脊髓小腦萎縮症31型相關基因連續TGGAA序列之結構和動力學研究
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2016
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脊髓小腦萎縮症31型 (Spinocerebellar ataxia 31, SCA31) 是一種神經遺傳性疾病，主要流行於日本。患者主要發病在晚年時期，會有許多神經功能障礙相關的症狀。先前的研究指出造成SCA31疾病產生的致病基因為染色體16號中的一段插入的重複(TGGAA)n序列。在先前的NMR研究中，發現(TGGAA)n會形成非沃森-克里克鹼基對 (Watson-Crick base pairs) 的雙股與單股髮夾似的結構，這種特殊的結構可能導致重複序列在DNA代謝過程中產生擴張，進而產生RNA聚集 (RNA foci)，最終導致細胞凋亡。本實驗使用單分子螢光共振能量轉移 (Single-molecule fluorescence resonance energy transfer, smFRET) 的技術，研究重複次數為2~6的(TGGAA)n重複序列的結構和動力學。結果顯示重複次數3以上的(TGGAA)n會形成單股髮夾似結構，重複次數為奇數的(TGGAA)n的結構為頭尾重複序列對齊的髮夾似結構，偶數則為奇數組的結構外加一段重複序列裸露在外。相同重複數目n的單股髮夾似結構的(TGGAA)n與互補序列(TTCCA)n的動力學實驗結果顯示，雜合 (hybridization) 反應速率顯示出超過一個數量級的差異。奇數與偶數的(TGGAA)n的雜合反應途徑因其結構而可能不同。
Spinocerebellar ataxia 31 (SCA31) is a neurodegenerative hereditary disease, mainly popular in Japan. The onset of the disease for SCA31 patients is predominantly in the old ages, accompanied with many symptoms of neurological dysfunction. Previous studies indicated that the pathogenic gene of SCA31 disease is the inserted by a (TGGAA)n tandem repeat on the chromosome 16 and the NMR studies has found that these tandem repeats form to non-Watson-Crick base pairing structures, a duplex by two identical sequences in an antiparallel configuration and a single-stranded hairpin-like structure. These special structures may lead to error-prone expansion and ultimately cause malfunction of the cells. Here, we present a single-molecule fluorescence resonance energy transfer (FRET) study on the (TGGAA)n, n = 2–6, sequences. The results show that the DNA molecules with more than two tandem repeats will fold into hairpin-like structures. Molecules with odd repeats is in an end-to-end aligned configuration while with the ones with even repeats are in a hairpin-like structure with one excess TGGAA overhang at one end. Our kinetic study on the hybridization of complimentary sequences with same length also shows a more than one order of magnitude difference in rate between the molecules with odd and even repeats. The significant difference suggests different hybridization pathways between odd and even repeats due to their difference in structure.
Spinocerebellar ataxia 31 (SCA31) is a neurodegenerative hereditary disease, mainly popular in Japan. The onset of the disease for SCA31 patients is predominantly in the old ages, accompanied with many symptoms of neurological dysfunction. Previous studies indicated that the pathogenic gene of SCA31 disease is the inserted by a (TGGAA)n tandem repeat on the chromosome 16 and the NMR studies has found that these tandem repeats form to non-Watson-Crick base pairing structures, a duplex by two identical sequences in an antiparallel configuration and a single-stranded hairpin-like structure. These special structures may lead to error-prone expansion and ultimately cause malfunction of the cells. Here, we present a single-molecule fluorescence resonance energy transfer (FRET) study on the (TGGAA)n, n = 2–6, sequences. The results show that the DNA molecules with more than two tandem repeats will fold into hairpin-like structures. Molecules with odd repeats is in an end-to-end aligned configuration while with the ones with even repeats are in a hairpin-like structure with one excess TGGAA overhang at one end. Our kinetic study on the hybridization of complimentary sequences with same length also shows a more than one order of magnitude difference in rate between the molecules with odd and even repeats. The significant difference suggests different hybridization pathways between odd and even repeats due to their difference in structure.
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脊髓小腦萎縮症31型, TGGAA序列, 重複序列, 雜合, 動力學, 互補股, 結構定性, 穩定性, Spinocerebellar Ataxia Type 31, TGGAA sequences, Tandem repeats, Hybridization, Kinetics, Complementary strand, Structural characterization, Stability