Jialong Fan

An end-point method based on graphene oxide for RNase H analysis and inhibitors screening

As a highly conserved damage repair protein, RNase H can hydrolysis DNA-RNA heteroduplex endonucleolytically and cleave RNA-DNA junctions as well. In this study, we have developed an accurate and sensitive RNase H assay based on fluorophore-labeled chimeric substrate hydrolysis and the differential affinity of graphene oxide on RNA strand with different length. This end-point measurement method can detect RNase H in a range of 0.01 to 1 units /mL with a detection limit of 5.0×10-3 units/ mL under optimal conditions. We demonstrate the utility of the assay by screening antibiotics, resulting in the identification of gentamycin, streptomycin and kanamycin as inhibitors with IC50 of 60±5µM, 70±8µM and 300±20µM, respectively. Furthermore, the assay was reliably used to detect RNase H in complicated biosamples and found that RNase H activity in tumor cells was inhibited by gentamycin and streptomycin sulfate in a concentration-dependent manner. The average level of RNase H in serums of HBV infection group was similar to that of control group. In summary, the assay provides an alternative tool for biochemical analysis for this enzyme and indicates the feasibility of high throughput screening inhibitors of RNase H in vitro and in vivo.

Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe

In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba2+, Co2+, Pb2+, As3+, and Cu2+ inhibited RNase A activity with IC50 values of 93.7 μM (Ba2+), 90.9 μM (Co2+), 110.6 μM (Pb2+), 171.5 μM (As3+), and 165.1 μM (Cu2+), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.

Increasing the sensitivity and selectivity of a GONS quenched probe for an mRNA assay assisted with duplex specific nuclease

The authors report a new graphene oxide nanosheet (GONS) based fluorescence method for mRNA assay with duplex-specific nuclease (DSN)-assisted signal amplification. Following hybridization between the target mRNA and its complementary DNA probe, the ssDNA in the DNA/mRNA hybrid is selectively cleaved by DSN to produce small fragments. The released mRNA molecule then initiates another cycle of hybridization and DSN digestion. In this manner, each mRNA molecule can specifically trigger many cycles of hybridization and DNA cleavages to produce numerous small DNA fragments. The short DNA fragments can exhibit strong fluorescence signals due to the weak adsorption of GONS to them. This fluorescence assay for mRNA with increased selectivity has a 1 fM detection limit under optimal conditions. Furthermore, this assay was successfully used for mRNA imaging in situ.

DNase-targeted natural product screening based on a sensitive and selective DNase I detecting system

As a widely used deoxyribonuclease, DNase I is involved in many physiological processes including tumor cell proliferation, metastasis and apoptosis. Furthermore, the level of this enzyme in serum can act as a functional biomarker for the therapeutic monitoring of systemic lupus erythematosus and other diseases. We report here a low cost and sensitive DNase I detecting system based on the single-stranded fluorogenic substrate and nanographene oxide (NGO) and use it for DNase-targeted natural product screening. The system with a detection limit of 0.005 U was then used to evaluate the effect of external factors on DNase I. The results show that Hg2+, As2+, Pb2+, Cd2+ and Cu2+ can inhibit DNase I activity in a concentration-dependent manner with IC50 values of 0.37 mM (Hg2+), 2.7 mM (As2+), 5 mM (Pb2+), 5.3 mM (Cd2+) and 7.8 mM (Cu2+), respectively. Meanwhile, 10 natural compounds isolated from Cyclocarya paliurus leaves were screened as DNase I inhibitors, while 5 compounds were identified as activators. Finally, the system was used to discriminate DNase activity of serum samples with and without HBV. The results showed that HBV infection significantly decreased the level of DNase I in serum samples. In summary, these data indicate that this method with the advantages of rapidity, low cost and high sensitivity is hopeful for DNase assay in biological samples as well as compound screening in vitro.

Synthesis of DNA-guided silver nanoparticles on a graphene oxide surface: enhancing the antibacterial effect and the wound healing activity

The occurrence of antibiotic resistance against pathogens is rapidly increasing and endangering the efficacy of antibiotics. Thus, finding a way to address this problem has become a major challenge due to the inability of conventional antibiotics to kill these multidrug-resistant bacteria. In order to further enhance the antibacterial ability and reduce the possibility of antibiotic resistance, we developed a simple two-step approach and synthesized a new nanocomposite by directly loading single-stranded DNA (ssDNA)-guided silver nanoparticles (AgNPs) on graphene oxide (ssDNA-AgNPs@GO). Through systematically evaluating the bactericidal activity and wound healing capability, we found that ssDNA-AgNPs@GO exhibited synergistic antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis with low minimum inhibitory concentrations (6.8 μg mL−1, 6.8 μg mL−1, 11.9 μg mL−1 and 10.2 μg mL−1, respectively) and large-diameter inhibition zones (12.83 ± 0.63 mm, 13.14 ± 0.37 mm, 8.6 ± 0.9 mm and 8.93 ± 0.47 mm, respectively). Furthermore, the wound healing experiment indicated that it has a striking ability to remedy wound infection caused by Staphylococcus aureus bacteria. In conclusion, the properties of ssDNA-AgNPs@GO with enhanced antibacterial and wound healing capability will give it broad applications in the future.

Quantitative Detection of miRNA-21 Expression in Tumor Cells and Tissues Based on Molecular Beacon

As a new tumor marker, the microRNA-21 (miRNA21) level can provide important information for early diagnosis, drug treatment, and prognosis of gastric cancer. With the tool of molecular beacons which can hybridize specifically with target miRNA-21 and generate fluorescence signal change, this paper develops a direct, simple, and rapid method for miRNA-21 detection with detection limit of 0.5 nM. Under the optimal conditions, the method was used to detect the expression of miRNA-21 in tumor cells and tissues. The results showed significant differences of miRNA-21 levels in tumor cells which have different origins and different degree of malignancy. In 8 cases of gastric cancer tissues and adjacent tissues, the level of miRNA-21 in 6 cases was higher than that in adjacent tissues, 1 case had lower expression level than that in adjacent tissues, and 1 case had no significant difference. Furthermore, qRT-PCR method was used to verify the detection results based on the fluorescent probe detection method. The consistent results show that the molecular beacon assay has a good prospect in direct and rapid detection of miRNA-21 expression and will be widely used in the functional research and clinical diagnosis of microRNA.