Search for the Structural Basis of Biomacromolecular Function and Activity

寻找生物大分子功能和活性的结构基础

• 批准号: 8348990
• 负责人: Yun Xing m wang
• 金额: $ 104.24万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8348990
• 关键词: Acids; Adenine; Amino Acids; Area; Bacteria; Biochemical; Biochemical Reaction; Biological; Biology; Biophysics; Cancer Biology; Cells; Chemicals; Chemistry; Clinic; Collaborations; Communities; Complex; Crystallization; DNA; Detection; Development; Diagnosis; Dimerization; Disease; Down-Regulation; Dyes; Energy Transfer; Environment; Event; Foxes; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genomics; HIV; HIV-1; Image; In Vitro; Indium; Individual; Isotope Labeling; Knowledge; Label; Laboratories; Legal patent; Licensing; Location; Malignant Neoplasms; Maps; Measurement; Messenger RNA; Methods; Microinjections; Molecular Biology; Monitor; Movement; Murine leukemia virus; NMR Spectroscopy; Nucleotides; Peptide Antibiotics; Peptide Synthesis; Peptides; Phase; Play; Process; Proteins; RNA; RNA Biochemistry; RNA chemical synthesis; Reagent; Relative (related person); Reporting; Research; Research Personnel; Resolution; Response Elements; Roentgen Rays; Role; Sampling; Signal Transduction; Site; Small RNA; Solid; Solutions; Staging; Structure; Therapeutic; Untranslated Regions; Up-Regulation; Vascular Endothelial Growth Factors; Viral; Viral Packaging; Virus; X-Ray Crystallography; aptamer; base; commercial application; dimer; disease-causing mutation; glutamyl-prolyl-tRNA synthetase; improved; malignant breast neoplasm; molecular marker; mutant; new therapeutic target; novel; professor; research study; response; rev Protein; single molecule; structural biology; three dimensional structure; time use; tool; tumor; viral RNA

During the past year, we have been focusing on two areas of research. 1. Study structural biology of VEGF 3-prime UTR RNA and its interaction with protein regulators; 2. Study the structure basis of the adenine riboswitch; 3. Develop tools and methods that facilitate the studies as well as have a significant impact to scientific communities and potential applications for imaging and disease detection. 1. The gene expression of VEGF is regulated at multiple stages, including but not limited to post-transcription and translational levels. The down-regulation of the VEGF expression has been mapped to specific interactions between RNA125 and one of proteins in the GAIT complex. An earlier study by Dr. Fox's laboratory showed that the down-regulation involves specific interactions between the linker domain of the glutamyl-prolyl tRNA synthetase (EPRS), which is one of components of the GAIT complex, and a 29-nt hairpin (H29) that is entirely composed of A or U nucleotides and is the part of the 125-nt RNA . The structural basis for this important interaction is unknown. The EPRS linker domain (ELD) consists of three dual-helical bundles and the interaction site for H29 has been mapped to the first two helical bundles, r1r2, by Dr. Fox's group using biochemical methods. r1r2 contains 114 amino acids and the three-dimensional structure of the first dual-helical bundle has been reported. We are currently investigating the structural basis of down- and up-regulation of the VEGF expression using combination of NMR and SAXS in a collaboration with Professor Paul Fox's group of the Cleveland Clinic. We have encountered a significant difficult to obtain highly pure RNA samples suitable for NMR study. Nevertheless, we have determined three-dimensional structures of r1r2 and the H29 RNA, and we are in the process to determine the complex structure between r1r2 and H29. 2. We have obtained samples of the adenine riboswitch and its mutants in large quantity and performed preliminary NMR experiments. However, we have to develop new methods for structure determination of this RNA. One of these new methods is described in the following section. 3. My group has developed a new method for selective labeling of RNA (SLOR) at designated residue(s) and/or segment(s) of large RNAs using solid-phase multi-cycle enzymatic reactions. The potential applications of SLOR are broad and far reaching, due to a wide range of roles that RNA plays in biology. The followings are just examples of a few areas. General RNA biochemistry, biophysics and molecular biology. The fluorescent labeled RNA molecules can be used to study interaction between/within RNAs, and between RNA and DNA, RNA and proteins in vitro or within the cellular environment following microinjection. For example, selectively labeled RNA can be used to study riboswitch mechanisms in regulation of gene expression. The fluorescent residues can be incorporated at two strategically locations in the riboswitch using SLOR in order to monitor the switching event that is directly synchronized with the relative movement between the aptamer and expression platform domains using time-resolved single molecule Forster Resonance Energy Transfer (FRET) experiments. Probing such an event has not been possible because of lack of the specifically fluorescent labeled riboswitch RNA molecules. RNA structural biology. RNA molecules alone are almost impossible to crystallize for structure determination. NMR spectroscopy is an ideal method for structure determination of RNAs since it is a solution-state method and does not require crystallization. However, it is limited to only small RNAs, up to 50 residues, because of the extensive overlaps of chemical shift signals and short lifetimes of NMR signals. With SLOR, selectively labeled RNAs at designated residue(s) and/or segment(s) can be used for recording NMR signals, resulting in greatly simplified NMR spectra for straightforward interpretation. Moreover, one can selectively deuterate designated residues/segments and record the signals from the remaining residues. The signals from the remaining residues will have a much longer lifetime, resulting in significant enhancement of both resolution and sensitivity of NMR signals. This enhancement will make it possible to determine high-resolution structures of much larger RNAs using NMR spectroscopy: this will revolutionize RNA structural biology. The SLOR method will have an immediate impact to several collaborations between my group and several other groups within NCI (those collaborations are part of reasons that I developed SLOR). For example, we are collaborating with Dr. Alan Reins group studying the dimerization of the genomic RNAs of HIV-1 and murine leukemia virus. Dimerization of these RNAs is critical for viral packaging and maturation. The minimum size of the viral RNA that behaves like the whole viral genomic RNA is ca. 170 nucleotides (nt). Using the SLOR method, we can place fluorescent residues at the selected locations to monitor the dimerization and viral packaging using FRET and imaging experiments. Moreover, we can determine the three-dimensional (3D) structure of the RNA dimer by recording NMR spectra of the selectively isotope-labeled RNA samples. Another example is our collaboration with Dr. LeGrices group to determine the 3D structure of the HIV Rev Response Element (RRE), a 267-nt RNA, and to study the interaction of RRE with the Rev protein. 3D structure determinations of both the RNAs (for encapsidation and for Rev recognition) have not been feasible because of their sizes and the limitations of NMR and X-ray crystallography. Moreover, in principle, the SLOR method may be useful in the research of any NCI intramural investigators who study RNA biology or use RNA-based probes for detection, imaging and therapeutic applications. Clinic application. The discovery of disease-causing mutations in RNAs is yielding a wealth of new therapeutic targets, and the growing understanding of RNA biology and chemistry provides new RNA-based tools for developing therapeutics. There is a boom in applications and development of RNA-based reagents for detection, diagnosis and therapy in the past two decades. Among those RNA-based reagents, RNA aptamers are particularly useful because of their wide range of application against a variety of targets, including but not limited to organic dyes and amino acids, antibiotics, peptides, proteins of various sizes, functions, whole cells, viruses as well as specific molecular markers in various cancers. Potentially, the labeled RNAs or RNA aptamers produced using SLOR can be used to identify or improve these interactions at a resolution of individual residues. Patent applications. The SLOR method and the SLOR synthesizer may have wide potential commercial applications and may be filed for two separate patent applications. Companies that provide DNA and/or RNA and/or peptide synthesis would be potential licensees for licensing the SLOR method; companies that specialized in RNA synthesis or manufacture DNA or peptide synthesizers may be potential licensees of the SLOR RNA synthesizer. The number of those companies ranges at least several tens in US alone.

在过去的一年里，我们一直专注于两个领域的研究。1. 研究VEGF 3-prime UTR RNA的结构生物学及其与蛋白调控因子的相互作用2. 腺嘌呤核开关的结构基础研究；3. 开发工具和方法，促进研究，并对科学界和成像和疾病检测的潜在应用产生重大影响。1. VEGF的基因表达受到多个阶段的调控，包括但不限于转录后和翻译水平。VEGF表达的下调已被定位为RNA125与步态复合体中的一种蛋白质之间的特定相互作用。Fox博士实验室的一项早期研究表明，下调涉及谷氨酰脯氨酸tRNA合成酶（EPRS）的连接域（EPRS是步态复合物的组成部分之一）与29 nt发夹（H29）之间的特定相互作用，H29完全由a或U核苷酸组成，是125 nt RNA的一部分。这种重要相互作用的结构基础尚不清楚。EPRS连接结构域（ELD）由三个双螺旋束组成，Fox博士的团队使用生化方法将H29的相互作用位点映射到前两个螺旋束r1r2上。R1r2含有114个氨基酸，第一个双螺旋束的三维结构已被报道。目前，我们正在与克利夫兰诊所Paul Fox教授的团队合作，利用NMR和SAXS联合研究VEGF表达下调和上调的结构基础。我们遇到了一个重要的困难，以获得高纯度的RNA样品适合核磁共振研究。尽管如此，我们已经确定了r1r2和H29 RNA的三维结构，我们正在确定r1r2和H29之间的复杂结构。2. 我们获得了大量的腺嘌呤核开关及其突变体的样本，并进行了初步的核磁共振实验。然而，我们必须开发新的方法来确定这种RNA的结构。下一节将描述其中一种新方法。3. 我的团队已经开发了一种新的方法来选择性标记RNA （SLOR）在指定残基(s)和/或区段(s)的大RNA使用固相多周期酶促反应。由于RNA在生物学中扮演着广泛的角色，SLOR的潜在应用是广泛而深远的。以下只是几个领域的例子。一般RNA生物化学，生物物理学和分子生物学。荧光标记的RNA分子可用于在体外或显微注射后的细胞环境中研究RNA之间/内部、RNA与DNA、RNA与蛋白质之间的相互作用。例如，选择性标记RNA可用于研究调控基因表达的核糖体开关机制。利用时间分辨单分子福斯特共振能量转移（FRET）实验，荧光残基可以在核糖开关的两个战略位置上结合，以便监测与适体和表达平台结构域之间的相对运动直接同步的开关事件。由于缺乏特异性荧光标记的核糖开关RNA分子，探测这样的事件是不可能的。RNA结构生物学。单独的RNA分子几乎不可能结晶以确定其结构。核磁共振波谱是一种理想的rna结构测定方法，因为它是一种溶液状态的方法，不需要结晶。然而，由于化学位移信号的广泛重叠和核磁共振信号的短寿命，它仅限于小rna，最多50个残基。使用SLOR，在指定残基和/或片段上选择性标记的rna可用于记录核磁共振信号，从而大大简化了核磁共振光谱，便于直接解释。此外，可以选择性地对指定的残基/段进行氘化，并记录来自剩余残基的信号。剩余残留物的信号将具有更长的寿命，从而显著提高核磁共振信号的分辨率和灵敏度。这种增强将使利用核磁共振波谱测定更大RNA的高分辨率结构成为可能：这将彻底改变RNA结构生物学。SLOR方法将对我的小组和NCI内其他几个小组之间的几个合作产生直接影响（这些合作是我开发SLOR的部分原因）。例如，我们正在与Alan Reins博士小组合作，研究HIV-1和小鼠白血病病毒基因组rna的二聚化。这些rna的二聚化对病毒的包装和成熟至关重要。表现得像整个病毒基因组RNA的病毒RNA的最小大小约为170个核苷酸（nt）。使用SLOR方法，我们可以将荧光残基放置在选定的位置，通过FRET和成像实验来监测二聚化和病毒包装。此外，我们还可以通过记录选择性同位素标记RNA样品的核磁共振光谱来确定RNA二聚体的三维结构。另一个例子是我们与LeGrices博士团队合作，确定HIV Rev Response Element （RRE）的3D结构，这是一种267-nt RNA，并研究RRE与Rev蛋白的相互作用。由于rna的大小和核磁共振和x射线晶体学的限制，这两种rna（用于封装和Rev识别）的三维结构测定还不可行。此外，原则上，SLOR方法可能对任何NCI内部研究人员研究RNA生物学或使用基于RNA的探针进行检测，成像和治疗应用的研究有用。临床应用。RNA致病突变的发现正在产生大量新的治疗靶点，对RNA生物学和化学的不断了解为开发治疗方法提供了新的基于RNA的工具。在过去的二十年中，基于rna的检测、诊断和治疗试剂的应用和发展迅速。在这些基于RNA的试剂中，RNA适体特别有用，因为它们广泛适用于各种靶标，包括但不限于有机染料和氨基酸、抗生素、肽、各种大小、功能的蛋白质、整个细胞、病毒以及各种癌症中的特定分子标记。潜在地，使用SLOR产生的标记RNA或RNA适体可用于在单个残基的分辨率上识别或改善这些相互作用。专利申请。SLOR方法和SLOR合成器可能具有广泛的潜在商业应用，并且可以提交两个单独的专利申请。提供DNA和/或RNA和/或肽合成的公司将是SLOR方法许可的潜在被许可方；专门从事RNA合成或制造DNA或肽合成器的公司可能是SLOR RNA合成器的潜在许可方。仅在美国，这类公司的数量就至少有几十家。