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.
Yang
Xianbin
X
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.
Bassett
Suzanne E
SE
Li
Xin
X
Luxon
Bruce A
BA
Herzog
Norbert K
NK
Shope
Robert E
RE
Aronson
Judy
J
Prow
Tarl W
TW
Leary
James F
JF
Kirby
Romy
R
Ellington
Andrew D
AD
Gorenstein
David G
DG
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
D017931:DNA Primers; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009705:Nucleosides; D009841:Oligonucleotides; D010710:Phosphates; D013873:Thionucleotides; C083120:phosphorodithioic acid