Biomedical subjects

Gang Bao

Publications and source records attributed to Gang Bao.

25 records · Page 2Linked to original sources

PubMed2003

Hybridization kinetics and thermodynamics of molecular beacons.

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem-loop structure of molecular beacons provides a competing reaction for probe-target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure-function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon-target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.

DNA Probes↗

PubMed2003

Shedding light on health and disease using molecular beacons.

The detection and identification of pathogens is often painstaking due to the low abundance of diseased cells in clinical samples. The genomic sequences of the pathogen can be amplified through methods such as the polymerase chain reaction and nucleic acid sequence-based amplification, but the nucleic acid targets are often lost among other unintended products of amplification. Novel nucleic acid probes known as molecular beacons have been developed allowing for the rapid and specific detection of genetic markers of a disease. Molecular beacons are hairpin-forming oligonucleotides labelled at one end with a quencher and at the other end with a fluorescent reporter dye. In the absence of target, the fluorescence is quenched. In the presence of target, the hairpin structure opens upon beacon/target hybridisation, resulting in the restoration of fluorescence. The ability to transduce target recognition into a fluorescence signal with high signal-to-background ratio, coupled with an improved specificity, has allowed molecular beacons to enjoy a wide range of biological and biomedical applications. Here, we describe the basic features of molecular beacons, review their applications in disease detection and diagnosis and discuss some of the issues and challenges of in vivo studies. The aim of this paper is to foster the development of new molecular beacon-based assays and to stimulate the application of this technology in laboratory and clinical studies of health and disease.

Animals↗

PubMed2003

An in vitro uniaxial stretch model for axonal injury.

We have developed a unique uniaxial stretching device to study axonal injury and neural cell death resulting from brain tissue deformations common in traumatic head injuries. Using displacement control rather than force control, this device is capable of achieving strains >70% and strain rates up to 90 s(-1), well above those currently used for studying axonal injury. We have demonstrated that the deformation of the specimen was uniaxial, uniform and highly reproducible; the prespecified displacement profiles could be realized almost precisely; and adequate cell adhesion could be achieved readily. The entire device can fit into a biological safety cabinet to maintain sterility, and the specimens are convenient for cell culture. This device can be used to investigate a wide range of biomechanical issues involved in diffuse axonal injury.

Animals↗

PubMed2003

[Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase in ameloblastoma].

OBJECTIVE: To study the expression of matrix metalloproteinase(MMP) and tissue inhibitor of metalloproteinase (TIMP) in ameloblastoma (AB) and to determine the relationship between biological behavior of AB and clinical pathology. METHODS: The specimens of 43 cases of AB, 10 cases of odontogenic keratocyst (OKC), 8 cases of normal oral mucosa were examined by streptavidin-biotin method. RESULTS: In 8 cases of normal oral epithelial, MMP-2 was negative or weak positive. In OKC, MMP-2 was extensively positive in stratum spinosum of 2 cases, and weekly or negative in stratum basale. MMP-2 was strongly expressed in the central and peripheral cells of the tumor islands in 28 cases of AB. There were difference among these three groups (P < 0.001). Comparing AB with normal oral mucosa and OKC, there was significant difference (P < 0.05). MMP was positive expressed in cells of stroma. The positively rate and intensity increased as AB recurrence and transformed malignantly, but were not associated with age, sex, pathological type and location. TIMP-1 was weakly or not expressed in normal oral mucosa, stoma, OKC and AB. CONCLUSIONS: The high expression of MMP-2 and MMP-9 is related to the biological behavior of AB. Imbalances of the expression of MMP-2 and MMP-9 protein maybe be one of the facts of the invasion of AB. The MMPs activation produced by stromal cells may be also be related to the invasion of AB.

Adolescent↗

PubMed2002

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets.

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.

DNA↗

PubMed2002

Structure-function relationships of shared-stem and conventional molecular beacons.

Molecular beacons are oligonucleotide probes capable of forming a stem-loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain flanked by two complementary short arm sequences that are independent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may discriminate between targets with a higher specificity. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe-target hybridization kinetics were similar for both classes of molecular beacons and were influenced primarily by the length and sequence of the stem. These findings should enable better design of molecular beacons for various applications.

Kinetics↗

PubMedDate not supplied

Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons.

The intracellular localization and specific organelle association of mRNA may reflect essential functions, stages, and stability of mRNA. We report the direct visualization of subcellular localization of K-ras and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs in live HDF cells using molecular beacons together with membrane-permeabilization and peptide-based delivery. Unexpectedly, we found that both K-ras and GAPDH mRNAs colocalize with mitochondria. Extensive control studies are performed, including the use of fluorescence in-situ hybridization (FISH), negative-control beacons, and the detection of colocalization of 28S ribosomal RNA with the rough endoplasmic reticulum (ER), suggesting that the mRNA localization and colocalization patterns observed in our study are true and specific. Our observation reveals intriguing subcellular associations of mRNA with organelles such as mitochondria, which may provide new insight into the transport, dynamics, and functions of mRNA and mRNA-protein interactions.

Cell Line↗