公开(公告)号：US20120270221A1

公开(公告)日：2012-10-25

申请号：US12931305

申请日：2011-01-28

申请人： John G. Bruno ,  Joseph Chanpong

发明人： John G. Bruno ,  Joseph Chanpong

IPC分类号： G01N21/64 ,  B82Y15/00

CPC分类号： C12N15/111 ,  C12N2310/16 ,  C12N2320/10 ,  G01N33/5308 ,  G01N33/533 ,  G01N33/542

摘要： The present invention describes methods for the production and selecting of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis. Alternatively, single DNA or RNA intrachain FRET-aptamers can be sequenced and the locations of F and Q within the structure can be determined by nanopore sequencing and the locations of F and Q within the structure can be verified by nucleic acid “combing” coupled to high-powered fluorescence microscopy.

摘要翻译： 本发明描述了在其结构内的各个位点生产和选择含有荧光团（F）和猝灭剂（Q）的单链（单链）荧光共振能量转移（FRET）DNA或RNA适体，使得当其 特异性匹配分析物结合，FRET适体被特定波长的光激发，系统的荧光强度与添加的分析物的量成比例地调节（增加或减少）。 F和Q与核苷酸三磷酸（NTPs）共价连接，其在聚合酶链反应（PCR）期间由各种核酸聚合酶例如Taq聚合酶掺入，然后通过亲和层析，大小排阻或分子筛选和荧光技术 。 通过离子对反相高效液相色谱（HPLC）或其他色谱法可以进一步分离相关的FRET-适体。 最后，通过用核酸外切酶消化和质谱核苷酸测序分析来测定FRET-适体结构中F和Q在FRET-适体结构中的特异性位置。 或者，可以对单个DNA或RNA链内FRET-适体进行测序，并且可以通过纳米孔测序确定结构内F和Q的位置，并且结构内的F和Q的位置可以通过核酸结合到高效液相色谱 动力荧光显微镜。

公开(公告)号：US20120135540A1

公开(公告)日：2012-05-31

申请号：US13199484

申请日：2011-08-31

申请人： John G. Bruno

发明人： John G. Bruno

IPC分类号： G01N33/53 ,  C07H21/04

CPC分类号： C12N15/115 ,  G01N33/5308 ,  G01N33/66 ,  G01N33/6887 ,  G01N33/82 ,  G01N2333/4712 ,  G01N2333/4737 ,  G01N2333/54 ,  G01N2333/5412 ,  G01N2333/61

摘要： Specific DNA sequences for binding various clinically relevant analytes from the human body are described. Each of these sequences or their linear, two- and three-dimensional linked sequences can function in varying assay and sensor formats with varying degrees of success. Linkage of the whole or partial DNA sequences (putative binding sites) can be used to enhance specificity and affinity towards complex targets, thereby improving assay selectivity and sensitivity in many instances. In addition, a FRET-based quantitative method is described for normalizing analyte data by assessing urine creatinine and urea levels. Finally, a method is described for removing creatinine or urea by size-exclusion chromatography prior to a FRET-based aptamer assay to avoid the denaturing effects of these compounds.

摘要翻译： 描述了用于结合来自人体的各种临床相关分析物的特异性DNA序列。 这些序列中的每一个或其线性，二维和三维连接的序列可以以不同的测定和传感器形式发挥不同程度的成功。 可以使用全部或部分DNA序列（推定的结合位点）的连接来增强对复杂靶标的特异性和亲和力，从而在许多情况下提高测定选择性和灵敏度。 此外，描述了基于FRET的定量方法，用于通过评估尿肌酐和尿素水平来归一化分析物数据。 最后，描述了在基于FRET的适体测定之前通过尺寸排阻色谱法除去肌酸酐或尿素的方法，以避免这些化合物的变性作用。

