title: Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing.
abstract: 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.
authors.affiliation: Sealy Center for Structural Biology and Department of Human Biological Chemistry and Genetics, 301 University Boulevard, The University of Texas Medical Branch at Galveston, TX 77555-1157, USA.
authors.forename: Xianbin
authors.initials: X
authors.lastname: Yang
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Bassett
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Luxon
authors.forename: Norbert K
authors.initials: NK
authors.lastname: Herzog
authors.forename: Robert E
authors.initials: RE
authors.lastname: Shope
authors.forename: Judy
authors.initials: J
authors.lastname: Aronson
authors.forename: Tarl W
authors.initials: TW
authors.lastname: Prow
authors.forename: James F
authors.initials: JF
authors.lastname: Leary
authors.forename: Romy
authors.initials: R
authors.lastname: Kirby
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
authors.forename: David G
authors.initials: DG
authors.lastname: Gorenstein
mesh_terms: D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D017931:DNA Primers; D008863:Microspheres; D016328:NF-kappa B / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D009705:Nucleosides / Q000737:chemistry; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
chemical_list: D017931:DNA Primers; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009705:Nucleosides; D009841:Oligonucleotides; D010710:Phosphates; D013873:Thionucleotides; C083120:phosphorodithioic acid