiotics deferentially showed an active effect on Fpg in vitro. In summary, these results show that the method not only provides an alternative for monitoring Fpg activity but also shows great potential for drug screening in the future.

A novel fluorescence method for activity assay and drug screening of T4 PNK by coupling rGO with ligase reaction.

T4 polynucleotide kinase (PNK) is the primary member of the 5'-kinase family that can transfer the γ-phosphate residue of ATP to the 5'-hydroxyl group of oligonucleotides. In this article, using the differential quenching ability of reduced graphene oxide (rGO) towards the fluorophore-labeled DNA probe, we propose a novel method for detecting T4 PNK activity assisted by ligase reaction. Under the optimized conditions, the detection limit of T4 PNK was estimated to be 0.0002 U µL-1 in the linear region of 0.001 U µL-1-0.1 U µL-1. Additionally, the developed method was used to screen regulators of T4 PNK from natural compounds. The compound f isolated from the root of Kadsura coccinea (Lem.) A.C. Smith was found to stimulate T4 PNK activity in a concentration-dependent manner in vitro. Finally, the method was used to monitor the relation of T4 PNK activity with pelvic inflammatory disease (PID). The results demonstrated that the development of this disease could inhibit T4 PNK activity to some extent. In summary, the above data indicate that the method not only provides a universal platform for monitoring T4 PNK activity, but also shows great potential to be used in drug screening and clinic diagnosis.

Bioinspired molecular engineering of bivalent aptamers by ligand-induced self-dimerization.

A bioinspired "point-and-shoot" molecular strategy is described for engineering noncovalent aptamers via K+-induced reassembly of a split G-quadruplex sequence at the end of monomeric aptamers. This design compensates for insufficient cell recognition of monovalent aptamers in biological environments and minimizes structure-induced nonspecific cell binding, expending the aptamer toolbox available for complex systems.

A graphene-based fluorescent nanoprobe for simultaneous imaging of dual miRNAs in living cells.

MicroRNAs (miRNAs) are regarded as important biomarkers for disease diagnostics and therapeutics due to their significant regulatory roles in physiologic and pathologic processes. Herein, a versatile nanoprobe based on reduced graphene oxide (rGO) and nucleic acid (DNA) probe was prepared for simultaneously visualize miR-451a and miR-214-3p in living cells. In vitro experiments demonstrated that the nanoprobe exhibits excellent selectivity and outstanding sensitivity as low as 1 nM towards miR-451a and miR-214-3p. Moreover, the detection signals of miRNAs have good linearity in their respective concentration ranges (miR-451a: 1-100 nM, Y1 = 9.3062X1+114.85 (R2 = 0.9965). miR-214-3p: 1-200 nM, Y2 = 1.4424X2+91.312 (R2 = 0.9961)). Finally, simultaneous dual-color imaging of miR-451a and miR-214-3p in human breast cancer cells (MDA-MB-231) was realized by exploiting the P1&P2@rGO nanoprobe. In summary, this simple and effective strategy provides a general sensing platform for highly sensitive detection and simultaneous imaging of dual miRNAs in living cells.

RNase A activity analysis and imaging using label-free DNA-templated silver nanoclusters.

A label-free, ultra-sensitive and turn-on method for detecting RNase A has been developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as the fluorescence probe. In this system, an RNA strand, which can perfectly hybridize with DNA template of nanocluster synthesis, was applied to lock the fluorescent signal of DNA-AgNCs by forming an RNA/DNA duplex. Meanwhile, the hybridized RNA/DNA duplex was used as the substrate of RNase A. The fluorescence signal of AgNCs was restored due to the degradation of RNA by RNase A. From the fluorescence signal change of this system caused by RNase A, it was found that the fluorescence signal showed a positive linear relation with RNase A concentration in the range from 0.2 pg/µL to 10 pg/µL with a detection limit of 0.098 pg/µL. Except for potential inhibitor screening and the kinetic study of this enzyme, this strategy was further used for monitoring dynamic change of RNase A in living cells successfully. In summary, the simple and sensitive method for RNase A assay can be hopefully used for drug screening in vitro and in vivo.

DNAzyme and rGO based fluorescence assay for Fpg activity analysis, drug screening, and bacterial imaging.

Due to the significant role of formamidopyrimidine DNA glycosylase (Fpg) in physiological processes and DNA oxidative damage-related diseases, it is essential to establish sensitive methods for monitoring the Fpg activity in vitro and in vivo so as to illustrate its concrete role in these events. In this work, a sensitive, simple and reliable fluorescence assay was developed by taking the advantages of DNAzyme assisted cascade signal amplification and ultra-high fluorescence quenching efficiency of reduced graphene oxide (rGO). This detection system consisted of DNAzyme, rGO and fluorescence probe allows the activity of Fpg to be detected in a linear range from 0 to 80 U/mL with a detection limit of 0.66 U/mL. With the help of this method, 11 natural compounds were screened, and 7 compounds were identified as activators of Fpg. More importantly, the developed assay was used to monitor the activity of Fpg through fluorescence imaging in living Escherichia coli for the first time. The imaging results visually demonstrated the dynamic activation effect of natural compound Ginsenoside Re on the Fpg of Escherichia coli. In summary, these results indicated that this DNAzyme and rGO based fluorescence assay provides a potent strategy for Fpg quantitative assay in vitro and real-time monitoring in living bacteria, which holds great potential for applying on biological study and Fpg-targeted drug screening.

Monitoring VEGF mRNA and imaging in living cells in vitro using rGO-based dual fluorescent signal amplification platform.

VEGF mRNA, as an important biomarker for disease diagnosis and therapeutics, has received extensive attention. However, how to monitor its mRNA levels rapidly and sensitively remains a challenge. Herein, a strategy was designed for facile and efficient detection of VEGF mRNA and imaging in living cells using a collaborative system of a fluorophore-labeled single-stranded probe (P), reduced graphene oxide (rGO) and double-specific nuclease (DSN). The combination of strong fluorophore-quenching ability of rGO with DSN assisted signal amplification contributes to the superior sensitivity of the assay for VEGF mRNA, which was reflected by the lower limit of mRNA detection of 100 fM obtained using dual signal amplification manner. Furthermore, the developed sensor was directly used for intracellular mRNA imaging in vitro without the assistance of transfection reagent. In summary, the simple, ultra-sensitive and cost-effective mRNA assay system, which provided a general analysis strategy for other mRNAs assay by replacing the sequence of the probe, is hopeful for applying on the clinical diagnosis and therapy.

An rGONS-based biosensor for simultaneous imaging of p53 and p21 mRNA in living cells.

Hepatocarcinoma is the second leading cause of cancer-related death worldwide accompanied by the aberrant expression of many genes. Among of them, p53 and p21 genes played a vital role in the development of liver cancer. Thus, monitoring mRNA levels of the two markers is urgently needed for early diagnosis and understanding the molecular mechanism of hepatocarcinoma development. Herein, a functional nanosystem for in situ and real-time monitoring levels of p53 and p21 mRNA was constructed using reduced graphene oxide nanosheet assisted fluorescence probes. Comparing with traditional methods, the new method indicated high sensitivity and outstanding selectivity towards p53 and p21 mRNA assay in vitro. Moreover, the functional nanosystem was successfully used for monitoring dynamic change of mRNAs in HepG2 cells caused by cisplatin treatment, the reliability of which was confirmed by RT-PCR. In summary, this strategy provides a promising tool to reveal p53 and p21 mRNA regulatory process in cancer cells, which is practicable for drug screening and therapy evaluation on clinic.