Biomedical subjects

Xianbin Yang

Publications and source records attributed to Xianbin Yang.

7 recordsLinked to original sources

PubMed2006

Identification of proteins bound to a thioaptamer probe on a proteomics array.

A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific "on-chip" captured proteins have been obtained that were pure enough to do "on-chip" digestion and the subsequent identification of the "on-chip" bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays.

Animals↗

PubMed2006

Delivery of double-stranded DNA thioaptamers into HIV-1 infected cells for antiviral activity.

Oligonucleotide agents (ODN) are emerging as attractive alternatives to chemical drugs. However, the clinical use of ODNs as therapeutics has been hindered by their susceptibility to degradation by cellular enzymes and their limited ability to penetrate intact cells. We have used various liposome-mediated transfection agents, for the in vitro delivery of DNA thioaptamers into U373-MAGI-CCR5 cells. Our lead thioaptamer, R12-2, targets the RNase H domain of the HIV-1 reverse transcriptase (RT) and inhibits viral infection in U373-MAGI-CCR5 cells. R12-2, a 62-base-pair, double-stranded DNA molecule with a monothio-phosphate modified backbone, was selected through a novel combinatorial selection method. We studied the use of oligofectamine (OF), TFX-20, Transmessenger (TM), and Gene Jammer (GJ) for transfection of the thio-modified DNA aptamers. OF-transfected U373-MAGI-CCR5 cells resulted in 68% inhibition of HIV infection in the treated cells compared to the untreated control. Inhibition was observed in a dose-dependent manner with maximal inhibition of 83%. In this report, we demonstrate that monothioate-modified DNA duplex oligonucleotides can be efficiently delivered into cells by liposome-based transfection agents to inhibit HIV replication.

Antiviral Agents↗

PubMed2004

Progress in thioaptamer development.

Thioaptamers are thiophosphate ester modified nucleic acids that are isolated via in vitro or bead-based thioaptamer selection against a target molecule such as a protein. Thioaptamers offer advantages over traditional aptamers in their enhanced affinity and specificity and higher stability, largely due to the properties of the sulfur backbone-modifications. An in vitro thioaptamer selection procedure that simultaneously selects for sequence and optimized hybrid phosphoromonothioate or phosphate backbone substitutions is outlined. A novel bead-based thioaptamer selection protocol that can produce mixed phosphorodithioate, phosphoromonothioate or phosphate hybrid backbones is also described. Several examples of thioaptamers targeting specific protein are provided. Such thioaptamers are shown to modulate protein activity in vivo.

Combinatorial Chemistry Techniques↗

PubMed2003

Immunofluorescence assay and flow-cytometry selection of bead-bound aptamers.

An immunofluorescence assay was developed to identify proteins specifically binding to oligonucleoside phosphorodithioate (ODN) aptamers from a bead-bound ODN library. Accordingly, NF-kappaB p50 protein was incubated with either bead-bound NF-kappaB consensus sequence or a bead-bound ODN combinatorial library and adsorption was then assessed using a specific primary antibody and a secondary antibody conjugated with Alexa 488 fluorescent dye. This assay avoids any problems related to fluorescently labeling target proteins. The method is straightforward and readily applicable to other transcription factors and proteins, and the feasibility of its application for high-throughput screening of large aptamer bead-based libraries by flow cytometry is demonstrated.

Binding, Competitive↗

PubMed2002

Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing.

Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries. Binding and selection of specific beads to the transcription factor NF-kappaB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3'-O-thioate/dithioate linkages was carried out by using a novel PCR-based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S-ODNs or S2-ODNs that bind to proteins.

Base Sequence↗

PubMed2002

Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion-exchange chromatography on a mono-q column.

A method using a strong anion-exchange liquid-chromatography column, Mono-Q, has been developed for high-resolution analysis and purification of oligonucleotide dithioates, which were synthesized by an automated, solid-phase, phosphorothioamidite chemistry. High-resolution separation of oligonucleotide phosphorodithioates from monothiophosphate impurities was obtained. High-resolution separation was also demonstrated at pH 8. The separation of oligonucleotide dithioates was found to be linearly dependent on the number of sulfurs for the same sequence length. Thiocyanate, SCN-, as eluting anion, can be used to purify oligonucleotides containing a high percentage of phosphorodithioate linkages in lower salt concentrations and provides better separation than chloride as eluting anion.

Anion Exchange Resins↗

PubMed2002

Solution structure and design of dithiophosphate backbone aptamers targeting transcription factor NF-kappaB.

A variety of monothio- and dithiosubstituted duplex aptamers targeting NF-kappaB have been synthesized and designed. The specificity and affinity of the dithioate aptamers of p50 and RelA(p65) NF-kappaB homodimers was determined by gel shift experiments. The NMR solution structures for several unmodified and dithioate backbone modified 14-base paired duplex aptamers have been determined by a hybrid, complete matrix (MORASS)/restrained molecular dynamics method. Structural perturbations of the dithioate substitutions support our hypothesis that the dithioate binds cations less tightly than phosphoryl groups. This increases the electrostatic repulsion across the B-form narrow minor groove and enlarges the minor groove, similar to that found in A-form duplexes. Structural analysis of modeled aptamer complexes with NF-kappaB homo- and heterodimers suggests that the dithioate backbone substitution can increase the aptamer's relative affinity to basic groups in proteins such as NF-kappaB by helping to "strip" the cations from the aptamer backbone.

Base Sequence↗