公开(公告)号：US20120123096A1

公开(公告)日：2012-05-17

申请号：US12716088

申请日：2010-03-02

申请人： John G. Bruno ,  Judson C. Miner

发明人： John G. Bruno ,  Judson C. Miner

IPC分类号： C07K14/435 ,  C07K14/765 ,  C07K14/745 ,  C07K14/79

CPC分类号： C07K14/00 ,  A61K47/549 ,  A61K47/64 ,  A61K47/643 ,  A61K47/645 ,  A61K47/6835 ,  C12N15/111 ,  C12N15/115 ,  C12N2310/16 ,  C12N2310/3513 ,  C12N2320/30 ,  C12N2320/51

摘要： Methods are described for improvement of the serum half life of therapeutic nucleic acids by 3′ conjugation to useful target proteins, or other large molecules with useful function. In one embodiment, a 3′ A, C or G overhang is added to ds-DNA and the primary amines conjugated using biocompatible bifunctional linkers to proteins. The resulting nucleic acid-3′ conjugates are serum nuclease-resistant and retained in vivo for long periods without rapid kidney clearance. Further, the choice of conjugate imparts additional functionality to the nucleic acid-3′ conjugate.

摘要翻译： 描述了通过3'缀合到有用靶蛋白或具有有用功能的其它大分子来改善治疗性核酸的血清半衰期的方法。 在一个实施方案中，将3'A，C或G突出端加入到ds-DNA中，并且使用生物相容性双功能接头与蛋白质缀合的伯胺。 所得到的核酸-3'缀合物是血清核酸酶抗性的并且长时间保留在体内而没有快速的肾清除。 此外，缀合物的选择赋予核酸-3'缀合物附加的功能。

公开(公告)号：US5972721A

公开(公告)日：1999-10-26

申请号：US816429

申请日：1997-03-14

申请人： John G. Bruno ,  Johnathan L. Kiel ,  John P. Kilian

发明人： John G. Bruno ,  Johnathan L. Kiel ,  John P. Kilian

IPC分类号： B03C1/01 ,  B03C1/032 ,  B03C1/033 ,  B03C1/034 ,  G01N33/533 ,  B03C1/00

CPC分类号： B03C1/032 ,  B03C1/01 ,  B03C1/0332 ,  B03C1/034 ,  Y10S436/824

摘要： An apparatus and method for immunomagnetic separation and concentration of target biological materials is disclosed. The immunomagnetic separation is performed by a magnetic flow cell, or filter block, as part of an automated mostly continuous immunomagnetic assay system. The magnetic flow cell has two bundles of ferromagnetic rods or pins positioned inside an internal chamber so that a fluid sample flowing through the flow cell passes through the pins. A pair of cobalt magnets flank the flow cell so that the pins concentrate and sufficiently increase the magnetic fields so that even nanometer size magnetic beads can be captured. The overall system combines a reaction subsystem for reacting coated magnetic beads with a sample, a collection subsystem for capturing magnetic beads, a rinsing subsystem for removing debris and a filtering subsystem for removing captured magnetic beads from the collection subsystem. The new magnetic flow filter is the key component for the collection and filtering subsystems.

摘要翻译： 公开了一种用于免疫磁性分离和靶向生物材料浓缩的装置和方法。 免疫磁性分离由磁流量池或过滤块进行，作为自动化大多数连续的免疫磁性测定系统的一部分。 磁流量计具有两束位于内部室内的铁磁棒或销，使​​得流过流动池的流体样品通过销。 在流动池侧面的一对钴磁体，使得引脚集中并充分增加磁场，使得可以捕获甚至纳米尺寸的磁珠。 整个系统结合了用于使涂覆的磁珠与样品反应的反应子系统，用于捕获磁珠的收集子系统，用于去除碎屑的冲洗子系统和用于从收集子系统中去除捕获的磁珠的过滤子系统。 新的磁流过滤器是收集和过滤子系统的关键组件。

