Multiplexed microcolumn-based process for efficient selection of RNA aptamers.
Multiplexed microcolumn-based process for efficient selection of RNA aptamers.
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DOI：
10.1021/ac400105e
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发表时间：
2013-03-19
影响因子：
7.4
通讯作者：
Craighead, Harold G.
中科院分区：
文献类型：
--
作者：
Latulippe, David R.;Szeto, Kylan;Ozer, Abdullah;Duarte, Fabiana M.;Kelly, Christopher V.;Pagano, John M.;White, Brian S.;Shalloway, David;Lis, John T.;Craighead, Harold G.
我们描述了一种可重复使用的微柱和方法，用于有效地发现多个靶分子的核酸适体。我们的装置的设计仅需要微升体积的亲和层析树脂--这是使靶结合序列相对于非靶结合序列的富集最大化的条件（即，背景）序列。此外，装置的模块化设计适应多重适体选择方案。我们优化了选择过程的性能，使用微柱填充绿色荧光蛋白（GFP）固定化树脂和监测，在广泛的实验条件下，富集一个已知的GFP结合RNA适体（GFPapt）对随机RNA适体库。我们通过在用GFP和其他蛋白制备物的从头选择实验中监测GFPapt的富集来验证多重方法。仅经过三轮选择后，在载有GFP的树脂上的累积GFPapt富集大于108，而没有富集其它非特异性靶标。我们使用这个优化的协议进行多重选择两个人热休克因子（hHSF）蛋白，hHSF 1和hHSF 2。使用高通量测序来鉴定每种蛋白质的适体，所述适体仅在三轮选择中优先富集，所述适体在五轮后被确认和分离。凝胶位移和荧光偏振测定显示，每个适体以高亲和力（KD < 20 nM）结合各自的靶标。我们的微柱与多重方法和高通量测序相结合，能够以高通量和有效的方式选择针对多个靶标的适体。
We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin—a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 108 with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.
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影响因子：
3.7
作者：
Gold L;Ayers D;Bertino J;Bock C;Bock A;Brody EN;Carter J;Dalby AB;Eaton BE;Fitzwater T;Flather D;Forbes A;Foreman T;Fowler C;Gawande B;Goss M;Gunn M;Gupta S;Halladay D;Heil J;Heilig J;Hicke B;Husar G;Janjic N;Jarvis T;Jennings S;Katilius E;Keeney TR;Kim N;Koch TH;Kraemer S;Kroiss L;Le N;Levine D;Lindsey W;Lollo B;Mayfield W;Mehan M;Mehler R;Nelson SK;Nelson M;Nieuwlandt D;Nikrad M;Ochsner U;Ostroff RM;Otis M;Parker T;Pietrasiewicz S;Resnicow DI;Rohloff J;Sanders G;Sattin S;Schneider D;Singer B;Stanton M;Sterkel A;Stewart A;Stratford S;Vaught JD;Vrkljan M;Walker JJ;Watrobka M;Waugh S;Weiss A;Wilcox SK;Wolfson A;Wolk SK;Zhang C;Zichi D
通讯作者：
Zichi D
影响因子：
14.9
作者：
Zhao X;Shi H;Sevilimedu A;Liachko N;Nelson HC;Lis JT
通讯作者：
Lis JT
影响因子：
2.9
作者：
Cox, JC;Rudolph, P;Ellington, AD
通讯作者：
Ellington, AD
影响因子：
3.5
作者：
JOYCE, GF
通讯作者：
JOYCE, GF
影响因子：
64.5
作者：
Dai, Chengkai;Whitesell, Luke;Lindquist, Susan
通讯作者：
Lindquist, Susan