Network of functions and structures of ribozymes

核酶的功能和结构网络

• 批准号: 14035224
• 负责人: INOUE Tan
• 金额: $ 41.22万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14035224/
• 关键词: RNA; ribozyme; synthetic biology; ligase; RNP; molecular design; molccular engineering; nano; ナノバイオ; 分子認識; 遺伝子; 核酸; 酵素; in vitroセレクション; self-folding RNA; ナオバイオ; self-foldingRNA; in vitro セレクション; P4-P6 RNA; 立体構造; 触媒部位

Onto an artificially designed and constructed RNA, a reaction site for RNA-RNA ligation and a catalytic module that conducts the ligation was installed. The catalytic module was prepared by conducting in vitro selection from a pool consisting of 30 nucleotides. The constructed RNA ligase ribozyme was further molecularly designed and engineered from cis to trans type ligase successfully. In addition, the ribozyme was transformed into a allosterically controllable ribozyme by installing receptor RNA unit(s) that alters their conformation due to the binding of a small molecule. The technique developed for constructing artificial RNA with defined 3D structure was further employed for design and construction of RNP (RNA-protein complex) by using RNA design and protein molecules with known 3D structures. An RNP that consists of a scaffold RNA and two protein molecules, CFP and YFP, attached to the RNA was constructed to test whether FRET can be observed between the two proteins. The FRET was observed as anticipated. It was also found that the FRET energy can be controlled by adjusting the distance between two proteins on the RNA by simply adjusting the size of the RNA. The molecular engineering that we have developed here should serve as a useful tool to develop a new field in Synthetic Biology.

在人工设计和构建的RNA上，安装了用于RNA-RNA连接的反应位点和进行连接的催化模块。通过从由30个核苷酸组成的库中进行体外选择来制备催化模块。进一步对构建的RNA连接酶核酶进行分子设计，成功地将其从顺式连接酶改造为反式连接酶。此外，通过安装受体RNA单元将核酶转化为变构可控的核酶，所述受体RNA单元由于小分子的结合而改变其构象。通过使用RNA设计和具有已知3D结构的蛋白质分子，将用于构建具有确定的3D结构的人工RNA的技术进一步用于RNP（RNA-蛋白质复合物）的设计和构建。构建了由支架RNA和附着于RNA的两种蛋白质分子CFP和YFP组成的RNP，以测试是否可以在两种蛋白质之间观察到FRET。如预期观察到FRET。还发现FRET能量可以通过简单地调节RNA的大小来调节RNA上两个蛋白质之间的距离来控制。我们在这里开发的分子工程应该成为开发合成生物学新领域的有用工具。

项目成果

リボザイムの分子設計法-RNA構造情報の分子デザインへの還元-

核酶的分子设计方法-将RNA结构信息还原为分子设计-

• 发表时间: 2004
• 期刊: 実験医学(増刊) 22・7
• 作者: 井川善也;井上丹
• 通讯作者: 井上丹

Shimizu, T., Shiraishi, H., Inoue, T.: "Cloning and characterization of novel extensin-like cDNAs that are expressed during late somatic cell phase in the green alga Volvox carteri"Gene. 284. 179-187 (2002)

Shimizu, T.、Shiraishi, H.、Inoue, T.：“在绿藻Volvox carteri 体细胞晚期表达的新型延伸蛋白样 cDNA 的克隆和表征”基因。

RNAシンセティックバイオロジー

RNA合成生物学

• 发表时间: 2007
• 作者: Kenmochi;N.;Nakao;A.;Nguyen;H. D. Yoshihama;M.;井上丹
• 通讯作者: 井上丹

Ikawa, Y., Sasaki, K., Tominaga, H., Inoue.T.: "P5 activator of a group IC ribozyme can replace P7.1/7.2 activator of a group IA ribozyme."J.Biochem.. 133. 665-670 (2003)

Ikawa, Y.、Sasaki, K.、Tominaga, H.、Inoue.T.：“IC 组核酶的 P5 激活剂可以替代 IA 组核酶的 P7.1/7.2 激活剂。”J.Biochem.. 133。

Nucleic Acid Switches and Sensors (edited by Scott K.Silverman)

核酸开关和传感器（由 Scott K.Silverman 编辑）

• 发表时间: 2006
• 作者: Tan Inoue*;Yoshiya Ikawa
• 通讯作者: Yoshiya Ikawa