22
L. Hao and H. Gu
that a single round of CE selection can, on average, enrich a randomer synthetic
DNA oligo mixture for thrombin binding activity from 0.4% aptamer content before
selection to >15% aptamer content. This level of enrichment will enable the NGS
platform to resolve, with very high probability, the best aptamers for targets of interest
in a variety of applications, including the discovery of new drug candidates and
diagnostic reagents.
Cancer Antigen 125 (CA125), the clinical gold standard biomarker for ovarian
cancer, is a mucin glycoprotein whose concentration in serum correlates with a
woman’s risk of developing ovarian cancer and also indicates response to therapy in
diagnosed patients. Scoville et al. reported on the use of One-Pot SELEX, in which
selection and amplification of candidate oligonucleotides occur in a single container,
to isolate ssDNA aptamers with affinity for CA125, followed by high-throughput
sequencing of the selected oligonucleotides [74] (Fig. 1.15). The entire selection
process, not merely its endpoint, was characterized by the bioinformatics analysis of
HTS results. After evaluating the binding affinities of aptamer candidates by using
fluorescence anisotropy and affinity probe capillary electrophoresis, two aptamers
were selected, which were found to bind to clinically relevant concentrations of the
protein target.
Fig. 1.15 Flowchart showing the steps involved in analyzing high-throughput sequence data
collected after a SELEX experiment [74]
L. Hao and H. Gu
that a single round of CE selection can, on average, enrich a randomer synthetic
DNA oligo mixture for thrombin binding activity from 0.4% aptamer content before
selection to >15% aptamer content. This level of enrichment will enable the NGS
platform to resolve, with very high probability, the best aptamers for targets of interest
in a variety of applications, including the discovery of new drug candidates and
diagnostic reagents.
Cancer Antigen 125 (CA125), the clinical gold standard biomarker for ovarian
cancer, is a mucin glycoprotein whose concentration in serum correlates with a
woman’s risk of developing ovarian cancer and also indicates response to therapy in
diagnosed patients. Scoville et al. reported on the use of One-Pot SELEX, in which
selection and amplification of candidate oligonucleotides occur in a single container,
to isolate ssDNA aptamers with affinity for CA125, followed by high-throughput
sequencing of the selected oligonucleotides [74] (Fig. 1.15). The entire selection
process, not merely its endpoint, was characterized by the bioinformatics analysis of
HTS results. After evaluating the binding affinities of aptamer candidates by using
fluorescence anisotropy and affinity probe capillary electrophoresis, two aptamers
were selected, which were found to bind to clinically relevant concentrations of the
protein target.
Fig. 1.15 Flowchart showing the steps involved in analyzing high-throughput sequence data
collected after a SELEX experiment [74]