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On the Multivalent Aptamer Selection, Design and Assays
|Publication Year :||2015|
|Abstract:||適體是一種由核酸或胜肽所構成的序列，其和蛋白質抗體一樣能對特定分子具有專一性的辨識功能。相對於抗體，其具有高環境穩定性、易生產、易修飾...等特性，且被視為具有取代抗體於相關診療應用的潛力。相對於抗體必須利用誘導生物體的免疫反應來產生，適體則是利用類似達爾文的演化觀點，由初始多樣的序列針對特定的標的物進行競爭與淘汰的機制來篩選。這種方法被稱為指數富集的配體系統進化技術(systematic evolution of ligands by exponential enrichment, SELEX)。
第一部分主要是建立低蛋白質消耗的適體篩選平台。對蛋白質的適體篩選技術已被探討多年，並發展出多種不同的平台系統。然而，要成功地篩選出適當的適體，往往要耗費mg等級的標的蛋白，且提高篩選成本。因此，建立經濟高效的篩選平台，有其發展必要性。本研究以前列腺癌生物標記α-甲基酰基輔酶A消旋酶(alpha-methylacyl-CoA racemase, AMACR)為標的，並將之固定在自製環氧基修飾之微米玻璃珠載體。在此利用單微珠SELEX技術進行適體篩選，且搭配即時定量聚合酶鏈鎖反應(real-time qPCR)進行篩選程序的監控。利用所建立的SELEX程序，有效地僅耗費45 ng之AMACR篩選出KD值達49 nM的之適體序列。此外本研究並同時以該適體序列開發出針對AMACR的螢光檢測平台，成功的於10−1 至 103 nM的動態區間內進行檢測，且其偵測極限可達0.44 nM之AMACR濃度。
第二部分則進行DNA序列庫多樣性對適體篩選影響之探討，並進一步探討多效價適體設計對適體與大分子標的物交互作用之影響。首先第一部分，採用三種不同DNA序列庫(A,B和C)以提升篩選序列的多樣性，且以上述建立之平台，進行適體的篩選。在此假設序列庫中的DNA引子鍵結區段會影響序列折疊構型的差異，且進一步影響篩選之結果。結果發現，引子確實在SELEX程序中扮演重要的角色。值得注意的是，利用A序列庫可成功地在3個篩選迴圈中，得到KD值達15 nM與22 nM之適體序列。此外，更進一步針對適體進行截短設計，並評估來自不同序列庫的適體在AMACR表面鍵結區位的差異。結果發現，由C序列庫篩選出來之序列，與AMACR抗體具有競爭作用。此結果暗示著DNA序列庫的組成可能會間接影響適體的演化進程，並且影響適體在標的物上的鍵結區位。第二部分，進一步利用截短序列進行二聚體設計，並探討此設計對適體親和性與專一性的影響。首先針對截短序列進行親和性評估，發現截短序列相對於原序列並無法表現更好的親和性。然而，經由二聚體設計，其親和性相對於原截短序列，則可提升2至4倍左右。有趣的是，藉由不同適體片段所形成之二聚體序列不只可以表現出良好的親和性，並且表現出比未截短之適體更好之專一性。
第三部分主要針對多效價適體與小分子標的物間交互作用之探討。氯化血紅素(hemin)與其適體會形成對過氧化氫具有催化能力的適酶單體。利用此特性，串連單體序列成多聚體構型，並針對其結構、催化效率與熱力學性質進行評估。結果發現，多效價適酶動力學表現會受多種因素組合影響(例如：親和性、結構穩定度與序列串連次數)，且hemin與多效價適體間的親和性主導了適酶催化的表現。經由測試，發現到三聚體構型最能有效提升適酶的催化表現，並且利用其搭配葡萄糖氧化酶在ABTS顯色系統中，成功的於40 至630 μM的線性區間內進行檢測，且其偵測極限可達10 μM之葡萄糖濃度。
In the definition, aptamers are oligonucleotides or peptide molecules that can bind to their targets with high specificity and affinity. Compared with antibodies, aptamers are easy to generate, chemically stable, capable of being chemically synthesized, and so on. Hence, aptamers are considered as an effective alternative to antibodies in medical and diagnostics applications. Unlike the preparation of antibodies, which relies on induction of an animal immune system, aptamers are screened by mimicking the natural selection process in laboratory, and the selection technique is called systematic evolution of ligands by exponential enrichment (SELEX).
