以核酸適合體與金奈米粒子抑制凝血酶與第一型人類免疫缺陷病毒反轉錄酶活性

Abstract

適合體是會形成特殊構形並且與特定分子有高親和力的單股寡核苷酸(oligonucleotide)。本論文主要探討表面修飾核酸適合體的金奈米粒子的製備，以及這些材料於調控酵素活性的應用。首先，將凝血酶核酸適合體以金–硫共價鍵結合到金奈米粒子表面，所製備出的金奈米複合物(TBA–Au NPs)能與凝血酶結合，進而抑制凝血酶與纖維蛋白原的反應，有效地延遲凝血的時間。抑制劑的活性可藉由改變金奈米粒子表面核酸適合體的密度調控，並且不受生物樣品中核酸酶的降解，能穩定存在於血漿中。以修飾有適合體互補序列的金奈米粒子做為解毒劑，能活化原先被抑制的凝血酶，凝血酶的活性可以藉由調控核酸適合體及其互補序列比例來調控。 在金奈米粒子表面修飾上一股互補用的序列(cDNA)，利用寡核苷酸具有鹼基互補而形成雙股螺旋的特性，將凝血酶核酸適合體以非共價鍵的形式修飾於金奈米粒子表面(hTBA15/hTBA29/cDNA–Au NPs)。所設計的核酸適合體部分與這段互補用序列形成雙股核酸，這種利用氫鍵形成鹼基互補的鍵結讓核酸適合體與凝血酶之間有更好的作用力，金奈米粒子表面的核酸適合體彼此間磷酸根負電排斥效應減小，可提高核酸適合體抗凝血的效率。同時施加兩股不同的序列的凝血酶核酸適合體，分別和凝血酶不同的結合位置作用，使得奈米材料與凝血酶的結合能力提高，可在血漿中延長凝血酶凝血時間。 最後，表面修飾核酸適合體的金奈米粒子可以有效抑制第一型人類免疫缺陷病毒反轉錄酶的活性。為了有效的抑制病毒複製，利用兩股核酸適合體(Aptpol、AptRH)–分別與反轉錄酵素的聚合酶(polymerase)及核糖核酸內切酶(RNase H)結合–修飾在金奈米粒子表面來抑制病毒複製。所製備的金奈米複合物在核酸酶存在下仍能抑制反轉錄酵素，並可穩定存在於細胞中。以慢病毒(lentivirus)為載體進行病毒抑制實驗，被感染的細胞會製造出綠色螢光蛋白。隨著接有核酸適合體的金奈米複合物濃度越高，以流式細胞儀(flow cytometer)所偵測到的帶有綠色螢光蛋白的細胞數越少，可有效降低病毒感染率。

Aptamers are single-stranded oligonucleotides which form specific structure when bind to certain targets with high affinities. This thesis focuses on preparation and application of apatmer conjugated gold nanoparticles (Au NPs, 13-nm in diameter) for enzyme activities modulation. First, thrombin-binding-aptamer conjugated gold nanoparticles (TBA–Au NPs) for highly effective control of thrombin activity towards fibrinogen. A 29-base long oligonucleotide (TBA29), has known no enzymatic inhibitory functions for thrombin-mediated coagulation, however, has been demonstrated an ultra-high anticoagulant potency of TBA29–Au NPs via the steric blocking effect, with two orders of magnitude higher than that of free TBA29. Second, The hTBA15/hTBA29/cDNA–Au NPs exhibit high anticoagulant activity as a result of inhibiting the thrombin-mediated cleavage of fibrinogen. Instead of directly conjugating functional aptamers onto Au NP surfaces through Au–S bonding, in this study hTBA15 and hTBA29 were hybridized with a complementary sequences that were themselves covalently bound to the Au NPs. The hTBA15/hTBA29/cDNA–Au NPs exert their high inhibitory effect toward thrombin through a combination of multivalent interactions and steric blocking effects. Finally, aptamer-conjugated gold nanoparticles (Apt–Au NPs) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognized to polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol–Au NPs and AptRH–Au NPs, respectively. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt–Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45–Au NPs show inhibitory efficiency in the retroviral replication cycle with a decreasing infectivity.