Abstract
Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (Kd) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a Kd of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer Kd.
Highlights
Aptamers [1], [2] are nucleic acid-based affinity reagents selected through an in vitro process that can bind to a range of molecular targets, including small molecules, proteins, and cells [3]
Due to the iterative nature of the aptamer selection process, the generation of aptamers requires a significant investment of time, labor and resources [5], and selections often fail to yield molecules with affinities suitable for molecular diagnostics or therapeutics
Using a common selection protocol, we have discovered new aptamer sequences that bind to PDGF-BB [9] and thrombin [10], [11] with higher affinities than those previously published
Summary
Aptamers [1], [2] are nucleic acid-based affinity reagents selected through an in vitro process that can bind to a range of molecular targets, including small molecules, proteins, and cells [3]. Microfluidic SELEX (M-SELEX) exerts stringent selection pressures by employing minimal amounts of target molecules and continuous washing to isolate high affinity aptamers in fewer rounds of selection compared to conventional methods [6], [7], [8]. To extend this method, in this work, we report a universal MSELEX process that can isolate high affinity aptamers for a range of protein targets with varying isoelectric points (pIs) within three selection rounds. To further explore this relationship, we performed microfluidic selections with PDGF-BB under varying pH conditions to alter its charge state, and report differences in the affinities of the resulting aptamer pools
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