Using nanobodies to increase the sensitivity and resolution of chromatin profiling through uliCUT&RUN

项目总结/摘要 DNA结合蛋白在所有DNA模板化过程中起着关键作用，如转录，剪接， 复制和DNA修复。DNA结合蛋白包括转录因子，其优先结合某些DNA结合蛋白。 DNA序列和组蛋白质，形成核小体的核心。重要的是， 在细胞类型中，不能仅通过DNA序列来预测因子或组蛋白。因此，蛋白质谱 技术用于鉴定功能性结合的细胞特异性特征。 DNA结合蛋白的重要性促使了实验性和 分析方法，以更好地识别和表征这些相互作用。通过ChIP-seq进行全基因组分析 这是一种广泛使用的技术，已协助表征无数染色质结合蛋白。 然而，该技术在表征具有小细胞的样品中的因子占用的能力方面是有限的。 数量以及特异性和稳健抗体的可用性。这些局限性使得 开发ChIP-seq的补充方法和扩展，以提供更完整的生物学分析。 细胞中的过程。最近，我们优化了CUT&RUN，一种新的本地化方法， 在极低的细胞群中占据，低至单细胞和单个小鼠胚泡胚胎 （称为uliCUT&RUN）。这一技术进步为分析因子占用率提供了机会， 罕见的细胞群体，如患者活检。此外，它允许测试细胞异质性， 在细胞群体中。然而，该技术的实际限制仍然包括抗体开发和 效率 骆驼科动物单链VHH抗体或纳米抗体（Nbs）是引人注目的新型抗体 其特征在于特别高的溶解度和热稳定性。我们最近开发了一个强大的管道 用于发现和表征高质量的抗原特异性Nb库。这条管道已经 针对十几种具有不同结构和免疫反应的抗原进行了广泛的测试和优化。与 通过这种方法，可以鉴定大量高质量构象Nb结合剂。 在这里，我们建议将我们在Nb开发方面的专业知识与uliCUT&RUN结合起来开发纳米抗体 针对低细胞群体的特异性CUT&RUN，并将该技术应用于单细胞和稀有细胞群体。 铌基uliCUT&RUN的开发和应用将广泛应用于社会，我们正在 鉴于我们对CUT&RUN的优化，我们已经准备好开发这项技术，这是第一次单细胞转录 我们在铌开发新领域的专业知识已经完成。此外，结果 将这项技术应用于样本将继续促进我们对正常细胞生物学的理解， 还提供了重要的信息，将有利于努力确定的原因和后果的异常 与疾病相关的细胞状态。

PROJECT SUMMARY/ABSTRACT DNA-binding proteins play crucial roles in all DNA templated processes, such as transcription, splicing, replication, and DNA repair. DNA binding proteins include transcription factors that bind preferentially to certain DNA sequences, and histone proteins that form the core of nucleosomes. Importantly, genomic location of factors or histone proteins cannot be predicted in cell types by DNA sequence alone. Therefore, protein profiling technologies are used to identify cell specific characteristics of functional binding. The importance of DNA-binding proteins has motivated the continued development of experimental and analytical methods to better identify and characterize these interactions. Genome-wide profiling by ChIP-seq is a widely-used technique that has assisted in the characterization of countless chromatin binding proteins. However, this technique is limited in its ability to characterize factor occupancy in samples with small cell numbers and by the availability of specific and robust antibodies. These limitations have necessitated the development of complementary methods and extensions of ChIP-seq to provide a more complete of biological processes in the cell. Very recently, we optimized CUT&RUN, a new localization method, to profile factor occupancy in extremely low cell populations, down to single cells and individual mouse blastocyst embryos (termed uliCUT&RUN). This technical advancement has opened the opportunity to profile factor occupancy in rare cell populations, such as patient biopsies. Furthermore, it permits for testing cell heterogeneity that occurs in cell populations. However, practical limitations of this technology still include antibody development and efficiency. Camelid single-chain VHH antibodies or Nanobodies (Nbs) are a compelling new class of antibodies characterized by exceptionally high solubility and thermostability. We have recently developed a robust pipeline for the discovery and characterization of high-quality antigen-specific Nb repertoires. This pipeline has been extensively tested and optimized for a dozen of antigens with different structures and immune responses. With this approach, a large cohort of high-quality conformational Nb binders can be identified. Here we propose to couple our expertise on Nb development and uliCUT&RUN to develop nanobody specific CUT&RUN for low cell populations and apply this technology to single cells and rare cell populations. The development and application of Nb-based uliCUT&RUN will be of wide use to the community and we are well poised to develop this technology given our optimization of CUT&RUN is the first time single cell transcription factor profiling has been accomplished and our expertise in the new field of Nb development. Further, results from applying this technology to samples will continue to further our understanding of normal cell biology, but also provide crucial information that will benefit efforts to determine the causes and consequences of abnormal cellular states that are associated with disease.