#文献分享# 适配体筛选的成败取决于什么？CE-SELEX大佬文章推荐

首页 ꄲ 文献分享 ꄲ #文献分享# 适配体筛选的成败取决于什么？CE-SELEX大佬文章推荐

推荐语

这是一篇研究筛选机理的文章，值得所有做筛选的小伙伴阅读。文章探讨的是如何确定筛选压力，才能实现文库的定向富集。Krylov S. N.教授是CE-SELEX领域的开创者，2002年就建立了CE-SELEX技术，过去20多年一直在研究CE-SELEX技术。为了向Krylov S. N.教授致敬，我们在文末附上了他的所有与aptamer相关的论文，供参考。

回到本篇论文，小编觉得论文写的不够简洁，作者引入BNR（binder-to-nonbinder ratio），3σBNR作为控制严谨性的参数等。我觉得由于binder占比非常少，譬如万分之一甚至更低，nonbinder几乎就等于全部的投入分子，所以nonbinder直接用投入的文库总分子数，BNR直接用保留率更简洁明了。文中的BNR也考虑了背景结合。

文章的结论用白话表达出来就是：有靶标筛选时的保留率，要比对照（无靶标仅有基质）要高3倍才有可能筛选成功。我们实验室一直在用保留率和有效保留率这样的参数来监测筛选的过程。团队大哥一直要求我们做实验，要做到心中有数，也就是每轮筛选要知道投入了多少分子，它们去了哪儿，最后幸存者有多少，以及反筛同样条件下会保留多少等等？

另外，文中还提到磁珠筛选重复性差的问题，其实，这就是手工操作所致。筛选步骤多，保证每次几十步操作一致是很难做到的。唯一的解决方法就是用自动化，我们开发的半自动化筛选仪，每次可以筛选1-16个靶标，非常适合多靶标不同条件的比较筛选，也非常适合类似本文的机理研究。近期签约购买，还有新年冲喜特价。

特别提示：小编认为，作者文章中考虑的因素并不全面，这点今后有机会再和大家分享。另外，本文中的MutS以及凝血酶都是非常容易筛选的蛋白，MutS也是作者实验室20年前就在筛选的蛋白。凝血酶我们一轮即可筛选成功。对这些容易筛选的蛋白，其实不需要套路，不需要复杂的方法。小编提示，请谨慎参考所有你读过的文章。

今天介绍一篇最新发表在JACS上的文章Quantitative Characterization of Partitioning Stringency in SELEX。主要介绍了 SELEX（指数富集适配体系统进化技术）中适配体与非适配体比例（BNR）与筛选严格性的定量关系，提出了一种新的衡量和控制筛选严格性的方法。

研究背景

SELEX 是一种用于从随机序列寡核苷酸文库中筛选适配体的技术，其过程包括反应、分离和扩增三个步骤，通过多轮迭代筛选出高亲和力的适配体。严格性是 SELEX 中的一个重要参数，但目前缺乏定量定义和测量方法，只能进行定性或比较性描述。增加严格性有助于筛选出强结合的适配体，但过高的严格性可能导致 SELEX 失败。

研究目的

对 SELEX（指数富集适配体系统进化技术）中的分配严格性进行定量表征。

研究内容

1. 严格性的定量测量

基于增加严格性会减少分离后适配体数量这一普遍效应，提出用适配体与非适配体比例（BNR）的倒数来定义严格性（S），即

其中B_out和N_out分别为分离后适配体和非适配体的数量。

给出了通过测量有无靶标时的输出文库量(L_out,+T和L_out,-T）来计算 BNR 和 S 的方法，即

这些量可通过定量 PCR（qPCR）测定，且部分 SELEX 从业者已常规测量。

2.SELEX 不失败准则

图 1. SELEX 过程的示意图（a）以及结合物数量随时间线的相关变化（b）。

理论上 SELEX 要成功 BNR 必须大于零，但实际中需考虑实验不确定性，因此提出 SELEX 不失败准则为BNR>n_σBNR（n 为置信水平，默认n=3），其中σBNR可通过误差传播规则计算，且主要由L(out,-T)的标准偏差决定。

