New Bioanalytical Methods Based on Next Generation Sequencing

描述（由申请人提供）：生物分子的检测和定量在生物医学研究和临床测定中至关重要。我们的目标是开发和应用新的生物分析方法，该方法将利用通常保留用于核酸分析的下一代测序（NGS）的能力，用于发现靶特异性多肽配体并高度平行地分析它们的相互作用。我们的假设是，通过在核糖体展示（RD）条件下的体外翻译，在肽序列和它们的编码RNA之间建立直接连接，定量分析数百万个肽序列的结合。 在单个实验中的肽将通过NGS实现。下一代测序（NGS）彻底改变了核酸分析。单个实验中的NGS分析可以提供关于样品中数百万个特定核酸序列的身份和相对丰度的信息。我们建议，NGS分析的非凡力量可以利用结合核糖体展示（RD）与NGS分析的多肽相互作用的分析。在RD中，编码肽序列的RNA在肽产物及其相应的RNA保持与核糖体结合的条件下体外翻译，有效地用独特的RNA序列标签标记每个肽，从而能够应用NGS分析。我们设想两个主要的应用，这种NGS增强RD（NGSERD）的方法。第一个是将其用作配体发现工具（目标1）。该NGSERD应用的一个特别的优势和独特的特征将是将计算工具应用于NGS数据的可能性，以使得能够发现复杂靶标的配体，其中识别所需靶标的真实配体必须从过量的假配体或非特异性配体中分选出来。NGSERD的第二个应用（目的2）是将其用作一种新型的高度平行的结合试验，其中NGS分析将提供独特的读数，其中RNA序列标签将识别配体，序列的读取计数将提供超灵敏的定量信号报告配体结合，从而能够在单个实验中分析大量（高达数百万）肽试剂。一旦NGSERD方法在目标1&2中被开发出来，我们将把它应用于人血清中疾病相关抗体的分析。识别、检测、特征分析 阻断抗体在自身免疫性疾病、感染性疾病、癌症和疫苗开发中具有巨大的研究、临床和治疗价值。为了建立基于NGSERD的抗体分析的实际应用的范例，我们将其应用于系统性红斑狼疮（SLE），一种自身免疫性疾病，其中产生的抗体对自身抗原负责的疾病的病理。该项目的影响在于NGSERD方法在研究、疾病诊断和预后、疾病早期检测、治疗剂设计和疫苗开发中的众多令人兴奋的应用。

DESCRIPTION (provided by applicant): Detection and quantitation of biomolecules is of central importance in biomedical research and in clinical assays. Our goal is to develop and apply novel bioanalytical methodology that will utilize the power of Next Generation Sequencing (NGS) that is normally reserved for analysis of nucleic acids, for discovery of target-specific polypeptide ligands and for highly parallel analysis of their interactions. Our hypothesis is that y establishing a direct link between peptide sequences and their coding RNA through in vitro translation under ribosome display (RD) conditions, quantitative analysis of binding of millions of peptides in a single experiment will be enabled by NGS. Next Generation Sequencing (NGS) has revolutionized analysis of nucleic acids. NGS analysis in a single experiment can provide information on identity and relative abundance of millions of specific nucleic acid sequences in a sample. We propose that the extraordinary power of NGS analysis can be harnessed for analysis of polypeptide interactions by combining ribosome display (RD) with NGS analysis. In RD, RNA encoding peptide sequences is translated in vitro under conditions where peptide products and its corresponding RNA's remain associated with the ribosomes effectively labeling each peptide with a unique RNA sequence tag enabling application of NGS analysis. We envision two major applications for this NGS-enhanced RD (NGSERD) approach. The first will be to use it as a ligand discovery tool (aim 1). A particular strength and unique feature of this NGSERD application will be the possibility of applying computational tools to NGS data to enable discovery of ligands for complex targets where authentic ligands recognizing desired target have to be sorted out from excess of spurious or nonspecific ligands. The second application for NGSERD (aim 2) will be to use it as a novel highly parallel binding assay in which NGS analysis will provide a unique readout where the RNA sequence tag will identify the ligand and the read count for the sequence will provide supersensitive quantitative signal reporting ligand binding enabling analysis of a large number (up to millions) of peptide reagents in a single experiment. Once the NGSERD methodology is developed in aims 1&2, we will apply it for the analysis of disease-related antibodies in human serum. Identification, detection, profiling and blocking antibodies has tremendous research, clinical and therapeutic values in autoimmune and infectious diseases, in cancer and in vaccine development. To establish a paradigm for practical applications of NGSERD-based antibody analysis, we will apply it to systemic lupus erythematosus (SLE), an autoimmune disease in which the antibodies produced to self-antigens are responsible for the pathology of the disease. The impact of this project lies in multitudes of exciting applications of NGSERD approach in research, in disease diagnosis and prognosis, in early detection of the disease, in design of therapeutic agents, and in vaccine development.