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21. 发明授权

US06699687B1 Circularly permuted fluorescent protein indicators 有权
标题翻译：循环置换荧光蛋白指标
公开(公告)号：US06699687B1
公开(公告)日：2004-03-02
申请号：US09316920
申请日：1999-05-21
申请人： Roger Y. Tsien , Geoffrey Baird
发明人： Roger Y. Tsien , Geoffrey Baird
IPC分类号： C12N1512
CPC分类号： C07K14/43595
摘要： The present invention provides polypeptide and polynucleotides encoding fluorescent indicators having inserted within a fluorescent moiety a sensor polypeptide. Also provided are methods of using the fluorescent indicator. Circularly permuted fluorescent polypeptides and polynucleotides are also provided.
摘要翻译：本发明提供编码在荧光部分内插入传感器多肽的荧光指示剂的多肽和多核苷酸。还提供了使用荧光指示剂的方法。还提供了环状置换的荧光多肽和多核苷酸。

22. 发明授权

US06472205B1 Cytosolic forms for &bgr;-lactamase and uses thereof 有权
标题翻译： β-内酰胺酶的胞质形式及其用途
公开(公告)号：US06472205B1
公开(公告)日：2002-10-29
申请号：US09481756
申请日：2000-01-11
申请人： Roger Y. Tsien , Gregor Zlokarnik
发明人： Roger Y. Tsien , Gregor Zlokarnik
IPC分类号： C12N500
CPC分类号： C07D501/36 , C07D501/00 , C07D505/00 , C07D519/00 , C12N9/86 , C12N15/85 , C12N2800/90 , C12N2830/002 , C12P17/184 , C12P17/188 , C12P35/00 , C12Q1/18 , C12Q1/34 , C12Q1/6897 , C12Q2334/00 , G01N2333/986 , G01N2415/00 , Y10S435/805
摘要： The present invention is directed to nucleic acid molecules that encode a cytosolic form of beta-lactamase, cells that include such nucleic acid molecules and methods for their use. These cells can be used in a variety of methods, such as methods for monitoring the expression of a gene or biochemical activity.
摘要翻译：本发明涉及编码胞内形式的β-内酰胺酶的核酸分子，包括这种核酸分子的细胞及其使用方法。这些细胞可以用于各种方法，例如用于监测基因表达或生化活性的方法。

23. 发明授权

US06451569B1 Synthetic molecules that specifically react with target sequences 有权
标题翻译：与靶序列特异性反应的合成分子
公开(公告)号：US06451569B1
公开(公告)日：2002-09-17
申请号：US09372338
申请日：1999-08-11
申请人： Roger Y. Tsien , B. Albert Griffin
发明人： Roger Y. Tsien , B. Albert Griffin
IPC分类号： C07K1113
CPC分类号： C07F9/74 , C07F9/80 , G01N33/532
摘要： The present invention features biarsenical molecules. Target sequences that specifically react with the biarsenical molecules are also included. The present invention also features kits that include biarsenical molecules and target sequences. Tetraarsenical molecules are also featured in the invention.
摘要翻译：本发明的特征在于双重分子。还包括与二元分子特异性反应的靶序列。本发明还包括包含二元分子和靶序列的试剂盒。特征在于本发明的特征。

24. 发明授权

US06221612B1 Photon reducing agents for use in fluorescence assays 失效
标题翻译：用于荧光测定的光子还原剂
公开(公告)号：US06221612B1
公开(公告)日：2001-04-24
申请号：US09122477
申请日：1998-07-23
申请人： Tom Knapp , Gregor Zlokarnik , Paul Negulescu , Roger Y. Tsien , Tim Rink
发明人： Tom Knapp , Gregor Zlokarnik , Paul Negulescu , Roger Y. Tsien , Tim Rink
IPC分类号： C12Q170
CPC分类号： G01N33/582 , C12Q1/682 , G01N33/5005 , G01N33/542 , Y10S436/80 , Y10S436/809 , Y10T436/13 , C12Q2563/107 , C12Q2527/125
摘要： The present invention provides a method for reducing undesirable light emission from a sample using at least one photon producing agent and at least one photon reducing agent (e.g. dye-based photon reducing agents). The present invention further provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one collisional quencher. The present invention also provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one quencher, such as an electronic quencher. The present invention also provides a system and method of screening test chemicals in fluorescent assays using photon reducing agents. The present invention also provides compositions, pharmaceutical compositions, and kits for practicing these methods.
摘要翻译：本发明提供一种使用至少一种光子产生剂和至少一种光子还原剂（例如基于染料的光子还原剂）来减少来自样品的不希望的光发射的方法。本发明进一步提供了用至少一种光子产生剂和至少一种碰撞猝灭剂减少来自样品（例如生物化学或细胞样品）的不希望的光发射的方法。本发明还提供了用至少一种光子产生剂和至少一种猝灭剂（例如电子猝灭剂）减少来自样品（例如，生物化学或细胞样品）的不希望的光发射的方法。本发明还提供了使用光子还原剂在荧光测定中筛选测试化学品的系统和方法。本发明还提供了用于实施这些方法的组合物，药物组合物和试剂盒。