图 2. 从非结合物（N）中分离结合物（B）的示意图。

该准则可用于判断 SELEX 是否会因严格性过高而失败，有助于研究者更合理地控制 SELEX 进程。

3.实验验证

以 His - tagged MutS 和 nontagged thrombin 为靶蛋白，使用单一 40 个随机核苷酸的 DNA 文库，通过改变靶浓度（500、100、10 和 1 nM）和分离时间，采用毛细管电泳（CE）进行分离，进行了多轮 SELEX 实验，并测定每轮的L_out,+T 、L_out,-T、k_N和 BNR，同时进行批量亲和力测定以跟踪 SELEX 进程。

4.实验结果

图3.在四种不同恒定靶标浓度下，对 MutS 和凝血酶进行 SELEX 筛选时的相关评估结果，包括 BNR 值（图 a）和以 R 值表示的整体亲和力（图 b）。

对于恒定靶浓度的 SELEX 实验，MutS 的k(N)值低于 thrombin，导致其 BNR 值更高；BNR 随靶浓度降低而降低，当 BNR 低于3(σBNR)时，后续轮次中未检测到 BNR 增加；BNR 值与亲和力成熟密切相关，较高的 BNR（>3σ）对应着明显的亲和力成熟。

图 4. 通过比较 BNR 值（a）和以 R 值表示的整体亲和力（b）来评估在逐轮降低靶标浓度下 MutS 和凝血酶的 SELEX 筛选结果。

在靶浓度逐轮降低的 SELEX 实验中，当 thrombin 的靶浓度降至 10 nM 时，BNR 低于3(σBNR)，亲和力成熟停止；而 MutS 在所有浓度下 BNR 均高于3(σBNR)，亲和力成熟持续。

研究结论

定量建立了 SELEX 严格性与 BNR 之间的关系，BNR 是一种实用、有效且通用的严格性测量方法。

提出的 SELEX 不失败准则（BNR > 0）得到实验支持，有助于降低 SELEX 失败风险。

采用逆 BNR 作为定量严格性测量方法，为优化 SELEX 进程提供了系统框架，也有望应用于其他涉及可量化适配体的人工进化技术。

以下是 Krylov, S. N. 教授有关CE-SELEX的相关论文，从2002到2024，供各位参考。

1 Berezovski, M. & Krylov, S. N. Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. J Am Chem Soc 124, 13674-13675, doi:ja028212e [pii] (2002).

2 Berezovski, M. & Krylov, S. N. Using DNA-binding proteins as an analytical tool. Journal of the American Chemical Society 125, 13451-13454, doi:10.1021/ja037186x (2003).

3 Berezovski, M., Nutiu, R., Li, Y. F. & Krylov, S. N. Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures. Analytical chemistry 75, 1382-1386, doi:10.1021/ac026214b (2003).

4 Berezovski, M. & Krylov, S. N. Using nonequilibrium capillary electrophoresis of equilibrium mixtures for the determination of temperature in capillary electrophoresis. Analytical chemistry 76, 7114-7117, doi:Doi 10.1021/Ac0489519 (2004).

5 Drabovich, A. & Krylov, S. N. Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis. J Chromatogr A 1051, 171-175 (2004).

6 Berezovski, M. et al. Nonequilibrium capillary electrophoresis of equilibrium mixtures: A universal tool for development of aptamers. Journal of the American Chemical Society 127, 3165-3171, doi:10.1021/ja042394q (2005).

7 Berezovski, M. & Krylov, S. N. Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures. Analytical chemistry 77, 1526-1529, doi:10.1021/ac048577c (2005).

8 Berezovski, M. V., Okhonin, V., Petrov, A. & Krylov, S. N. in Progress in Biomedical Optics and Imaging - Proceedings of SPIE.

