利用RNA干擾進行基因功能分析的高效率篩選策略

An efficient screening strategy for gene functional analysis using RNA interference

張智宏(Chih-Hung Chang)

指導教授 ： 唐傳義

國立清華大學/電機資訊學院/資訊工程學系/博士(2013年)

https://doi.org/10.6843/NTHU.2013.00142

摘要

RNA干擾(RNAi)常被應用在基因體規模的基因功能篩選上。然而，針對每一個基因做一對一的RNAi分析非常耗費經費與成本。先前的研究已經指出siRNAs也可以影響與其近似互補的RNAs序列，並且這一現象被稱為脫靶效應(off-target effect)。此一效應也說明了，我們可以利用一細心設計的siRNA同時抑制多個基因的表現。
在此論文中，我們提出了一個啟發式演算法(heuristic algorithm)來設計可同時抑制多個基因的siRNAs。並且結合了群試(group testing)的概念，我們也提出了一個策略，以減少在大規模RNAi分析實驗中所需的RNAi實驗次數。為了驗證此一策略的效能，我們以蘭花的表現序列標籤(expressed sequence tag, EST)資料做為測試資料，模擬如何篩選與植物抗病反應相關的轉錄因子(transcription factor)。根據我們的計算，只需94個siRNAs，即足夠對這229個預測出來的轉錄因子進行初步篩選。在這94個siRNAs中，有一siRNAs在計算上可以同時抑制編號15與編號21的轉錄因子(TF15, TF21)，而TF15已被證實與植物抗病反應有關，因此，我們利用此一siRNA進行RNAi實驗，以驗證我們所提出的方法之可行性。而實驗結果，不僅確認了此siRNA可以同時抑制TF15與TF21，以及我們的策略可以成功地證實TF15與植物抗病反應有關，同時，我們也發現TF21也與植物抗病反應有關。
我們的計算結果顯示出，比起利用一對一RNAi的篩選，利用較少的實驗也能對全部的基因進行篩選。並且，我們也證實了，我們所提出的策略可以成功地協助找出與某一性徵相關的基因。

關鍵字

RNA干擾 ； RNA篩選 ； siRNA設計 ； 基因功能分析 ； 群試

並列摘要

RNA interference (RNAi) has been widely applied in genome-scale gene functional screens. Traditional one-on-one RNAi analysis works well but is cost-ineffective and laborious. Previous studies have indicated that siRNAs actually can simultaneously affect RNAs that are near-perfectly complementary to the induced siRNAs, and this phenomenon has been termed as off-target effect. Off-target effect implies that it is possible to silence several genes simultaneously with a carefully designed siRNA.
We first proposed a heuristic algorithm to design suitable siRNAs that can target multiple genes, and then adopted group testing method to present a gene functional analysis strategy. Combining these two work can help to reduce the number of required RNAi experiments in a large-scale RNAi analysis. To verify the efficacy of our strategy, we used the Orchid expressed sequence tag data as a case study to screen the putative transcription factors that are involved in plant disease responses. According to our computation, 94 qualified siRNAs were sufficient to examine all of the predicated 229 transcription factors. In addition, among the 94 computer-designed siRNAs, an siRNA that targets both TF15 (a previously identified transcription factor that is involved in the plant disease-response pathway) and TF21 was introduced into orchids. The experimental results showed that this siRNA can simultaneously silence TF15 and TF21, and application of our strategy successfully confirmed that TF15 is involved in plant defense responses. Interestingly, our second-round analysis, which used an siRNA specific to TF21, indicated that TF21 is a previously unidentified transcription factor that is related to plant defense responses.
Our computational results showed that it is possible to screen all genes with fewer experiments than would be required for the traditional one-on-one RNAi screening. We also verified that our strategy is capable of identifying genes that are involved in a specific phenotype.

並列關鍵字

RNA interference ； RNAi screening ； SiRNA design ； Gene functional analysis ； Group testing

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