Generation of highly specific aptamers via micromagnetic selection.
Aptamers are nucleic acid-based reagents that bind to target molecules with high affinity and specificity. However, methods for generating aptamers from random combinatorial libraries (e.g., systematic evolution of ligands by exponential enrichment (SELEX)) are often labor-intensive and time-consuming. Recent studies suggest that microfluidic SELEX (M-SELEX) technology can accelerate aptamer isolation by enabling highly stringent selection conditions through the use of very small amounts of target molecules. We present here an alternative M-SELEX method, which employs a disposable microfluidic chip to rapidly generate aptamers with high affinity and specificity. The micromagnetic separation (MMS) chip integrates microfabricated ferromagnetic structures to reproducibly generate large magnetic field gradients within its microchannel that efficiently trap magnetic bead-bound aptamers. Operation of the MMS device is facile and robust and demonstrates high recovery of the beads (99.5%), such that picomolar amounts of target molecule can be used. Importantly, the device demonstrates exceptional separation efficiency in removing weakly bound and unbound ssDNA to rapidly enrich target-specific aptamers. As a model, we demonstrate here the generation of DNA aptamers against streptavidin in three rounds of positive selection. We further enhanced the specificity of the selected aptamers via a round of negative selection in the same device against bovine serum albumin (BSA). The resulting aptamers displayed dissociation constants ranging from 25 to 65 nM for streptavidin and negligible affinity for BSA. Since a wide spectrum of molecular targets can be readily conjugated to magnetic beads, MMS-based SELEX provides a general platform for rapid generation of specific aptamers.
Oh
Seung Soo
SS
Materials Department, [corrected] University of California, Santa Barbara, California 93106, USA.
Qian
Jiangrong
J
Lou
Xinhui
X
Zhang
Yanting
Y
Xiao
Yi
Y
Soh
H Tom
HT
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D008280:Magnetics; D044085:Microfluidics / Q000379:methods*; D008969:Molecular Sequence Data; D052156:SELEX Aptamer Technique / Q000295:instrumentation / Q000379:methods*; D019809:Streptavidin / Q000032:analysis*; D013379:Substrate Specificity
D052157:Aptamers, Nucleotide; D019809:Streptavidin