透過「指數富集式配體系統進化技術」篩選與Integrin α2 I domain高度專一性之適體

Abstract

分子影像為精準醫療中一個重要的方法與工具，透過會與生物標記有專一性結合的標定探針，人們可以更準確地也更早地檢測、定位與診斷疾病。Integrin α2β1是最主要的collagen結合受體，先前的研究顯示攝護腺癌與大腸癌細胞過量表現integrin α2β1時，癌細胞更容易轉移至骨髓或肝臟這些富含collagen的環境中，因此本研究的目的是為了找出與重組integrin α2 I domain有專一結合性的適體 (aptamer)，並且將它應用在分子影像上。 SELEX (Systematic evolution of ligands by exponential enrichment) 是一個生產高親和性適體的技術，適體library會與目標分子作用以進行篩選，經過多個輪次的篩選後，存留下的適體可以與目標分子專一結合。本篇論文在SELEX中利用兩種不同的策略生產單股DNA: 非對稱PCR (asymmetric PCR) 與streptavidin beads配合鹼處理的方法。然而非對稱PCR的策略耗時又難以掌控，所以我們最終並沒有以這種方法完成篩選。我們以saturation binding assay 測試YCP-11與YCP-12這兩種候選適體的專一性結合能力，結果發現這兩種適體都不會與我們的目標蛋白質結合，更進一步，我們對不同篩選輪次的適體池 (aptamer pool) 進行定序，發現了library多樣性的流失可能是導致SELEX失敗的主因，我們認為在篩選的過程中適體的反應濃度太低或洗的條件太過嚴苛都可能造成library多樣性的流失，因此建議將篩選時的適體濃度提升至500 nM或是調整洗的條件為每次洗的體積為200 μl且一次30秒將可以改進目前的SELEX方法。

Molecular imaging is a powerful method and technique in precision medicine. With a labeled probe which is specific to the corresponding biomarker, people could easily detect, locate and diagnose disease in a more accurate way. Integrin α2β1 was identified as a major collagen binding receptor. Previous research indicated that prostate and colon cancers which overexpressed integrin α2β1 are more possible to metastasize to bone marrow and liver because of abundant collagen in the microenvironment. Therefore, the purpose of this research is to identify the DNA aptamer which could specifically bind to Integrin α2β1 and used as a probe in molecular imaging. SELEX (Systematic evolution of ligands by exponential enrichment) is the technique for producing high affinity aptamer. The aptamer library would incubate with target molecules for selection. After several rounds of selection, the remaining aptamer could specifically bind to target molecule. In this thesis, two strategies for single-stranded DNA generation were used in SELEX: asymmetric PCR and streptavidin beads with alkaline treatment. However, the strategy of asymmetric PCR was time-consuming and hard to control as a result we did not finish our selection with this method. The binding affinity of two aptamer candidates, YCP-11 and YCP-12, were tested by saturation binding assay but the result showed that these aptamers were not specific binding to our target protein. Further, from the sequencing result of different round aptamer pool, we found that the losing of library diversity might be the main reason that led to the failure of SELEX. We thought that two possible reasons might cause the losing of library diversity: the concentration of aptamer was low in the selection step or the washing condition was too harsh. We suggested that increasing the concentration of aptamer to 500 nM and adjusting washing condition to 200 μl in volume with 30 seconds every time might improve the present method of SELEX.