利用可重複使用的矽奈米線場效電晶體偵測及鑑定雙股小片段核醣核酸

Abstract

矽奈米線場效電晶體 (SiNW-FET) 現今已廣泛用做於生物感測器，應用在各種偵測上，像是去氧核醣核酸雜交 (DNA hybridization)，蛋白質，病毒，或是小分子。由於SiNW-FET有高靈敏度，選擇性，免標記與及時偵測的優點，我們可以利用可重複使用的SiNW-FET去偵測小片段的核醣核酸。 後轉錄時期基因沉默 (post-transcriptional gene silencing；PTGS) 為植物抵抗外來病毒感染的一個策略，但是病毒會產生抑制蛋白，以防止這種攻擊病毒的-siRNA) 有很好的結合力。在本篇論文中，我們運用奈米科技而發展出修飾有P19的矽奈米線場效電晶體 (P19/SiNW-FET) 可以偵測基因沉默機制，番茄叢矮病毒 (Tombusvirus) 含有的抑制蛋白- P19 (分子量19 kDa)，具有長度專一性的鑑別率，並與21 nt雙股小干擾核醣核酸 (double-stranded short-interfering RNA；dsds-siRNA。P19/SiNW-FET具有極高的靈敏度，不只可以偵測到500 pM的21 nt ds-siRNA，還可以分辨不同二級結構的ds-siRNA，像是不同長度或是未完全配對的ds-siRNA。再者， P19/SiNW-FET抓到的未知雙股核醣核酸 (dsRNA) ，在偵測實驗完後可以被沖提出來並回收，以進行下一步的鑑定，像是即時聚合酶連鎖反應 (real-time polymerase chain reaction；real time PCR)。從這些測量結果可以了解到SiNW-FET提供很好的平台去篩選多種蛋白質-核醣核酸之間的交互作用；另一個特點則是我們可以將這些小片段核醣核酸從P19/SiNW-FET給沖提出來，並且做進一步的定量分析而能了解其資訊。

Silicon nanowire field-effect transistors (SiNW-FETs) have been extensively used as biosensors in a variety of detections, ranging from DNA hybridization, proteins, virus, and small molecules. Taking advantages of the ultrahigh sensitivity, selectivity, label-free and real-time detection capabilities of a SiNW-FET, we have applied reusable SiNW-FETs for the study of protein-small RNA interactions. Post-transcriptional gene silencing (PTGS) is an antiviral strategy in plants to avoid the virus infection. However, the virus can produce a viral suppressor protein to suppress the PTGS in order to prevent the silencing machinery from attacking virus. P19 of Tombusvirus (with 19 kDa molecular weight) has been identified as a viral suppressor that can specifically bind 21 nucleotide (nt) double-strand short-interfering RNA (ds-siRNA) with high affinity and size selection. In this study, we reported the application of the nanotechnology to develop a highly sensitive SiNW-FET biosensor immobilized with P19 protein (referred to as P19/SiNW-FET) for the detection of interacting siRNAs. This P19/SiNW-FET biosensor is capable of not only detecting 21 nt ds-siRNAs to a 500 pM level, but also distinguishing the various secondary structures of ds-siRNAs, such as their size, and the mismatch between P19 and ds-siRNAs. Moreover, unknown ds-siRNAs captured by P19/SiNW-FET in the sensing measurement can be recovered after detection for the further identification with the polymerase chain reaction (PCR) technique. From these sensing measurements, we demonstrate that SiNW-FET biosensors provide an excellent platform for high-throughput screening protein-RNA interactions. In particular, the interacting siRNAs recorvered from a specific protein-modified SiNW-FET can be identified to reveal their profile.