利用叉狀DNA 與去氧核醣核酸酶恆溫訊號放大機制進行流感病毒亞型鑑定

Abstract

有鑑於流感病毒的亞型鑑定對於臨床診斷及病毒傳播監控之重要性，本論文的研究目的在發展一流感病毒亞型鑑定的平台，結合叉狀DNA 的應用與去氧核醣核酸酶(DNAzyme)截切反應，以同時偵測流感病毒兩種重要抗原[血球凝集素Hemagglutinin (HA)與神經胺酸酶 Neuraminidase (NA)]基因片段。 本研究利用簡單邏輯閘(logic gate)的概念，在H5 與N2 基因同時存在下才會產生單一螢光訊號，可快速提供檢驗結論與降低人為誤判的機會。在針對流感病毒的H5 基因做偵測時，首先將兩段DNA 引子加入病毒cDNA 中，使之形成叉狀DNA，再加入酵素在叉狀DNA 上進行鏈置換擴增反應(strand displacement amplification)，進而產生多段去氧核醣核酸酶(DNAzyme)。DNAzyme 可將核酸基質由莖環結構水解為兩段單股核酸，而此產物其中一股帶有螢光染劑；同時，針對病毒的N2 基因亦進行同樣的反應。最後藉由H5 與N2 基因所誘發的反應分別產生之帶有螢光染劑的單股核酸進行雜合反應，最後產生螢光共振能量轉移(FRET)訊號。為了瞭解鏈置換擴增反應中兩種酵素作用的最佳條件，我們探討了兩種酵素間的比例與濃度對DNAzyme 產物生成的影響；為了減少在DNA 聚合酶反應過程中常見的非特異性產物的產生，我們也探討了DNA 引子的修飾及結構對於非特異性擴增現象的影響。實驗結果顯示，病毒cDNA 與引子所形成的叉狀DNA 確實可與酵素進行鏈置換擴增反應，並產出有功能的DNAzyme，且此反應有部分訊號放大的效果；而所產出的DNAzyme 亦可再進行接下來的基質截切反應，在此步驟中，因為DNAzyme 可循環利用故也有訊號放大的功能；最後由H5 與N2 基因誘發的反應所分別產生帶有螢光染劑的基質截切後產物也能順利的進行雜合，並得到FRET 訊號。本系統有良好的選擇性，在H5 與N2 基因序列同時存在下的FRET訊號能夠明顯的與其他三種陰性組合(無H5 與N2 基因、只有H5 基因、只有N2 基因)區別。在靈敏度方面，本系統對於H5 與N2 基因同時存在時的偵測極限約為3 nM。本設計的優點在於：(1) 整體反應可在恆溫環境下進行而不需昂貴的溫控裝置；(2) 整體反應時間只需一小時，大幅提升了目標偵測的便利性；(3) 相較於一般蛋白質酵素，DNAzyme 的活性穩定且易於保存；(4) 在鏈置換擴增反應及訊號輸出部分的序列設計彈性較大，不需受限於偵測目標的序列。鑒於此項篩檢技術的高應用性與功能性，可望有效應用於多種的流感病毒亞型鑑定。

Subtyping of influenza virus is essential for its treatment, diagnosis and surveillance. We herein report a sensor design that integrates DNA three-way junction and DNAzyme catalytic activities to sensitively and specifically identify dual influenza viral nucleic acid sequences, hemagglutinin (HA) and neuraminidase (NA) genes, simultaneously. Employing a simple concept of logic gate, output signal can only be obtained when both H5 and N2 gene exist. This design is able to derive a rapid result exclusive of potential personal error made in data interpretation. To detect viral H5 gene, we first mixed two synthetic DNA primers with viral cDNA to form DNA three-way junction. Subsequently, two enzymes, DNA polymerase and Nicking enzyme were added to initiate strand displacement amplification reaction, leading to the production of deoxyribozyme (DNAzyme). As a consequence, the functional DNAzyme could hydrolyze stem-loop substrates, resulting in two separated single-stranded DNA, one of which was modified with a fluorophore. Parallelly another set of this isothermal amplification could also be induced by N2 gene. Consequently the Cy3- and Cy5-labled single-stranded DNA, generated from the reactions initiated by H5 and N2 gene, respectively, hybridized to form duplexes. Due to the proximity of the two dyes, fluorescence signal could be detected as a result of Förster resonance energy transfer (FRET). To verify the optimal condition for two enzymes which participated in strand displacement amplification, we studied the influence of the concentrations and ratio between DNA polymerase and Nicking enzyme on production of DNAzyme. To reduce the generation of non-specific products, which often appear in DNA polymerase-related reaction, we also investigated the effects of phosphate group-modified 3-way junction template and primer conformation (linear vs. hairpin) on non-specific signal amplification. The results revealed that the DNA three-way junction composed of viral cDNA and synthesized DNA primers can be successfully extended by DNA polymerase and specifically nicked by nicking enzyme to produce functional DNAzyme. In addition, the as-generated DNAzyme products could effectively catalyze the hydrolysis of nucleic acids substrates. By taking advantage of the enzymatic property of DNAzyme, an excess amount of desired products could be acquired, which could further amplify the signal. Finally the two fluorophore-labeled products of DNAzyme catalyzed reactions, each generated from the reactions initiated by H5 and N2 gene, can hybridize successfully and produce FRET signal. This system has remarkable selectivity, the FRET signal acquired in the simultaneous presence of both target segments is differentiated from the other three negative combinations (i.e., without H5 and N2 gene sequences, only with either H5 or N2 sequences). The limit of detection (LOD) of this system was calculated as 3 nM when both H5 and N2 segments exist. Our newly designed sensor features (i) the reaction could proceed isothermally, without need of expensive temperature-controlling equipment, (ii) the entire time needed for our detection platform is 1 hour, which dramatically enhance the convenience of target detection, (iii) the chemical and thermal stability of DNAzyme is superior than conventional protein-based enzymes, and (iv) the whole design for signal amplification and outputting are universal, that can be readily extended to other potential target sequences. On account of the elegant signal amplification scheme and broad bio-applicability, this screening platform technology is expected to be a promising subtyping tool for variable influenza viruses.