25. 发明授权

US6054271A Methods of using synthetic molecules and target sequences 失效
标题翻译：使用合成分子和靶序列的方法
公开(公告)号：US6054271A
公开(公告)日：2000-04-25
申请号：US955050
申请日：1997-10-21
申请人： Roger Y. Tsien , B. Albert Griffin
发明人： Roger Y. Tsien , B. Albert Griffin
IPC分类号： C07F9/80 , G01N33/532 , G01N33/566 , C12N15/09 , C12N15/64
CPC分类号： C07F9/80 , G01N33/532
摘要： The present invention features biarsenical molecules and target sequences that specifically react with the biarsenical molecules. Methods of using the biarsenical molecules, tetraarsenical molecules and the target sequences are included.
摘要翻译：本发明的特征在于与两性分子特异性反应的双链分子和靶序列。包括双缩醛分子，四元分子和靶序列的使用方法。

26. 发明授权

US5932474A Target sequences for synthetic molecules 失效
标题翻译：合成分子的靶序列
公开(公告)号：US5932474A
公开(公告)日：1999-08-03
申请号：US955206
申请日：1997-10-21
申请人： Roger Y. Tsien , B. Albert Griffin
发明人： Roger Y. Tsien , B. Albert Griffin
IPC分类号： C07F9/80 , G01N33/532 , C12N15/63
CPC分类号： C07F9/80 , G01N33/532
摘要： The present invention features biarsenical molecules and target sequences that specifically react with the biarsenical molecules. Bonding partners that include target sequences, vectors that include nucleic acid sequences that encode the target sequences and host cells that include the target sequences are also featured in the invention.
摘要翻译：本发明的特征在于与两性分子特异性反应的双链分子和靶序列。包括靶序列的结合伙伴，包括编码靶序列的核酸序列的载体和包含靶序列的宿主细胞也在本发明中。

27. 发明授权

US5925558A Assays for protein kinases using fluorescent protein substrates 失效
公开(公告)号：US5925558A
公开(公告)日：1999-07-20
申请号：US680876
申请日：1996-07-16
申请人： Roger Y. Tsien , Andrew B. Cubitt
发明人： Roger Y. Tsien , Andrew B. Cubitt
IPC分类号： C07K14/435 , C12N1/21 , C12Q1/48 , G01N33/58 , C12N15/62 , C07K19/00
CPC分类号： G01N33/582 , C07K14/43595 , C12Q1/48 , G01N2333/91205
摘要： This invention provides assays for protein kinase activity using fluorescent proteins engineered to include sequences that can be phosphorylated by protein kinases. The proteins exhibit different fluorescent properties in the non-phosphorylated and phosphorylated states.

28. 发明授权

US5374710A Compounds which release nitric oxide upon illumination 失效
标题翻译：照射时释放一氧化氮的化合物
公开(公告)号：US5374710A
公开(公告)日：1994-12-20
申请号：US67657
申请日：1993-05-26
申请人： Roger Y. Tsien , Lewis R. Makings
发明人： Roger Y. Tsien , Lewis R. Makings
IPC分类号： A61K31/13 , A61K31/135 , A61P9/00 , A61P25/00 , A61P43/00 , C07C291/08 , C07C245/02 , C01B21/24 , C07C245/24
CPC分类号： C07C291/08
摘要： Chemical derivatives of nitric oxide are provided which are stable indefinitely in oxygen-containing solutions until photolysis, whereupon they release NO. These compounds have the general formulaA--N.sup.+ (O.sup.-)=N--O--B (I)wherein A is typically a nitrogen- or oxygen-containing substituent and B is a group lablie to photolysis. The compounds are stable and inert in oxygenated aqueous solutions, but release NO upon illumination. Given the ease with which the intensity, timing and location of illumination may be controlled, these compounds are particularly useful in investigating the biological effects of NO with much higher spatial or temporal resolution than heretofore possible.
摘要翻译：提供一氧化氮的化学衍生物，其在含氧溶液中无限制地稳定直至光解，于是它们释放NO。这些化合物具有通式A-N +（O - ）= N-O-B（I）其中A通常是含氮或含氧取代基，B是不溶于光解的基团。化合物在氧化水溶液中是稳定的和惰性的，但在照射时释放NO。鉴于可以轻松控制照明的强度，时间和位置，这些化合物特别适用于调查NO的生物效应，空间或时间分辨率比以前更高。