Unlike immune response, which can generate antibodies with two or more binding-sites for epitopes insertion, the conventional SELEX has a strong tendency to yield an aptamer with a single binding site. In previous study, the affinity of IgM antibodies per combining site has been reported to be low. However, the multivalent binding property of IgM might lead a high binding energy between an antibody and antigen, so IgM can be effective in immune response. Besides, it was also reported that if an antigen is recognized by two distinct antibodies, the specificity of the antigen detection can be improved. In recently, the concept of multivalent is wildly discussed in aptamer researches. Based on this reason, the author aims at discussing the effect of the “multivalent design” on the performance of aptamers against macro- and small molecular targets. To present clear picture of this dissertation, the main contents are summarized as follows.
In the 1st part, the author tries to establish an aptamer discovery platform with low protein consumption. Although protein-SELEX has been discussed for several years, an ordinary SELEX protocol might consume milligram amounts of protein to accomplish all SELEX rounds, and it could cause the cost increase for aptamer screening. Hence, it becomes an important issue to establish an efficient platform for SELEX. In this work, the author uses alpha-methylacyl-CoA racemase (AMACR), a prostate cancer biomarker, as a target, and fixes the target on homemade epoxide-functionalized glass microbeads. The AMACR aptamers are in vitro selected using a single-microbead SELEX approach, which uses real-time quantitative PCR (qPCR) to monitor the evolution of aptamers during the SELEX rounds. The author successfully picks up an aptamer features an apparent KD value of 49 nM by just consuming ca. 45 ng AMACR. Moreover, an aptamer-based fluorescent AMACR assay is also demonstrated. The assay features a wide dynamic range (from 10-1 to 103 nM of AMACR), and a low detection limit of 0.44 nM.
In the 2nd part, the author tries to explore the effect of diversity of the DNA library on aptamer screening, and further discuss the effect of the “multivalent design” on the performance of macromolecular targets (AMACR as the model target). In the 1st issue, the author uses single-bead SELEX for aptamer selection, and utilizes three different DNA libraries (A, B and C) to increase the diversity of the initial pool. Here, the author assumes different primer-binding regions of the DNA library fragments could increase the diversity of DNA conformation, and further affect the SELEX result. From the result, it is found the composition of the DNA library indeed plays an important role for aptamer discovery. It's worth noting that aptamers with KD values of 15 and 22 nM can be picked up from Library A by just 3 rounds of SELEX. Moreover, the author also tries to minimize the aptamers and identifies the aptamer-binding locations on AMACR. Interestingly, it is observed that aptamers from Library C could bind near the AMACR antibody-binding epitope on AMACR. It implies the primer regions of DNA library fragments could cause aptamers binding to different locations on AMACR. In 2nd issue, the author uses the truncated aptamers to compose bivalent aptamers, and compares the affinity and specificity of these aptamers to the full length aptamers. From the result, it is found the truncated aptamers cannot present better affinity than their full length aptamers. However, the affinity of truncated aptamers can be increased to 2-4 folds after dimerization. Interestingly, it is found that linking different truncated aptamers, which could bind to different locations on AMACR, not only can enhance the AMACR binding affinity but also present higher specificity than the full length aptamers.
In the 3rd part, the author discusses the effect of the “multivalent design” on the performance of small molecular targets (hemin as the model target). The hemin-aptamer complex, called aptazyme, can perform H2O2 catalytic activity. In this work, the author chooses a well-proven hemin-aptamer,CatG4, as the model and investigates the circular dichroism (CD) characteristics, H2O2 catalysis kinetics and thermodynamic properties for the dimers, trimers, and tetramers of CatG4, with or without oligo-dT adapting linkers. With many aspects of assessments, the author observes that the aptazyme kinetic properties are affected by multifactor (affinity, structural stability, and tandem repeat times) and the binding stability between the hemin and G-quadruplex dominates the improvement of aptazyme catalytic performance in tandem repeat design. Then, a sensitive colorimetric glucose assay is demonstrated with the collaboration of the trivalent aptazyme, glucose oxidase, and ABTS reporter. The linear detection range is between 2 to 31.3 nmoles for a 50 μL sample (40 to 630 μM), and the LOD is 0.5 nmoles for a 50 μL sample (10 μM).
Based on above studies, the author hopes and believes that the results can be applied as the guidelines for designing or developing multivalent aptamers for medical and diagnostics applications.
|Appears in Collections:||生物機電工程學系|
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