Huimin Li

Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction

Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction. This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase. Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase. Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction, a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 × 10−15 M.

Atomic force microscopy investigation of the characteristic effects of silver ions on Escherichia coli and Staphylococcus epidermidis

The influence of silver ions on Escherichia coli and Staphylococcus epidermidis has been investigated by atomic force microscopy (AFM). Both single cell and cell communities were visualized, and the nanoscale ultrastructure images of these two bacteria strains before and after stimulation of silver ions were obtained. The results showed that in the case of E. coli after treatment with silver ions, vesicles appeared on the cell walls, and the size of vesicles became larger with the increase of the incubation time. However, in the case of S. epidermidis after treatment with silver ions, irregular and deep grooves appeared on the cell walls, and the cytoplasm membrane shrank and became separated from the cell wall. The significant differences in cell wall changes between E. coli and S. epidermidis after treatment with silver ions were related to their structural characters. According to those results, it is shown that AFM can provide new insight into the antimicrobial mechanism of silver ions, and show a new methodological approach to understand the relationship between structure and function of microbial cell when it was exposed to external stimulation.

Real‐Time Monitoring of Nucleic Acid Dephosphorylation by Using Molecular Beacons

Dephosphorylation of the 3’ termini of nucleic acids is important for cellular events, such as DNA replication, recombination, and repair of DNA damage induced by a variety of genotoxic agents, which include ionizing radiation, certain antineoplastic alkylating agents, bleomycin, and topoisomerase inhibitors. Dephosphorylation of the 3’ phosphate is usually catalyzed by various repair enzymes, which include the commonly used T4 polynucleotide kinase (T4 PNK). Since the identification of T4 PNK over 40 years ago, T4 PNK has exemplified a family of bifunctional enzymes with 5’-kinase and 3’phosphatase activities, and has become a molecular biology workhorse as well as an invaluable research tool in biology and bioengineering. Its 3’-phosphatase activity allows T4 PNK to dephosphorylate the 3’ termini of DNA molecules. This converts the DNA to a 3’hydroxyl substrate that is required for DNA polymerases and ligases, which play important roles in RNA and DNA repair. Traditionally, P radiolabeling, PAGE, and autoradiography are used to assay the dephosphorylation of nucleic acids. These methods are discontinuous, time consuming, laborious, require radiolabeled substrates, and cannot be used to analyze the rapid continuous dephosphorylation processes. Recently, Dobson and Allinson used fluorescent probes in combination with denaturing PAGE to study human PNK, but this method also suffers from being discontinuous. There are many advantages to using real-time assays instead of discontinuous ones, particularly in dissecting the molecular mechanisms of enzymes and their regulation. Continuous assays are relatively less laborious than discontinuous assays and they save time. Furthermore, they can be easily adjusted and optimized for high-throughput systems. Therefore, to investigate the dephosphorylation of nucleic acids in real time it is necessary to develop simple and sensitive methods that do not require isotopic labeling. In this paper, a novel method is described for real-time monitoring of the dephosphorylation process by using molecular beacon (MB) DNA probes. Since they were first reported in 1996, MBs have become a class of DNA probes that are widely used in chemistry, biology, biotechnology, and medical sciences for biomolecular recognition, due in part to their high sensitivity and excellent specificity. Previously, we developed techniques for real-time monitoring of nucleic-acid ligation and phosphorylation using MBs; these provided powerful systems for exploring the interactions between nucleic acids and proteins (enzymes). However, these methods are complex. Here, we describe the development of a new technique based on a polymerase-extension reaction to monitor nucleic acid dephosphorylation with MBs in real time. This approach is very simple, rapid, cost effective, and requires only one oligonucleotide. This method is schematically represented in Scheme 1.