9 Drabovich, A., Berezovski, M. & Krylov, S. N. Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). J Am Chem Soc 127, 11224-11225, doi:10.1021/ja0530016 (2005).

10 Krylov, S. N. Smart aptamers: The proof of principle. Abstr Pap Am Chem S 229, U147-U147 (2005).

11 Petrov, A., Okhonin, V., Berezovski, M. & Krylov, S. N. Kinetic capillary electrophoresis (KCE): A conceptual platform for kinetic homogeneous affinity methods. Journal of the American Chemical Society 127, 17104-17110, doi:Doi 10.1021/Ja0562321 (2005).

12 Berezovski, M., Musheev, M., Drabovich, A. & Krylov, S. N. Non-SELEX selection of aptamers. J Am Chem Soc 128, 1410-1411, doi:10.1021/ja056943j (2006).

13 Berezovski, M. V., Musheev, M. U., Drabovich, A. P., Jitkova, J. V. & Krylov, S. N. Non-SELEX: Selection of aptamers without intermediate amplification of candidate oligonucleotides. Nature Protocols 1, 1359-1369, doi:10.1038/nprot.2006.200 (2006).

14 Drabovich, A. P., Berezovski, M., Okhonin, V. & Krylov, S. N. Selection of smart aptamers by methods of kinetic capillary electrophoresis. Analytical chemistry 78, 3171-3178, doi:10.1021/ac060144h (2006).

15 Krylov, S. N. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM): A novel method for biomolecular screening. Journal of Biomolecular Screening 11, 115-122, doi:Doi 10.1177/1087057105284339 (2006).

16 Musheev, M. U. & Krylov, S. N. Selection of aptamers by systematic evolution of ligands by exponential enrichment: Addressing the polymerase chain reaction issue. Analytica Chimica Acta 564, 91-96, doi:10.1016/j.aca.2005.09.069 (2006).

17 Okhonin, V., Petrov, A. P., Berezovski, M. & Krylov, S. N. Plug-plug kinetic capillary electrophoresis: Method for direct determination of rate constants of complex formation and dissociation. Analytical chemistry 78, 4803-4810, doi:Doi 10.1021/Ac060108i (2006).

18 Drabovich, A. P., Okhonin, V., Berezovski, M. & Krylov, S. N. Smart aptamers facilitate multi-probe affinity analysis of proteins with ultra-wide dynamic range of measured concentrations. Journal of the American Chemical Society 129, 7260-+, doi:10.1021/ja072269p (2007).

19 Krylov, S. N. Kinetic CE: Foundation for homogeneous kinetic affinity methods. Electrophoresis 28, 69-88, doi:10.1002/elps.200600577 (2007).

20 Berezovski, M. V., Lechmann, M., Musheev, M. U., Mak, T. W. & Krylov, S. N. Aptamer-facilitated biomarker discovery (AptaBiD). Journal of the American Chemical Society 130, 9137-9143, doi:10.1021/ja801951p (2008).

21 Krylov, S. N. in Aptamers in Bioanalysis 181-212 (2008).

22 Musheev, M. U., Javaherian, S., Okhonin, V. & Krylov, S. N. Diffusion as a tool of measuring temperature inside a capillary. Analytical chemistry 80, 6752-6757, doi:Doi 10.1021/Ac8009406 (2008).

23 Javaherian, S., Musheev, M. U., Kanoatov, M., Berezovski, M. V. & Krylov, S. N. Selection of aptamers for a protein target in cell lysate and their application to protein purification. Nucleic Acids Research 37, doi:ARTN e62

10.1093/nar/gkp176 (2009).

24 Krylova, S. M., Okhonin, V. & Krylov, S. N. Transverse diffusion of laminar flow profiles - A generic method for mixing reactants in capillary microreactor. Journal of separation science 32, 742-756, doi:10.1002/jssc.200800671 (2009).

25 Yunusov, D. et al. Kinetic capillary electrophoresis-based affinity screening of aptamer clones. Analytica Chimica Acta 631, 102-107, doi:10.1016/j.aca.2008.10.027 (2009).

