Array-based discovery of aptamer pairs.
Array-based discovery of aptamer pairs.
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DOI：
10.1021/ac504076k
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发表时间：
2015-01-06
影响因子：
7.4
通讯作者：
Soh, H. Tom
中科院分区：
文献类型：
--
作者：
Cho, Minseon;Oh, Seung Soo;Nie, Jeff;Stewart, Ron;Radeke, Monte J.;Eisenstein, Michael;Coffey, Peter J.;Thomson, James A.;Soh, H. Tom
识别目标蛋白上不同表位的亲和试剂对可以极大地提高分子检测的灵敏度和特异性。重要的是，这样的对可以连接起来产生试剂，实现两个位置的“双齿”目标识别，亲和力大大超过任何一种单价成分。DNA适配子特别适合这种结构，因为它们可以通过标准的合成技术连接在一起，而不需要化学连接。不幸的是，适配子对很难产生，主要是因为传统的选择方法优先产生识别主导的“热点”表位的适配子。我们的基于阵列的多价适配子发现平台(AD-MAP)克服了这一问题，实现了高效的适配子对发现。我们使用微流控选择和高通量测序来获得丰富的适体序列池。接下来，我们根据这些序列合成一个定制的阵列，并进行平行的亲和力测量，以确定目标蛋白的最高亲和力适配子。我们使用这个适配子形成封闭靶标上主要结合位点的复合体，然后用这些复合体筛选相同的阵列来识别与二级表位结合的适配子。我们使用AD-MAP发现DNA适配子对以高亲和力结合人血管生成素-2上的不同位置，即使在未稀释的血清中也是如此。据我们所知，这是第一次使用阵列发现新的适配子对。随后，我们将这些适配子与一种灵活的连接物连接起来，构建了超高亲和力的双齿试剂，其平衡解离常数低至97 pm：>；200倍于任一组分适配子。功能研究证实，这两个适配子都对这种超高亲和力起到了关键作用，突显了此类试剂在研究和临床应用方面的前景。
Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site “bidentate” target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant “hot spot” epitope. Our array-based discovery platform for multivalent aptamers (AD-MAP) overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM: >200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultrahigh affinity, highlighting the promise of such reagents for research and clinical use.
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DOI：
10.3390/s8021090
发表时间：
2008-02-19
期刊：
Sensors (Basel, Switzerland)
影响因子：
--
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
4.5
作者：
Nimjee, Shahid M.;Oney, Sabah;Sullenger, Bruce A.
通讯作者：
Sullenger, Bruce A.
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