高親和性標籤在單分子螢光標記和生物巨分子固定上之應用

Abstract

單分子螢光共振能量轉移 (Single Molecule Fluorescence Resonance Energy Transfer, smFRET) 顯微術中，需將生物巨分子均勻分散且穩定地固定於樣本槽的玻璃表面上，便於長時間量測 FRET pair 的螢光強度。將 RNA polymerase II(Pol II) 固定於鋪有攜鈣素 (Calmodulin) 的玻璃界面時，注入至樣品槽的 Pol II 的濃度範圍通常為 10-12 M 。但 Pol II 次單元上的 Calmodulin binding peptide (CBP tag) 與攜鈣素的解離平衡常數 (equilibrium dissociation constant) KD 值約為10-9 M，造成 CBP tagged Pol II 與攜鈣素之偶合效率較低，成像範圍內的 FRET pair 較少。 為了提升 CBP tagged Pol II 與攜鈣素之偶合效率，透過定點突變修改 CBP tag 的 DNA 序列，分別獲得胺基酸序列 N9A 和 S14A 突變的 CBP tag (NCBP tag)，期許能得到對 Calmodulin 具較高親和力(解離平衡常數約10-12 M) 的 NCBP tag。 藉由生物分子交互作用分析系統，生物薄膜干涉儀 (Biolayer Interferometry) 與微量熱泳動儀 (Microscale Thermophoresis) 兩種方式，驗證 NCBP tagged Pol II 對攜鈣素的親和力改善之情形，然而因為 Pol II 對 NeutrAvidin 的非專一性吸附及Pol II 複合體本身的複雜性，兩種方法均無法有效的獲得正確的 KD 值範圍。除了上述兩方法，亦使用單分子螢光顯微鏡分別對 Pol II 與攜鈣素成像，藉由單分子影像位置之映對 (mapping) ，做 colocalization 分析以測定 Pol II 與攜鈣素的結合率。 CBP tagged Pol II 對 Calmodulin 的結合率最佳會落在 1 nM 的濃度範圍，結合率可達 41% ，而兩種 NCBP tagged Pol II 與攜鈣素的結合率分別為 25% (N9A) 和 12% (S14A) 。 本文找到合適的CBP tagged Pol II濃度範圍，能提供穩定且有效率的單分子成像條件進行後續的量測。Biocytin 與 Biotin-BSA的添加可有效地減緩 RNA polymerase II 對 NeutrAvidin 的非專一性吸附，得以在相對單純的環境中分析 CBP tagged Pol II 與攜鈣素間的生物分子交互作用。

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to study complex biological interactions and dynamics with sub-nanometer sensitivity. In smFRET technique, biomolecule must be immobilized sparsely and stably on a substrate for measuring the intensity of time trajectories of donors and acceptors. In this study, RNA polymerase II (Pol II) on which a calmodulin binding peptide tag (CBP tag) fused was immobilized on a glass surface with the aid of biotinylated-calmodulin and NeutrAvidin system. In the chamber, CBP tagged Pol II concentration range is usually 10-12 pM , but the dissociation constant KD between CBP tag and calmodulin-Ca2+ complex is about 10-9 M. Because the concentration of CBP tagged Pol II is smaller than KD value, the number of FRET pair is low and the binding between donors and acceptors becomes unstable. To improve the affinity between CBP tagged Pol II and calmodulin, we used site-directed mutagenesis to modify CBP tag. The alanine substitution within CBP tag resulted Asn9Ala and Ser14Ala (N9A/S14A) mutants which may improve affinity from nano- to pico-molar range. Both modified CBP tags are called as NCBP tags. The improved affinity can stabilize the binding between donors and acceptors and then the measurements of FRET efficiencies is more efficient. We used Biolayer Interferometry and Microscale Thermophoresis to quantify the affinity between NCBP tagged Pol II and calmodulin. However, the results showed unreliable KD range due to the nonspecific binding between Pol II and NeutrAvidin and maybe the complexity of Pol II complex. Besides, we setup a total internal reflection fluorescence microscope to directly measure the binding ratio between CPB tagged Pol II and calmodulin by colocalized two channel images of different color. The highest binding ratio of CBP tagged Pol II is 41% as Pol II is at 1 nM ; however, at the same Pol II concentration, the binding ratio of two kinds of NCBP tagged Pol II, N9A and S14A, are 25% and 12%, respectively. In summary, we tried to improve the affinity between CBP tagged Pol II and calmodulin with the substitution of NCBP tag to get more stable FRET pair and optimize the efficiency of FRET measurements. Although the affinity improvement of NCBP tag is not obvious, the experimental results suggest an optimized Pol II concentration to obtain the best FRET efficiency. Furthermore, buffer additives like Biocytin and Biotin-BSA can effectively reduce the nonspecific binding between Pol II and NeutrAvidin.