Probing the limits of aptamer affinity with a microfluidic SELEX platform.
Probing the limits of aptamer affinity with a microfluidic SELEX platform.
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DOI：
10.1371/journal.pone.0027051
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Soh HT
中科院分区：
文献类型：
--
作者：
Ahmad KM;Oh SS;Kim S;McClellen FM;Xiao Y;Soh HT
相对于抗体和其他基于蛋白质的亲和试剂，基于核酸的适体提供了许多潜在的优点，包括容易的化学合成、可逆折叠、改善的热稳定性和较低的成本。然而，它们的选择需要大量的时间和资源，并且选择通常不能产生具有足以用于分子诊断或治疗的亲和力的分子。为了获得一种能够高效、可重复地产生高性能适体的选择技术，我们开发了一种微流控选择方法（M-SELEX），该方法可用于获得针对不同蛋白质靶标的高亲和力适体。在这里，我们使用一个共同的协议，针对三个蛋白质的目标，不同的等电点（pI）分离的DNA适体。仅经过三轮筛选，我们就发现了与血小板衍生生长因子B（PDGF-BB; pI = 9.3）和凝血酶（pI = 8.3）结合的新型适体序列，其解离常数（Kd）分别为0.028 nM和0.33 nM，均上级先前报道的针对这些靶点的适体。 同时，我们发现了一种新的适体，它与载脂蛋白E3（ApoE; pI = 5.3）结合，Kd为3.1 nM。 此外，我们观察到蛋白质的净电荷可能会对所选择的适体的亲和力产生影响。为了进一步探索这种关系，我们使用相同的选择方案在不同pH条件下对PDGF-BB进行选择，并报告蛋白质电荷和适体Kd之间的负相关性。
Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (Kd) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a Kd of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer Kd.
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DOI：
10.1016/j.str.2009.09.011
发表时间：
2009-11-11
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Huang RH;Fremont DH;Diener JL;Schaub RG;Sadler JE
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Sadler JE
DOI：
10.3390/molecules15010001
发表时间：
2009-12-24
期刊：
Molecules (Basel, Switzerland)
影响因子：
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作者：
Avci-Adali M;Paul A;Wilhelm N;Ziemer G;Wendel HP
通讯作者：
Wendel HP
影响因子：
3.7
作者：
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通讯作者：
Zichi D
影响因子：
5.6
作者：
Tasset, DM;Kubik, MF;Steiner, W
通讯作者：
Steiner, W
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