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2015 ; 87
(1
): 821-8
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Array-based discovery of aptamer pairs
#MMPMID25495696
Cho M
; Oh SS
; Nie J
; Stewart R
; Radeke MJ
; Eisenstein M
; Coffey PJ
; Thomson JA
; Soh HT
Anal Chem
2015[Jan]; 87
(1
): 821-8
PMID25495696
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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 ultrahigh affinity, highlighting the
promise of such reagents for research and clinical use.
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