點擊化學策略在磁珠上引入醣基的研究

Abstract

腫瘤相關醣抗原（tumor-associated carbohydrate antigens, TACAs） 在人類癌細胞表面有過度表現的現象。因此，如何辨識這些 TACAs 已成為生物分析與藥物開發中極需解決的問題。適配體（aptamer），即短鏈 DNA 或 RNA 寡核苷酸，近年來已成為一種常用的生物感測工具。其獨特的核酸序列可藉由 體外篩選技術（in vitro selection） 產生，並透過特殊的三維複雜摺疊結構，對特定標的物（如小型生物分子與胜肽）展現高度的專一性與親和力。 本實驗室欲取得特定適配體序列以辨認以下三種特定腫瘤相關抗原：Tn、sTn 與 Tf。，以利後續應用。為了進行體外篩選，需要一種固相載體來在多輪篩選過程中捕捉序列。磁珠（magnetic beads, MBs）對大多數化學物質具有正交性，且操作便利，因此是一種強而有力的固相載體。在本研究以兩步驟的方式將 sTn 抗原導入磁珠。第一步引入使帶有 BCN官能基的spacer修飾於氨基磁珠（NH2-MBs）上，第二步引入使帶有疊氮尾部的抗原與經過 BCN 活化的磁珠以環張力促進叢集炔-疊氮點擊反應（SPAAC）的方式進行偶聯。 為了驗證磁珠上成功引入的化學修飾，本研究使用茚三酮（ninhydrin）測試得知NH2-MB氨基含量。BCN spacer 的完全轉換則透過茚三酮測試中吸光度下降的反推來驗證，並藉由紅外光譜（IR spectra）的比較來確認 BCN spacer 的成功導入。最後，利用 Shukla’s assay 測得磁珠上唾液酸（sialic acid）的負載量。

Tumor-associated carbohydrate antigens (TACAs) are overexpressed on the surface of human carcinoma cells. Therefore, recognizing these TACAs is a prompt issue for bioanalysis and drug development. Aptamer, a short DNA or RNA oligonucleotide have become a popular biosensor in recent years. The unique sequence of the nucleic acid can be generated by a technique called in vitro selection to exhibit high affinity and selectivity toward the particular target such as small biomolecules and peptides by the unique three-dimensional complex folding. Our laboratory aimed to obtain the specific aptamer sequences that could recognize three tumor-associated antigens: Tn, sTn, and Tf, for subsequent applications. To perform in vitro selection, a solid support is requested to capture the sequences through multiple rounds. The magnetic beads (MBs) can be the powerful solid support in this job for its orthogonality to most of the chemicals and the convenience to operate. In this work, we demonstrated a two-step process of introducing the sTn onto the MBs. The first step involved the introduction of the BCN-functionalized spacer to modify the amino magnetic beads (NH2-MBs). In the second step, the azide-modified antigen would be coupled to the BCN-activated beads through strain-promoted alkyne–azide cycloaddition (SPAAC). To verify the successful chemical modifications introduced onto the MBs, the NH2 loading of the starting MBs was determined by the ninhydrin test. The complete conversion of the BCN spacer on the MBs would be verified by the reverse engineering of the absorbance loss of the ninhydrin test. We had also verified the successful introduction of BCN spacer by the comparison of the IR spectra. Finally, the sialic acid loading of the final target beads would be measured by the Shukla’s assay.