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

描述(申请人提供)：先天性免疫系统利用胚线编码的模式识别受体，如Toll样受体(TLRs)，来检测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。抗病毒反应需要TLR9。尽管TLR9与病毒DNA的识别有关，但该受体如何区分病毒和宿主DNA尚不清楚。直到最近，TLR9还被认为只能识别DNA中的未甲基化的CpG基序，这一特征在病毒DNA中很常见，但在细胞DNA中很少见。然而，这一模型主要是从使用带有修饰骨架的DNA的研究中得出的，最近受到了一份报告的挑战，该报告表明，无论是否存在CpG基序，天然(未修饰的)DNA都能刺激TLR9。此外，我们合作者实验室最近的工作表明，TLR9在合成后经过酶处理，产生一种截断形式，与内体和溶酶体中的DNA结合。初步数据表明，全长TLR9(以前所有研究都是为了研究TLR9与DNA之间的分子相互作用)和TLR9的生理切割形式可能与ODN有明显的亲和力。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。在这项提案中，我们的目标是利用微流控技术识别TLR9裂解形式最能识别的所有序列(多达8个聚体，超过65,000个可能性)。然后我们将确定哪些序列刺激或抑制TLR9的活性。最后，正如我们的初步数据所表明的，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不足。重要的是，高度刺激和抑制基序的识别将是设计和/或改进基于DNA的疫苗和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。当与DNA相互作用时，这种受体在内小体和溶酶体中被激活。在分子水平上，TLR9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丰富，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挑战。一种认为TLR9与DNA的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形式并不是全长的(如之前所认为的)，而是一种截短的形式，缺少其胞外结构域的很大一部分。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8个)，并确定这些序列的频率是否在DNA病毒中受到调节。

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.