Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing.
Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing.
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DOI：
10.1371/journal.pone.0019395
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Borer PN
中科院分区：
文献类型：
--
作者：
Kupakuwana GV;Crill JE 2nd;McPike MP;Borer PN
适配子是以高亲和力和选择性结合蛋白质和其他靶标的寡核苷酸。二十年前，为了区分随机序列文库中的适配子，自然选择的元素被适应于体外选择。传统适体发现的主要瓶颈是多个周期的体外进化。我们表明，适体文库中序列的过度表示和深度测序使适体的无环鉴定成为可能。我们通过分离已知的人类α-凝血酶适配子家族证明了这一点。在包含每个可能的随机15聚体片段的平均56,000个拷贝的文库中发现了适体。在200-600万次阅读中，高亲和力序列的计数比背景高出许多倍。对具有10个以上计数的序列的聚类分析区分了两个具有高丰度候选序列的序列基序。Motif I包含之前观察到的共识15聚体、Thb1(46,000个计数)和相关的变异体，主要是G/T替换；二次分析表明对凝血酶的亲和力与丰度相关(Thb1的KD = 12 nM)。在这个实验中，Thb1的信噪比约为10,000：1。Motif II与Thb1无关，领先候选者(29,000个)是刀豆素A的储存和洗脱缓冲液中针对己糖的新型适配子(KD = 0.5µM代表α-甲基甘露糖苷)；ConA用于固定化α-凝血酶。过度表达和深度测序可以极大地缩短发现过程，区分与靶标具有广泛亲和力的适体，允许在简化的文库中穷尽地搜索序列空间，减少所需的靶标的数量，消除循环伪影，并且应该允许对测序实验和靶标进行多路复用。
Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2–6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (Kd = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (Kd = 0.5 µM for α-methyl-mannoside); ConA was used to immobilize α-thrombin. Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets.
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影响因子：
5.6
作者：
Kelly, JA;Feigon, J;Yeates, TO
通讯作者：
Yeates, TO
DOI：
10.1073/pnas.1009331107
发表时间：
2010-08-31
影响因子：
11.1
作者：
Cho, Minseon;Xiao, Yi;Soh, H. Tom
通讯作者：
Soh, H. Tom
影响因子：
3.5
作者：
Cox, JC;Ellington, AD
通讯作者：
Ellington, AD
影响因子：
4.1
作者：
Golden, MC;Collins, BD;Koch, TH
通讯作者：
Koch, TH
影响因子：
15
作者：
Drabovich, A;Berezovski, M;Krylov, SN
通讯作者：
Krylov, SN