Atomic Force Microscopy Study of the Effect of Pulsed Electric Field on Staphylococcus epidermidis

The effect of a pulsed electric field (PEF) on Staphylococcus epidermidis was investigated by using an atomic force microscopy (AFM) image and force measurement under liquid. The changes in the bacterial envelope were probed in situ before and after applying different dosages of PEF. Our results indicated that PEF induced the changes of bacterial peptidoglycan layer and the exposure of plasma membrane. This conclusion was further confirmed by two control experiments: the effect of the lysozyme or heat on the bacterial envelope. Furthermore, our results demonstrated that AFM analysis including image and force measurement is helpful to explain the relationship between the chemical composition change of the cellular envelope and the external stimulation.

RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon.

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.

Fidelity genotyping of point mutation by enhanced melting point difference using DNA ligase

Melting curve analysis is a powerful tool for detecting single-base mutations that may be linked to genetic diseases. Current existing methods provide insignificant melting point difference for some point mutations with the risk of wrong genotyping results, causing great limitations to their applications in clinic diagnosis. Here, we have developed an enhanced melting point difference approach to genotype single-base mutations using DNA ligase. Ligase covalently joins an allele-specific discriminating probe and a signal probe flanked the mutation site to form a long duplex, resulting in an enhanced melting temperature. CD17 and Ivs-2-654 point mutations of beta-globin gene in thalassemia disease were identified by using this approach, and the homozygotes and heterozygotes were scored accurately and conveniently. To the best of our knowledge, the use of ligase to improve the differences of melting temperature between various genotypes has not been reported. This method will provide a promising tool for clinical diagnosis of gene-mutant diseases.

Using force spectroscopy analysis to improve the properties of the hairpin probe

The sensitivity of hairpin-probe-based fluorescence resonance energy transfer (FRET) analysis was sequence-dependent in detecting single base mismatches with different positions and identities. In this paper, the relationship between the sequence-dependent effect and the discrimination sensitivity of a single base mismatch was systematically investigated by fluorescence analysis and force spectroscopy analysis. The same hairpin probe was used. The uneven fluorescence analysis sensitivity was obviously influenced by the guanine-cytosine (GC) contents as well as the location of the mismatched base. However, we found that force spectroscopy analysis distinguished itself, displaying a high and even sensitivity in detecting differently mismatched targets. This could therefore be an alternative and novel way to minimize the sequence-dependent effect of the hairpin probe. The advantage offered by force spectroscopy analysis could mainly be attributed to the percentage of rupture force reduction, which could be directly and dramatically influenced by the percentage of secondary structure disruption contributed by each mismatched base pair, regardless of its location and identity. This yes-or-no detection mechanism should both contribute to a comprehensive understanding of the sensitivity source of different mutation analyses and extend the application range of hairpin probes.

Preparation of PEGylated Paclitaxel Liposomes and Tissue Distribution Study in Mice

A long circulation paclitaxel liposome was prepared by dispersion of film method. The size of liposomes was around 207 nm and drug encapsulation efficiency was 96.5%. The PEGylated paclitaxel liposomes were very stable when stored in PBS, pH 6.5 at 4degC. After 22 days, leakage of drug from liposomes was only 6%. In vivo biodistribution of paclitaxel liposomes and free paclitaxel were compared in prostate tumor-bearing mice by intravenous injection. Drug concentration of paclitaxel liposomes in tumor was remarkably higher than that of free paclitaxel. These results suggest that this stable, long circulation paclitaxel- containing liposome may be a feasible targeting delivery system for paclitaxel and other hydrophobic or toxic compounds.

Application of fluorescence labeled liposome nanoparticles in the cell imaging

Fluorescence labeled liposome nanoparticles were prepared by dispersion of film method. The size of nanoparticles was around 50 nm. DPPE-FITC synthesized in our lab was used to label the liposome nanoparticles. Anti-cytokeratins 19 antibody was connected to the surface of the fluorescence liposome nanoparticles. After incubation with MGC cells and COS-7 cells for 30 min, MGC cells were selectively recognized by anti-cytokeratins 19 antibody modified liposome nanoparticles and well imaged under laser confocal microscope. This fluorescence labeled liposome nanoparticles is expected to have good applications in cell recognition and tumor diagnosis.