Speeding up in vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation.

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ID: 59775
2019
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Abstract
We report here a modified aptamer selection method, magnetic cross-linking precipitation (MCP)-SELEX, for highly efficient library enrichment and aptamer isolation. MCP-SELEX isolates bound aptamers via highly efficient chemical cross-linking between amino groups of target proteins and activated carboxylic acid groups on magnetic beads (> 90% coupling efficiency). Importantly, MCP-SELEX avoids surface interferences in conventional target-fixed methods and substantially minimizes nonspecific binding. The enrichment efficiencies of MCP-SELEX for various proteins (PD-L1, ubiquitin, thrombin, and HSA) were all greatly higher than those of the conventional target-bound magnetic bead based-SELEX (MB-SELEX). Anti-thrombin aptamer with KD of 33 nM was successfully isolated by four rounds of MCP-SELEX. MCP-SELEX also enabled the efficient aptamer isolation by coupling with MB-SELEX or falling-off-SELEX. We identified structure-switching aptamers (SSAs) that specifically bind to HSA with low nanomolar dissociation constant via 3 rounds of MCP-SELEX and 1 round of falling-off-SELEX. Our HSA SSAs also have ~ 3-fold higher specificity against streptavidin relative to thrombin SSAs discovered through falling-off-SELEX only. The enriched library has ~78-fold higher signal-to-noise ratio (the number of DNAs eluted by 50 nM HSA divided by the number of DNAs self-dissociated in blank buffer) than that obtained by 4 rounds of direct falling-off-SELEX. We finally demonstrated the application of the selected SSA in fluorescent detection of HSA in urine with diagnostic required sensitivity and dynamic range. We expect that MCP-SELEX may be coupled with other selection methods to substantially accelerate aptamer discovery.
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qiao2019speedinganalytical Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Qiao, Na;Li, Jing;Wu, Xiao;Diao, Donglin;Zhao, Jiaxing;Li, Jiyuan;Ren, Xijiao;Ding, Xiaofan;Shangguan, Dihua;Lou, Xinhui;
Journal Analytical chemistry
Year 2019
DOI
10.1021/acs.analchem.9b00081
URL
Keywords
phytoremediation soil contamination chemical fractionation phytostabilization pseudomonas spp.

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