26 Kanoatov, M., Javaherian, S. & Krylov, S. N. Selection of aptamers for a non-DNA binding protein in the context of cell lysate. Analytica Chimica Acta 681, 92-97, doi:10.1016/j.aca.2010.09.044 (2010).

27 Musheev, M. U., Filiptsev, Y. & Krylov, S. N. Noncooled capillary inlet: A source of systematic errors in capillary-electrophoresis-based affinity analyses. Analytical chemistry 82, 8637-8641, doi:10.1021/ac1018364 (2010).

28 Bao, J. Y., Krylova, S. M., Reinstein, O., Johnson, P. E. & Krylov, S. N. Label-Free Solution-Based Kinetic Study of Aptamer-Small Molecule Interactions by Kinetic Capillary Electrophoresis with UV Detection Revealing How Kinetics Control Equilibrium. Analytical chemistry 83, 8387-8390, doi:10.1021/ac2026699 (2011).

29 Cherney, L. T., Kanoatov, M. & Krylov, S. N. Method for Determination of Peak Areas in Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures. Analytical chemistry 83, 8617-8622, doi:10.1021/ac2027113 (2011).

30 de Jong, S. & Krylov, S. N. Protein Labeling Enhances Aptamer Selection by Methods of Kinetic Capillary Electrophoresis. Analytical chemistry 83, 6330-6335, doi:10.1021/ac201242r (2011).

31 Kanoatov, M. & Krylov, S. N. DNA Adsorption to the Reservoir Walls Causing Irreproducibility in Studies of Protein-DNA Interactions by Methods of Kinetic Capillary Electrophoresis. Analytical chemistry 83, 8041-8045, doi:10.1021/ac202048y (2011).

32 Krylova, S. M. et al. DNA Aptamers as Analytical Tools for the Quantitative Analysis of DNA-Dealkylating Enzymes. Analytical biochemistry, doi:10.1016/j.ab.2011.03.010 (2011).

33 Krylova, S. M. et al. DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes. Analytical biochemistry 414, 261-265, doi:10.1016/j.ab.2011.03.010 (2011).

34 Bao, J. Y. & Krylov, S. N. Volatile Kinetic Capillary Electrophoresis for Studies of Protein-Small Molecule Interactions. Analytical chemistry 84, 6944-6947, doi:10.1021/ac301829t (2012).

35 de Jong, S., Epelbaum, N., Liyanage, R. & Krylov, S. N. A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis. Electrophoresis 33, 2584-2590, doi:10.1002/elps.201200153 (2012).

36 Krylova, S. M., Koshkin, V., Bagg, E., Schofield, C. J. & Krylov, S. N. Mechanistic studies on the application of DNA aptamers as inhibitors of 2-oxoglutarate-dependent oxygenases. Journal of Medicinal Chemistry 55, 3546-3552, doi:10.1021/jm300243h (2012).

37 Cherney, L. T., Obrecht, N. M. & Krylov, S. N. Theoretical Modeling of Masking DNA Application in Aptamer-Facilitated Biomarker Discovery. Analytical chemistry 85, 4157-4164, doi:10.1021/ac400385v (2013).

38 Yufa, R. et al. Emulsion PCR Significantly Improves NECEEM-based Aptamer Selection, Allowing for Efficient and Rapid Selection of Aptamer to Unmodified ABH2 Protein. Anal Chem, doi:10.1021/ac5044187 (2014).

39 Agostino, F. J. & Krylov, S. N. Advances in steady-state continuous-flow purification by small-scale free-flow electrophoresis. Trac-Trends in Analytical Chemistry 72, 68-79, doi:10.1016/j.trac.2015.03.023 (2015).

40 Kanoatov, M. et al. Using Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) for Simultaneous Determination of Concentration and Equilibrium Constant. Analytical chemistry 87, 3099-3106, doi:10.1021/acs.analchem.5b00171 (2015).

