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

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，如Toll样受体（TLR），来感知微生物病原体中常见但宿主中罕见或不存在的分子模式。TLR 9是抗病毒反应所必需的。尽管TLR 9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，TLR 9被认为只识别DNA中未甲基化的CpG基序，这是病毒DNA中常见但在细胞DNA中罕见的特征。然而，这个模型，主要来自使用具有修饰骨架的DNA的研究，最近受到一份报告的挑战，该报告表明天然（未修饰的）DNA刺激TLR 9，而不管CpG基序的存在。此外，我们合作者实验室的最新工作表明，TLR 9在合成后被酶促加工，以产生在核内体和溶酶体中结合DNA的截短形式。初步数据表明，全长TLR 9（用于所有以前的研究，旨在表征TLR 9和DNA之间的分子相互作用）和TLR 9的生理切割形式可能具有不同的亲和力ODNs。因此，被识别为TLR 9的序列，特别是天然DNA中的生理（截短）形式，仍有待确定。在这项提案中，我们的目标是使用微流体技术识别所有最能被TLR 9切割形式识别的序列（高达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TLR 9活性。最后，我们将研究刺激序列是否在DNA病毒基因组中统计学上代表性不足，正如我们的初步数据所表明的那样。重要的是，高度刺激性和抑制性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：TLR 9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。该受体在与DNA相互作用后在核内体和溶酶体中被激活。在分子水平上，TLR 9被认为识别核酸修饰和核酸序列（特别是未甲基化的CpG基序），这些修饰和序列在病毒中富集但在宿主基因组中被抑制。然而，最近的两份报告对这一模式提出了质疑。其中一个表明TLR 9-DNA相互作用在很大程度上是序列独立的。第二篇论文表明，与DNA相互作用的TLR 9形式不是全长（如先前所认为的），而是缺乏大部分胞质外结构域的截短形式。在这里，我们建议确定所有的序列基序（高达8聚体），刺激或抑制TLR 9的生理（截短）形式，并确定这些序列的频率是否在DNA病毒调制。

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.