29. 发明授权

US5141627A Chelators whose affinity for calcium ion is increased by illumination 失效
标题翻译：通过照明增加对钙离子的亲和力的螯合剂
公开(公告)号：US5141627A
公开(公告)日：1992-08-25
申请号：US412852
申请日：1989-09-25
申请人： Roger Y. Tsien , Stephen R. Adams
发明人： Roger Y. Tsien , Stephen R. Adams
IPC分类号： C07C245/18
CPC分类号： C07C245/18
摘要： The present invention relates to a group of organic chelators whose affinity for calcium ion in solution is increased by electromagnetic radiation. Specifically, the chelators are related to BAPTA and utilize the addition of an electron-withdrawing group (e.g., diazocarbonyl) to a ring of BAPTA, para to the amino group. Photochemical rearrangement of the diazoacetyl group converts the group to the electron-donating carboxymethyl group, causing the calcium ion efficiency to increase 25 to 50 fold. These chelators when incorporated into rat fibroblasts either by microinjection or by incubation as the membrane-permeable, enzymatically-labile tetraacetoxymethyl ester and flash-photolyzed cause a drop in intracellular free calcine ion to or below resting valves of about 10.sup.-7 M. These chelators are used to generate controlled fast removal of intracellular free calcium ion to mimic or modulate a number of important cellular responses, especially in nerve or muscle.
摘要翻译：本发明涉及通过电磁辐射增加溶液中对钙离子的亲和力的一组有机螯合剂。具体地说，螯合剂与BAPTA有关，并利用向吸附对氨基的BAPTA环中加入吸电子基团（如重氮羰基）。重氮乙酰基的光化学重排将该基团转移到给电子的羧甲基基团，导致钙离子效率增加25-50倍。这些螯合剂通过显微注射或通过孵育作为膜可渗透的，酶促不稳定的四乙酰氧基甲基酯并且闪光光引发而引入大鼠成纤维细胞时，导致细胞内游离的煅烧离子降至约10-7M的静止阀或其下面。这些螯合剂用于产生受控的快速去除细胞内游离钙离子以模拟或调节许多重要的细胞反应，特别是在神经或肌肉中。

30. 发明授权

US4603209A Fluorescent indicator dyes for calcium ions 失效
标题翻译：用于钙离子的荧光指示剂染料
公开(公告)号：US4603209A
公开(公告)日：1986-07-29
申请号：US648649
申请日：1984-09-07
申请人： Roger Y. Tsien , Grzegorz Grynkiewicz
发明人： Roger Y. Tsien , Grzegorz Grynkiewicz
IPC分类号： A61K38/00 , A61K38/22 , A61P5/00 , C07C229/18 , C07C229/52 , C07C237/30 , C07C255/59 , C07C309/49 , C07C311/39 , C07C317/32 , C07D209/08 , C07D209/14 , C07D215/40 , C07D307/79 , C07D307/82 , C07D413/04 , C07K7/23 , G01N21/78 , G01N33/84 , C07C101/447 , C07D307/87
CPC分类号： C07D307/79 , C07D209/08 , C07D215/40 , C07D413/04 , Y10S436/80
摘要： The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, "quin2", the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca.sup.2+ binding, slightly lower affinities for Ca.sup.2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca.sup.2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca.sup.2+, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca.sup.2+ specific fluorescent indicators.
摘要翻译：本发明公开了一类对钙离子具有特异性的高荧光指示剂染料。新的荧光指示剂染料将二苯乙烯型荧光团与具有EGTA和BAPTA特征的连接组的八配位图案的四羧酸母体Ca 2+螯合化合物结合。优选的形式包含额外的杂环桥，以加强1,2-二苯乙烯的烯键并降低疏水性。与其广泛使用的前身“quin2”相比，新型染料提供高达三十倍的荧光，Ca2 +结合时的波长（不仅仅是强度）的主要变化，Ca2 +的亲和力略低，激发波长稍长，提高Ca2 +对其他二价阳离子的选择性。这些性质，特别是对Ca2 +的波长敏感性，使得染料对于许多细胞内应用尤其是在单细胞，贴壁细胞层或体组织中是有用的指标。本发明还公开了一种合成α-酰氧基烷基溴化物的改进方法，其中如此合成的溴化物没有污染的双（1-溴烷基）醚。改进的方法在本文中示例性地合成乙酰氧甲基溴，其是可用于制备本文公开的乙酰氧基甲酯作为新型Ca 2+特异性荧光指示剂的化合物。

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