41 Yufa, R. et al. Emulsion PCR Significantly Improves Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures-Based Aptamer Selection: Allowing for Efficient and Rapid Selection of Aptamer to Unmodified ABH2 Protein. Analytical chemistry 87, 1411-1419, doi:10.1021/ac5044187 (2015).

42 Kanoatov, M. & Krylov, S. N. Analysis of DNA in Phosphate Buffered Saline Using Kinetic Capillary Electrophoresis. Analytical chemistry 88, 7421-7428, doi:10.1021/acs.analchem.6b02117 (2016).

43 Norris, V., Krylov, S. N., Agarwal, P. K. & White, G. J. Synthetic, Switchable Enzymes. J Mol Microb Biotech 27, 117-127, doi:10.1159/000464443 (2017).

44 Beloborodov, S. S. et al. Aptamer facilitated purification of functional proteins. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 1073, 201-206, doi:10.1016/j.jchromb.2017.12.024 (2018).

45 Beloborodov, S. S., Krylova, S. M. & Krylov, S. N. Spherical-Shape Assumption for Protein-Aptamer Complexes Facilitates Prediction of Their Electrophoretic Mobility. Anal Chem, doi:10.1021/acs.analchem.9b02019 (2019).

46 Le, A. T. H., Krylova, S. M., Kanoatov, M., Desai, S. & Krylov, S. N. Ideal-Filter Capillary Electrophoresis (IFCE) Facilitates the One-Step Selection of Aptamers. Angewandte Chemie-International Edition 58, 2739-2743, doi:10.1002/anie.201812974 (2019).

47 Le, A. T. H., Krylova, S. M. & Krylov, S. N. Determination of the Equilibrium Constant and Rate Constant of Protein-Oligonucleotide Complex Dissociation under the Conditions of Ideal-Filter Capillary Electrophoresis. Anal Chem 91, 8532-8539, doi:10.1021/acs.analchem.9b01801 (2019).

48 Le, A. T. H., Wang, T. Y., Krylova, S. M., Beloborodov, S. S. & Krylov, S. N. Quantitative Characterization of Partitioning in Selection of DNA Aptamers for Protein Targets by Capillary Electrophoresis. Analytical chemistry 94, 2578-2588, doi:10.1021/acs.analchem.1c04560 (2022).

49 Teclemichael, E., Le, A. T. H., Krylova, S. M., Wang, T. Y. & Krylov, S. N. Bulk Affinity Assays in Aptamer Selection: Challenges, Theory, and Workflow. Analytical chemistry, doi:10.1021/acs.analchem.2c03173 (2022).

50 Wang, T. Y. et al. Transient Incomplete Separation of Species with Close Diffusivity to Study the Stability of Affinity Complexes. Analytical chemistry 94, 15415-15422, doi:10.1021/acs.analchem.2c03313 (2022).

51 Le, A. T. H., Krylova, S. M. & Krylov, S. N. Kinetic capillary electrophoresis in screening oligonucleotide libraries for protein binders. TrAC - Trends in Analytical Chemistry 162, doi:10.1016/j.trac.2023.117061 (2023).

52 Norris, V., Oláh, J., Krylov, S. N., Uversky, V. N. & Ovádi, J. The Sherpa hypothesis: Phenotype-Preserving Disordered Proteins stabilize the phenotypes of neurons and oligodendrocytes. Npj Syst Biol Appl 9, doi:ARTN 31

10.1038/s41540-023-00291-8 (2023).

53 Le, A. T. H., Teclemichael, E., Krylova, S. M. & Krylov, S. N. Quantitative Characterization of Partitioning Stringency in SELEX. Jacs Au, doi:10.1021/jacsau.4c00890 (2024).

54 Riesen, A. J. V. et al. Tuning phenolic hemicyanines for ratiometric DNA sensing and live cell nuclear bioimaging applications. Canadian Journal of Chemistry, doi:10.1139/cjc-2024-0014 (2024).