公开(公告)号：US08648181B2

公开(公告)日：2014-02-11

申请号：US13199082

申请日：2011-08-18

申请人： John G. Bruno

发明人： John G. Bruno

IPC分类号： C07H21/04

CPC分类号： C12N15/115 ,  C07H21/04 ,  C12N2310/16

摘要： Specific DNA ligand sequences for binding various arthropod-borne pathogens including arboviruses, rickettsia and parasites are described. Each of these sequences or their linear, two-and three-dimesional linked sequences can function in varying assay and sensor formats with varying degrees of success. Linkage of the whole or partial DNA sequences (putative binding sites) can be used to enhance specificity and affinity towards complex targets, thereby improving assay selectivity and sensitivity in many instances. In addition, the DNA sequences may bind and neutralize or prevent infection from arthropod-borne viruses, rickettsia and Leishmania or other parasites.

摘要翻译： 描述了用于结合各种节肢动物传播的病原体的具体DNA配体序列，包括罗非鱼病毒，立克次体和寄生虫。 这些序列中的每一个或其线性，二维和三维连锁序列可以以不同的测定和传感器形式发挥不同程度的成功。 可以使用全部或部分DNA序列（推定的结合位点）的连接来增强对复杂靶标的特异性和亲和力，从而在许多情况下提高测定选择性和灵敏度。 此外，DNA序列可以结合并中和或防止节肢动物传播的病毒，立克次体和利什曼原虫或其他寄生虫的感染。

公开(公告)号：US20120219961A1

公开(公告)日：2012-08-30

申请号：US12931309

申请日：2011-01-28

申请人： John G. Bruno ,  Joseph Chanpong

发明人： John G. Bruno ,  Joseph Chanpong

IPC分类号： G01N21/64

CPC分类号： C12N15/111 ,  C12N2310/16 ,  C12N2320/10 ,  G01N33/5308 ,  G01N33/533 ,  G01N33/542

摘要： The present invention describes methods for the production and use of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis.

摘要翻译： 本发明描述了在其结构内的各种基因座上生产和使用含有荧光团（F）和猝灭剂（Q）的单链（单链）荧光共振能量转移（“FRET”）DNA或RNA适体的方法，使得 当其特异性匹配分析物结合并且FRET适体被特定波长的光激发时，与添加的分析物的量成比例地调节（增加或减少）系统的荧光强度。 F和Q与核苷酸三磷酸（NTPs）共价连接，其在聚合酶链反应（PCR）期间由各种核酸聚合酶例如Taq聚合酶掺入，然后通过亲和层析，大小排阻或分子筛选和荧光技术 。 通过离子对反相高效液相色谱（HPLC）或其他色谱法可以进一步分离相关的FRET-适体。 最后，通过用核酸外切酶消化和质谱核苷酸测序分析来测定FRET-适体结构中F和Q在FRET-适体结构中的特异性位置。

公开(公告)号：US20120149889A1

公开(公告)日：2012-06-14

申请号：US13199082

申请日：2011-08-18

申请人： John G. Bruno

发明人： John G. Bruno

IPC分类号： C07H21/04

CPC分类号： C12N15/115 ,  C07H21/04 ,  C12N2310/16

摘要： Specific DNA ligand sequences for binding various arthropod-borne pathogens including arboviruses, rickettsia and parasites are described. Each of these sequences or their linear, two- and three-dimesional linked sequences can function in varying assay and sensor formats with varying degrees of success. Linkage of the whole or partial DNA sequences (putative binding sites) can be used to enhance specificity and affinity towards complex targets, thereby improving assay selectivity and sensitivity in many instances. In addition, the DNA sequences may bind and neutralize or prevent infection from arthropod-borne viruses, rickettsia and Leishmania or other parasites.

