1. 利用光鉗研究核酸結構生成的動力學及熱力學

2. 利用光鉗研究核醣體轉譯及調控的機制

近年來，單分子 (single-molecule) 技術逐漸被用來研究生物巨分子 ( 包括 DNA, RNA, proteins) 的結構變化及其相關的動力學與熱力學。另一個普遍的應用則是在觀察並測量酵素分子在其介質或模板 (templates) 上面的移動情形，或是分子間的交互作用。簡單而言，單分子技術就是利用雷射光鉗 (optical tweezers) 或螢光等等的方法，同時且獨立地觀測一個至數百個分子。

為什麼我們要獨立地觀測個別的分子呢？一般的化學或酵素反應都俱有隨機性 (stochastic) ，也就是說，對同一個分子 ( 或不同的分子間 ) ，兩個連續反應的時間間隔通常是不一樣的，所造成的結果也可能不同，而這些差異可能與其功能的調控有關。舉例來說， RNA polymerase 在反應過程中偶爾會有長短不一的停滯 (pausing) 現象，並沿著其 DNA 模板倒退 (backtracking) 一小段距離。這個現象極可能是因為 polymerase 把錯的鹼基加到新合成的 RNA 上後，嘗試去修改這個錯誤所造成的，這正顯示出 RNA polymerase 所擁有的校正 (proof-reading) 的功能。若是用傳統的實驗方法，這些偶發現象的訊號便會被掩蓋掉而難以偵測。

我的主要研究興趣便是利用雷射光鉗的單分子技術來探討核醣體 (ribosomes) 的轉譯機制。核醣體存在於各類原核及真核細胞中，是細胞內最大的酵素之一，其功能為讀取 mRNA 的訊息以合成蛋白質，為分子生物學中心法則 (Central Dogma) 的核心成員。從發現至今，對核醣體的研究已經超過 50 年，但由於其巨大的結構及複雜的反應，我們對這整個過程的了解仍是相當有限。最近由於技術上的突破，我們已經能夠利用雷射光鉗即時地 (real-time) 追蹤單一個核醣體在其 mRNA 模板上移動的情形，並測量在每一步 (one codon) 所停留的時間。這一技術對研究轉譯動力學及調控非常有幫助。例如，許多病毒的 mRNA 在特定的位置存在特定的序列及結構，這些安排是為了能夠在轉譯過程中調控病毒蛋白的表現而設計的。當碰到這些調控點時核醣體所作的反應，便是我們要觀測的重點。值得一提的是，許多類似的調控只適用於病毒蛋白，宿主細胞本身的蛋白表現並不採用，所以這方面的研究或許有助於我們思考如何抑制病毒的繁衍。

Reference papers:

1. Wen, J.-D., Kuo, S.T. & Chou, H.-H.D. The diversity of Shine-Dalgarno sequences sheds light on the evolution of translation initiation. RNA Biol., Published online: 21 Dec 2020 (DOI: 10.1080/15476286.2020.1861406).

2. Kuo, S.-T., Jahn, R.-L., Cheng, Y.-J., Chen, Y.-L., Lee, Y.-J., Hollfelder, F., Wen, J.-D., and Chou, H.-H.D. Global fitness landscapes of the Shine-Dalgarno sequence. Genome Res. 30, 711-723 (2020).

3. Chang, K.-C., Salawu, E.O., Chang, Y.-Y., Wen, J.-D., & Yang, L.-W. Resolution-exchanged structural modeling and simulations jointly unravel that subunit rolling underlies the mechanism of programmed ribosomal frameshifting. Bioinformatics 35, 945-952 (2019).
4. Chen, Y.-T., Chang, K.-C., Hu, H.-T., Chen, Y.-L., Lin, Y.-H., Hsu, C.-F., Chang, C.-F., Chang, K.-Y., and Wen, J.-D. (2017). Coordination among tertiary base pairs results in an efficient frameshift-stimulating RNA pseudoknot. Nucleic Acids Res. 45, 6011-6022 (2017).
https://doi.org/10.1093/nar/gkx134
5. Chang, K.-C., Wen, J.-D., Yang, L.-W. Functional Importance of Mobile Ribosomal Proteins. BioMed Research International volume 2015, ID: 539238 (2015)
6. Yan, S., Wen, J.-D., Bustamante, C. & Tinoco, I., Jr. Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways. Cell 160, 870-881 (2015)
7. Liu, T., Kaplan, A., Alexander, L., Yan, S., Wen, J.-D., Lancaster, L., Wickersham, C. E., Fredrik, K., Noller, H., Tinoco, I., Jr. & Bustamante, C. Direct measurement of the mechanical work during translocation by the ribosome. eLife 3, e03406 (2014)
8. Wu, Y.-J., Wu, C.-H., Yeh, A. Y.-C. & Wen, J.-D. Folding a stable RNA pseudoknot through rearrangement of two hairpin structures. Nucleic Acids Res. 42, 4505-4515 (2014)
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24. Shin, S.-J., Lai, F.-J., Wen, J.-D., Lin, S.-R., Hsieh, M.-C., Hsiao, P.-J. & Tsai, J.-H. Increased nitric oxide synthase mRNA expression in the renal medulla of water-deprived rats. Kidney Int. 56, 2191-2202 (1999)
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