Probing the Limits of Aptamer Affinity with a Microfluidic SELEX Platform

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.

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

“…SOMAmer structures reveal a strong reliance on hydrophobic interactions, and in this sense their binding to proteins more closely resembles typical protein-protein interactions. Consistent with this observation, the affinity of SL5 for PDGF shows virtually no decrease across a broad range of salt concentrations (0.1-1.0 M NaCl) or pH values (5.0-8.8), in contrast to the effects seen with traditional aptamers (14,34).…”

“…Reducing the disulfide bonds of PDGF-BB with DTT diminished binding affinity by >100-fold, showing that SL1 selectively recognizes the native conformation of its target. Overall, SL1 binds to PDGF-BB with affinity that compares favorably to previously reported PDGF aptamers (13,14). SL1 potently inhibits PDGF-BB-induced phosphorylation of PDGF β-receptors (PDGFRβ) in cultured human fibroblasts (Fig.…”

Quantitative selection and parallel characterization of aptamers

“…We performed four rounds of M-SELEX using the micromagnetic separation (MMS) device previously developed by our group (16), which allowed us to uniformly control the selection and washing stringency (15)(16)(17)(18). Although the MMS device is capable of performing highly stringent washing, we used moderate selection conditions for this experiment.…”

“…For example, novel separation methodologies such as capillary electrophoresis (13,14) and microfluidic devices (15)(16)(17) can isolate high-affinity aptamers after a minimal number of selection rounds. This increased efficiency is achieved through tighter control over selection stringency by applying rigorous washing to isolate aptamers with slow off-rates or by using minimal target quantities to establish a highly competitive binding environment.…”

Particle Display: A Quantitative Screening Method for Generating High‐Affinity Aptamers

“…We chose thrombin and ApoE in order to compare the affinity of our aptamers to previously published aptamers for these targets [20][21][22] . PAI-1 and 4-1BB were chosen because, although RNA aptamer have been reported [23,24] revious attempts at generating natural DNA aptamers for these proteins via SELEX were unsuccessful and ultimately required the use of chemically modified bases [8,23,24] , suggesting that natural DNA may lack the chemical and/or structural diversity to yield useful aptamers for these two proteins.…”