黏蛋白-1適體篩選與分析

Abstract

黏蛋白-1 (mucin-1，簡稱MUC1)是一種在多數腺癌細胞表面上過表現的穿膜醣蛋白，可作為一癌症指標，不論在診斷或治療上皆具有重要意義。由於目前仍未有以MUC1部分開放閱讀框 (ORF)之序列 (315 a.a. - 420 a.a.) 為目標的適體。根據其未來之應用性，本研究欲篩選一可適用於生物感測的適體，在修飾之下仍可與MUC1結合。因此，本研究將以此序列片段作為目標，進行MUC1之適體篩選。篩選利用的方法為系統性配位子指數增益演繹技術，以單顆玻璃珠作為反應基材，人類血清白蛋白作為反向篩選的目標，藉由多回合的擇選與擴增，得到與MUC1具親和性與選擇性的特定單股 DNA群；並以MEME分析軟體將其定序結果進行分析與挑選，最後利用表面電漿共振 (surface plasmon resonance，簡稱SPR) 之親和力測試，找出與MUC1親和力最高的序列，也就是DNA適體。篩選以演化樹來表示其序列演化的過程，其中包含K1、K2、K3三個分支。篩選過程中發現非專一性序列於序列群中的數量，可以作為演化過程中優勢序列的預測指標。根據SPR中每一個序列與MUC1反應的訊號及其組成，發現GC/AT比與序列之親和性具有中度相關 (r = 0.499)。本研究成功篩選出一個MUC1適體，研究中將其命名為K1R4.2，此適體不會因為加上修飾物而改變其結合能力，於含有Fe(CN)63-/4之條件下仍可與MUC1結合。此外，經由SPR動態分析得到適體K1R4.2與MUC1結合之kon¬ (1.33×10-4¬¬ sec-1nM-1)、koff¬ (7.05×10-3¬ sec-1)¬，並計算求得其解離常數 (KD¬) 為53.0 nM；且於穩態時根據不同濃度的適體K1R4.2結合於MUC1上的量，可計算得其KD值為55.1 nM。此適體與MUC1具有高親和力，且序列中GC含量高 (65.6%)，表示其DNA密度及對熱與鹼的穩定性較高，根據這些特性優勢，未來可將其應用於適體感測器的開發，甚至是醫學上生物性治療的發展。

Mucin-1 (MUC1) is a transmembrane glycoprotein that is overexpressed on the surface of most adenocarcinoma cells, and it is served as a tumor marker. It is of great significance in both diagnosis and treatment. There is currently no aptamer selected targeting the MUC1 partial ORF (315 a.a. - 420 a.a.). It has MUC1 binding functionality after modification. According to its future application, this study intends to select a suitable aptamer for electrochemical sensing. Therefore, this sequence fragment is used as the target for MUC1 aptamer selection in this study. The selection method is called the systematic evolution of ligands by exponential enrichment (SELEX), with the use of single glass bead as the reaction substrate and human serum albumin as the target of reverse selection. Through multiple rounds of selection and amplification, a specific single-strand DNA pool with selectivity and affinity to MUC1 is obtained. For sequence analysis and selection, an analysis software MEME Suite is used. Finally, to find a sequence with the highest affinity to MUC1, which is the DNA aptamer, the affinity test was conducted by surface plasmon resonance (SPR). The evolution process of the sequences during the selection is shown by an evolution tree, which includes three branches, K1, K2, and K3. The number of non-specific sequences found in the pool during the selection can be used as a predictor of the dominant sequences during the evolution process. According to the SPR binding signal of the sequences to MUC1, and the composition of each sequence, it was found that the GC/AT ratio is moderately related to the affinity of the sequence (r = 0.499). In this study, a MUC1 aptamer was successfully found, which is named K1R4.2. This aptamer will not change its binding ability after modification, and it can also bind to MUC1 in the presence of Fe(CN)63-/4. Besides, the kon¬ (1.33×10-4¬¬ sec-1nM-1) and the koff¬ (7.05×10-3¬ sec-1) of aptamer K1R4.2 binding to MUC1 were obtained through SPR dynamic analysis, and the calculated KD is 53.0 nM. According to the amount of aptamer K1R4.2 bound to MUC1 at steady state, the obtained KD is 55.1 nM. This aptamer has a strong affinity to MUC1, and the GC content in the sequence is high (65.6%). This indicates that its DNA density and stability to heat and alkali are both high. Based on these characteristics and advantages, it can be applied to the development of aptasensors in the future, and even the progress of medical biological treatment.