摘要翻译： 描述了用于结合各种节肢动物传播的病原体的具体DNA配体序列，包括罗非鱼病毒，立克次体和寄生虫。 这些序列或其线性，二维和三维连锁序列中的每一个可以以不同的测定和传感器形式发挥不同程度的成功。 可以使用全部或部分DNA序列（推定的结合位点）的连接来增强对复杂靶标的特异性和亲和力，从而在许多情况下提高测定选择性和灵敏度。 此外，DNA序列可以结合并中和或防止节肢动物传播的病毒，立克次体和利什曼原虫或其他寄生虫的感染。

公开(公告)号：US20120071639A1

公开(公告)日：2012-03-22

申请号：US13136820

申请日：2011-08-11

申请人： John G. Bruno

发明人： John G. Bruno

IPC分类号： C07H21/04

CPC分类号： G01N33/56916 ,  C12N15/115 ,  C12N2310/16 ,  C12Q1/689 ,  G01N33/56922 ,  Y02A50/451

摘要： Specific DNA sequences for binding various foodborne and waterborne pathogens and biotoxins are described. Each of these sequences can function in varying assay and sensor formats with varying degrees of success.

摘要翻译： 描述了用于结合各种食源性和水源性病原体和生物毒素的特异性DNA序列。 这些序列中的每一个可以以不同的测定和传感器形式起作用，具有不同程度的成功。

公开(公告)号：US07906280B2

公开(公告)日：2011-03-15

申请号：US11433009

申请日：2006-05-12

申请人： John G. Bruno ,  Joseph Chanpong

发明人： John G. Bruno ,  Joseph Chanpong

IPC分类号： C12Q1/68 ,  G01N33/53

CPC分类号： C12N15/111 ,  C12N2310/16 ,  C12N2320/10 ,  G01N33/5308 ,  G01N33/533 ,  G01N33/542

摘要： The present invention describes methods for the production and use of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis.

摘要翻译： 本发明描述了在其结构内的各种基因座上生产和使用含有荧光团（F）和猝灭剂（Q）的单链（单链）荧光共振能量转移（“FRET”）DNA或RNA适体的方法，使得 当其特异性匹配分析物结合并且FRET适体被特定波长的光激发时，与添加的分析物的量成比例地调节（增加或减少）系统的荧光强度。 F和Q与核苷酸三磷酸（NTPs）共价连接，其在聚合酶链反应（PCR）期间由各种核酸聚合酶例如Taq聚合酶掺入，然后通过亲和层析，大小排阻或分子筛选和荧光技术 。 通过离子对反相高效液相色谱（HPLC）或其他色谱法可以进一步分离相关的FRET-适体。 最后，通过用核酸外切酶消化和质谱核苷酸测序分析来测定FRET-适体结构中F和Q在FRET-适体结构中的特异性位置。

公开(公告)号：US20080161236A1

公开(公告)日：2008-07-03

申请号：US11735221

申请日：2007-04-13

申请人： John G. Bruno ,  Judson C. Miner

发明人： John G. Bruno ,  Judson C. Miner

IPC分类号： A61K38/16 ,  C07K2/00 ,  A61P31/00 ,  A61P35/04

CPC分类号： C12N15/115 ,  A61K47/549 ,  A61K47/64 ,  A61K47/643 ,  A61K47/645 ,  A61K47/6835 ,  C12N15/111 ,  C12N2310/3513 ,  C12N2320/30 ,  C12N2320/51

摘要： Methods are described for improvement of the serum half life of therapeutic nucleic acids by 3′ conjugation to useful target proteins, or other large molecules with useful function. In one embodiment, a 3′ A, C or G overhang is added to ds-DNA and the primary amines conjugated using biocompatible bifunctional linkers to proteins. The resulting nucleic acid-3′-conjugates are serum nuclease-resistant and retained in vivo for long periods without rapid kidney clearance. Further, the choice of conjugate imparts additional functionality to the nucleic acid -3-conjugate.

摘要翻译： 描述了通过3'缀合到有用靶蛋白或具有有用功能的其它大分子来改善治疗性核酸的血清半衰期的方法。 在一个实施方案中，将3'A，C或G突出端加入到ds-DNA中，并且使用生物相容性双功能接头与蛋白质缀合的伯胺。 所得的核酸-3'-缀合物是血清核酸酶抗性的并且长时间保留在体内，没有快速的肾清除。 此外，缀合物的选择赋予核酸-3缀合物更多的功能。