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公开(公告)号：US09441263B2
公开(公告)日：2016-09-13
申请号：US14111947
申请日：2012-04-14
申请人： Scott C. Blanchard , Michael Brian Feldman , Leyi Wang , James H. Doudna Cate , Arto Pulk , Roger B. Altman , Michael R. Wasserman
发明人： Scott C. Blanchard , Michael Brian Feldman , Leyi Wang , James H. Doudna Cate , Arto Pulk , Roger B. Altman , Michael R. Wasserman
IPC分类号： C12Q1/68 , G01N33/542 , G01N33/94
CPC分类号： C12Q1/68 , G01N33/542 , G01N33/9446 , G01N2333/245
摘要： The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.
摘要翻译： 本发明涉及使用完整的细菌核糖体的结构鉴定在细菌核糖体的新霉素结合口袋中结合的分子的方法，其揭示了核糖体如何通过x射线晶体学确定的两个功能不同的状态结合tRNA。 一个国家在肽基-tRNA结合位点定位tRNA。 第二个完全旋转状态通过核糖体循环因子（RRF）稳定，并在杂交肽基/出口（P / E）位点中以高度弯曲的构象结合tRNA。 此外，本发明涉及各种测定法，包括用于核糖体回收的单分子测定法，以及通过使用核糖体上已知和新的FRET对检测新鉴定的中间FRET状态来鉴定干扰核糖体功能的化合物的方法。 本发明还提供具有N-末端标记的S13蛋白的载体和组合物。
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公开(公告)号：US20140127682A1
公开(公告)日：2014-05-08
申请号：US14111947
申请日：2012-04-14
申请人： Scott C. Blanchard , Michael Brian Feldman , Leyi Wang , James H. Doudna Cate , Arto Pulk , Roger B. Altman , Michael R. Wasserman
发明人： Scott C. Blanchard , Michael Brian Feldman , Leyi Wang , James H. Doudna Cate , Arto Pulk , Roger B. Altman , Michael R. Wasserman
IPC分类号： C12Q1/68
CPC分类号： C12Q1/68 , G01N33/542 , G01N33/9446 , G01N2333/245
摘要： The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein
摘要翻译： 本发明涉及使用完整的细菌核糖体的结构鉴定在细菌核糖体的新霉素结合口袋中结合的分子的方法，其揭示了核糖体如何通过x射线晶体学确定的两个功能不同的状态结合tRNA。 一个国家在肽基-tRNA结合位点定位tRNA。 第二个完全旋转状态通过核糖体循环因子（RRF）稳定，并在杂交肽基/出口（P / E）位点中以高度弯曲的构象结合tRNA。 此外，本发明涉及各种测定法，包括用于核糖体回收的单分子测定法，以及通过使用核糖体上已知和新的FRET对检测新鉴定的中间FRET状态来鉴定干扰核糖体功能的化合物的方法。 本发明还提供具有N-末端标记的S13蛋白的载体和组合物
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3.发明申请SIGNAL AMPLIFICATION METHODS FOR THE IMAGING OF PROTEIN SYNTHESIS AND NEUROTRANSMITTER TRANSPORT 有权用于蛋白质合成和神经递质运输成像的信号放大方法公开(公告)号：US20140127681A1
公开(公告)日：2014-05-08
申请号：US14110372
申请日：2012-04-06
IPC分类号： G01N33/68
摘要： The present invention describes the synthesis of biological samples that can be used for the purpose of enhancing the signal-to-noise ratios achievable during the imaging of protein synthesis, amino acid transport and neurotransmitter transport, particularly in applications where single-molecule resolution is demanded. The present invention provides quencher-labeled elongation factor (EF-Tu) and fluorophore-labeled tRNA. When these molecules are present in a ternary complex with GTP, the fluorescently-labeled tRNA is quantitatively quenched. Once the tRNA is incorporated into an actively translating ribosome, however, a burst of fluorescence is released and can be detected by a variety of techniques, including smFRET imaging. The invention further provides novel EF-Tu constructs for achieving quencher labeling at high levels while retaining native or near native activity in the translation reactions, as well as methods for preparing stable ternary complexes, methods of protein sequencing, methods of detecting amino acid transport using a proteoliposome assay system and the proteoliposomes systems and methods of imaging translation events in single living cells. The present invention should have an immediate impact on next-generation sequencing technologies and the detection of neurotransmitter transporter activities in both in vitro and in vivo settings, a critical component of drug activity/screening assays targeting this important class of molecules.
摘要翻译： 本发明描述了可用于增强蛋白质合成，氨基酸转运和神经递质转运成像期间可实现的信噪比的生物样品的合成，特别是在需要单分子分辨率的应用中 。 本发明提供淬灭标记的延伸因子（EF-Tu）和荧光团标记的tRNA。 当这些分子与GTP存在于三元复合物中时，荧光标记的tRNA被定量淬灭。 然而，一旦将tRNA并入活跃的转录核糖体中，就会释放出一阵荧光，并可通过各种技术（包括smFRET成像）来检测。 本发明还提供了用于以高水平实现猝灭标记的新型EF-Tu构建体，同时在翻译反应中保留天然或接近天然活性，以及制备稳定三元复合物的方法，蛋白质测序方法，使用 蛋白脂质体测定系统和单个活细胞中翻译事件成像的蛋白脂质体系统和方法。 本发明应该对下一代测序技术立即产生影响，并且在体外和体内环境中检测神经递质转运蛋白活性，这是靶向该重要类别分子的药物活性/筛选试验的关键组分。
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4.发明授权公开(公告)号：US09625466B2
公开(公告)日：2017-04-18
申请号：US14110372
申请日：2012-04-06
摘要： The present invention describes the synthesis of biological samples that can be used for the purpose of enhancing the signal-to-noise ratios achievable during the imaging of protein synthesis, amino acid transport and neurotransmitter transport, particularly in applications where single-molecule resolution is demanded. The present invention provides quencher-labeled elongation factor (EF-Tu) and fluorophore-labeled tRNA. When these molecules are present in a ternary complex with GTP, the fluorescently-labeled tRNA is quantitatively quenched. Once the tRNA is incorporated into an actively translating ribosome, however, a burst of fluorescence is released and can be detected by a variety of techniques, including smFRET imaging. The invention further provides novel EF-Tu constructs for achieving quencher labeling at high levels while retaining native or near native activity in the translation reactions, as well as methods for preparing stable ternary complexes, methods of protein sequencing, methods of detecting amino acid transport using a proteoliposome assay system and the proteoliposomes systems and methods of imaging translation events in single living cells. The present invention should have an immediate impact on next-generation sequencing technologies and the detection of neurotransmitter transporter activities in both in vitro and in vivo settings, a critical component of drug activity/screening assays targeting this important class of molecules.
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