Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing
AUTOR(ES)
Yang, Xianbin
FONTE
Oxford University Press
RESUMO
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-κB 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=137987Documentos Relacionados
- Immunofluorescence assay and flow-cytometry selection of bead-bound aptamers
- Rapid PCR-based characterization of sequences flanking microsatellites in large-insert libraries.
- A PCR-based method for high stringency screening of DNA libraries.
- A Rapid PCR-Based DNA Test for Enterotoxic Bacillus cereus
- Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds