Defining TLR9 regulatory sequences in natural DNA; implication for vaccine develo

定义天然 DNA 中的 TLR9 调控序列；

• 批准号: 7712519
• 负责人: LAURENT COSCOY
• 金额: $ 22.48万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7712519
• 关键词: Accounting; Adenoviruses; Affinity; Antiviral Response; Base Composition; Base Sequence; Binding; Bioinformatics; Cell Culture System; Cells; Characteristics; Cleaved cell; DNA; DNA Binding; DNA Viruses; Data; Deoxyribonucleases; Discrimination; Elements; Endosomes; Family; Frequencies; Gene Transduction Agent; Genome; Genomics; Herpesviridae; Human; Immune response; Immune system; Immunologic Receptors; Inflammation; Laboratories; Length; Lysosomes; Microfluidics; Modeling; Modification; Molecular; Mus; Nature; Nucleic Acids; Nucleic acid sequencing; Oligonucleotides; Paper; Pattern; Pattern recognition receptor; Phagocytosis; Physiological; Poxviridae; Process; Reporting; Technology; Toll-like receptors; Vaccines; Vertebral column; Viral; Virus; Work; base; design; gene therapy; immunogenicity; microbial; pathogen; phosphodiester; phosphorothioate; public health relevance; receptor; response; sugar; vector; viral DNA

DESCRIPTION (provided by applicant): The innate immune system utilizes germline-encoded pattern recognition receptors, such as Toll like receptors (TLRs), to sense molecular patterns that are common in microbial pathogens but rare or non-existent in the host. TLR9 is required for antiviral responses. Although TLR9 has been implicated in the recognition of viral DNA, it is not clear how this receptor discriminates between viral and host DNA. Until recently, TLR9 was thought to exclusively recognize unmethylated CpG motifs in DNA, a characteristic frequent in viral DNA but rare in cellular DNA. However, this model, drawn mostly from studies using DNA with modified backbone, has recently been challenged by a report indicating that natural (unmodified) DNA stimulates TLR9 regardless of the presence of CpG motifs. Moreover, recent work from the laboratory of our collaborator shows that TLR9 is enzymatically processed after synthesis to generate a truncated form that binds DNA in the endosome and lysosome. Preliminary data indicate that the full-length TLR9 (used in all the previous studies aimed at characterizing molecular interactions between TLR9 and DNA) and the physiological cleaved form of TLR9 might have distinct affinities for ODNs. Thus, the sequences that are recognized as TLR9, and in particular the physiological (truncated) form in natural DNA, remain to be determined. In this proposal, we aim to identify all the sequences (up to 8mers, over 65,000 possibilities) that are best recognized by the cleaved form of TLR9 using microfluidic technology. We will then determine which sequences stimulate or repress TLR9 activity. Finally, we will examine whether stimulatory sequences are statistically underrepresented in the genome of DNA viruses, as suggested by our preliminary data. Importantly, the identification of highly stimulatory and inhibitory motifs will be key information for the design and/or improvement of DNA-based vaccines as well as gene therapy vectors. PUBLIC HEALTH RELEVANCE: TLR9 has been implicated in the recognition of different families of viruses including herpesviruses, poxviruses and adenoviruses. This receptor is activated in the endosomes and lysosomes upon interaction with DNA. At the molecular level, TLR9 is thought to recognize nucleic acid modifications and nucleic acid sequences (in particular unmethylated CpG motifs), which are enriched in viruses but suppressed in host genomes. However, two recent reports have challenged this model. One suggests that TLR9-DNA interaction is largely sequence-independent. A second paper showed that the TLR9 form that interacts with DNA is not full length (as previously thought) but a truncated form lacking a large portion of its extracytoplasmic domain. Here, we propose to identify all sequence motifs (up to 8mers) that stimulate or inhibit the physiological (truncated) form of TLR9 and determine whether the frequency of these sequences are modulated in DNA viruses.

描述（由申请人提供）：先天免疫系统利用种系编码的模式识别受体，例如像受体（TLRS）一样，以感知在微生物病原体中常见但在宿主中罕见或不存在的分子模式。 TLR9是抗病毒反应所必需的。尽管TLR9与病毒DNA的识别有关，但尚不清楚该受体如何区分病毒和宿主DNA。直到最近，TLR9被认为仅识别DNA中未甲基化的CpG基序，这是病毒DNA中经常的特征，但在细胞DNA中很少见。但是，该模型主要来自使用带有修饰主链的DNA的研究，最近受到一份报告的挑战，表明自然（未修改）DNA刺激TLR9，无论CPG基序的存在如何。此外，我们合作者实验室的最新工作表明，在合成后，对TLR9进行了酶促处理，以生成截短的形式，该形式结合了内体和溶酶体中的DNA。初步数据表明，全长TLR9（用于表征TLR9和DNA之间的分子相互作用的所有先前研究）和TLR9的生理切割形式可能对ODN具有不同的亲和力。因此，被识别为TLR9的序列，尤其是天然DNA中的生理（截短）形式。在此提案中，我们旨在确定使用微流体技术的TLR9裂解形式最能识别的所有序列（最多8mers，超过65,000种可能性）。然后，我们将确定哪些序列刺激或抑制TLR9活性。最后，我们将检查刺激序列是否在DNA病毒的基因组中统计上的代表性不足，如我们的初步数据所示。重要的是，鉴定高度刺激和抑制基序将是设计和/或改善基于DNA的疫苗以及基因治疗载体的关键信息。公共卫生相关性：TLR9与识别不同的病毒家族有关，包括疱疹病毒，痘病毒和腺病毒。与DNA相互作用后，该受体在内体和溶酶体中被激活。在分子水平上，TLR9被认为可以识别核酸的修饰和核酸序列（尤其是未甲基化的CpG基序），这些序列富含病毒，但在宿主基因组中受到抑制。但是，最近的两份报告对该模型提出了挑战。人们认为TLR9-DNA相互作用在很大程度上是序列无关的。第二篇论文表明，与DNA相互作用的TLR9形式不是全长（如前所述），但是截短的形式缺乏其大部分外质质结构域。在这里，我们建议识别所有刺激或抑制TLR9生理（截断）形式的序列基序（最多8mers），并确定这些序列的频率是否在DNA病毒中调节。