Accession Number:

AD1045708

Title:

Array based Discovery of Aptamer Pairs (Open Access Publisher's Version)

Descriptive Note:

Journal Article - Open Access

Corporate Author:

CALIFORNIA UNIV SANTA BARBARA SANTA BARBARA United States

Report Date:

2014-12-11

Pagination or Media Count:

8.0

Abstract:

Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site bidentate target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant hot spot epitope. Our array based discovery platform for multivalent aptamers AD-MAP overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM 200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultra high affinity, highlighting the promise of such reagents for research and clinical use.

Subject Categories:

  • Biochemistry

Distribution Statement:

APPROVED FOR PUBLIC RELEASE