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.

Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: Current and developing technologies

“…However, fluidic ELISA-based assays capable of identifying unique bound sequences in a multiplexed oligonucleotide binding reaction have been recently developed. Gorenstein, et al [68,69] created a polystyrene bead library consisting of unique bead collections synthesized with a single species of nuclease-resistant synthetic phosphodiester-modified oligonucleotides known as thioaptamers. A mixture of differently labeled thioaptamer coated beads was incubated with NF-κB p50 protein, and then the bound protein was detected with a fluorescently labeled anti-p50 antibody.…”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…Therefore, a natural modification of these systems involves replacing cells with beads. Selections with beads that are each bound to only a single type of nucleic acid sequence have been demonstrated where high affinity beads are identified by incubating them with their target and imaging the level of binding with fluorescently labeled targets 86 or antibodies (Figure 4(b)). 87 This method is unique in that the highest affinity aptamers are assayed and identified directly by the brightest beads.…”

Ultra high‐speed sorting

“…5). Application of high-speed flow cytometry and sorting to bead-based combinatorial chemistry libraries has been demonstrated in two recent papers (30,31). speed sorting experiments of 10 h or longer.…”