Document Detail


Generation of highly specific aptamers via micromagnetic selection.
MedLine Citation:
PMID:  19480397     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Seung Soo Oh; Jiangrong Qian; Xinhui Lou; Yanting Zhang; Yi Xiao; H Tom Soh
Related Documents :
17911257 - Guanine riboswitch variants from mesoplasma florum selectively recognize 2'-deoxyguanos...
8180157 - 1h nmr studies of the high-affinity rev binding site of the rev responsive element of h...
23632627 - The hemoglobin system of the serpent eel ophisurus serpens: structural and functional c...
15854817 - Analytical applications of aptamers.
10395457 - Nucleotide and mg2+ induced conformational changes in groel can be detected by sulfhydr...
1737937 - Analysis of the ap-1 sites in the il-2 promoter.
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Analytical chemistry     Volume:  81     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2009 Jul 
Date Detail:
Created Date:  2009-06-30     Completed Date:  2009-09-29     Revised Date:  2014-09-21    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5490-5     Citation Subset:  IM    
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Aptamers, Nucleotide / chemistry*
Base Sequence
Magnetics*
Microfluidics / methods*
Molecular Sequence Data
SELEX Aptamer Technique / instrumentation,  methods*
Streptavidin / analysis*
Substrate Specificity
Grant Support
ID/Acronym/Agency:
R21 EB008215/EB/NIBIB NIH HHS; R21 EB008215-02/EB/NIBIB NIH HHS; R21 EB009518/EB/NIBIB NIH HHS; R21 EB009518-01/EB/NIBIB NIH HHS
Chemical
Reg. No./Substance:
0/Aptamers, Nucleotide; 9013-20-1/Streptavidin
Comments/Corrections
Erratum In:
Anal Chem. 2011 Mar 1;83(5):1866

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Evaluation of use of a dicationic liquid stationary phase in the fast and conventional gas chromatog...
Next Document:  Single-molecule fluorescence imaging of peptide binding to supported lipid bilayers.