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Toll样受体（TLR）来感知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 DNA 和宿主 DNA。直到最近，TLR9 还被认为能够专门识别 DNA 中的非甲基化 CpG 基序，这是病毒 DNA 中常见的特征，但在细胞 DNA 中很少见。然而，该模型主要取自使用具有修饰主链的 DNA 的研究，最近受到一份报告的挑战，该报告表明，无论 CpG 基序是否存在，天然（未修饰）DNA 都会刺激 TLR9。此外，我们合作者实验室最近的工作表明，TLR9 在合成后经过酶促加工，生成截短形式，与内体和溶酶体中的 DNA 结合。初步数据表明，全长 TLR9（用于之前所有旨在表征 TLR9 和 DNA 之间分子相互作用的研究）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，特别是天然 DNA 中的生理（截短）形式，仍有待确定。在本提案中，我们的目标是使用微流体技术识别 TLR9 切割形式最能识别的所有序列（最多 8 聚体，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在 DNA 病毒基因组中是否在统计上代表性不足，正如我们的初步数据所表明的那样。重要的是，高度刺激和抑制基序的鉴定将是设计和/或改进基于 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 与不同病毒家族的识别有关，包括疱疹病毒、痘病毒和腺病毒。该受体在内体和溶酶体中与 DNA 相互作用后被激活。在分子水平上，TLR9被认为可以识别核酸修饰和核酸序列（特别是未甲基化的CpG基序），这些修饰和核酸序列在病毒中富集，但在宿主基因组中受到抑制。然而，最近的两份报告对这一模型提出了挑战。有人认为 TLR9-DNA 相互作用在很大程度上与序列无关。第二篇论文表明，与 DNA 相互作用的 TLR9 形式不是全长（如之前认为的），而是一种截短形式，缺乏大部分胞质外结构域。在这里，我们建议鉴定所有刺激或抑制 TLR9 生理（截短）形式的序列基序（最多 8 聚体），并确定这些序列的频率是否在 DNA 病毒中受到调节。