Development of enhanced gene specific technology for the isolation of proteins binding at a single locus in vivo.

开发增强型基因特异性技术，用于分离体内单个位点结合的蛋白质。

• 批准号: BB/K013785/1
• 负责人: Kevin Ford
• 金额: $ 15.36万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK013785%2F1
• 关键词: Development; enhanced; gene; specific; technology

The identification of proteins that bind to and regulate gene expression within the cell is an important, ongoing but time consuming process. Current methodologies, such as chromatin immunoprecipitation (ChIP) require foreknowledge that a particular protein may be binding to a particular region of DNA in the cell. This protein is then used as a 'handle' to isolate all the DNA in the cell to which that protein might be binding. (This is generally achieved by using antibodies specific for the particular protein). Then an assessment of this DNA is carried out to determine whether the original DNA of interest is enriched relative to other sequences. The method can be extended somewhat by using the protein 'handle' as a means to isolate other additional proteins that might be interacting with the target protein, thus some kind of picture can be laboriously assembled as to the general protein occupancy of a given promoter. The methodology however suffers from several flaws:- 1) one needs to identify a specific protein as a starting point, 2) one needs to have access to high quality antibodies specific for the target protein, 3) such antibodies differ in their affinity/specificity between individual batches and between protein targets, leading to potentially large variations in results between experiments, 4) ALL the DNA regions that are bound by the target protein throughout the cell will be isolated, along with a majority of the other proteins that it interacts with at these other DNA loci. Thus identifying DNA region specific binding proteins via follow on experiments such as mass spectrometry is impossible.One clever approach to circumvent these problems has been to use small DNA molecules (oligonucleotides) complementary to the DNA region of interest. These have a single chemical handle attached (des-thiobiotin), so that when these oligonucleotides bind to their target DNA sequence they can be retrieved from the cell, along with those proteins that have remained bound to the target DNA. So far this method has only been used to examine proteins bound to DNA that is repetitive, thus artificially boosting the sensitivity of the approach. Also, one of the key steps in this method is denaturing (melting) the cell's DNA to allow the oligonucleotide to bind it. This represents a possible downside, in that some of the DNA:protein interactions we were hoping to capture may be lost during the process. We propose to develop a method that circumvents these issues, delivering a reagent that will have applications for scientists throughout the world, in identifying in one go, all of the proteins that are associated with a specific, individual gene. Our approach is to use zinc finger proteins (or similar acting TALE proteins), that can be designed to bind virtually any DNA sequence, linked to multiple small tags (biotin acceptor peptides) that become modified in vivo, and that will provide an incredibly strong 'handle' for DNA isolation. We suggest that this approach, which does not require any DNA denaturation and can have multiple 'handles' to increase sensitivity, will provide a versatile and highly sensitive route for the isolation of individual DNA regions throughout the genome and the proteins that bind them. Additional routes will involve enhancing the 'handle' efficacy of these enzymes within the cell via increasing their degree of modification in vivo. The ultimate aim will be to enable scientists to rapidly and confidently build up a picture of events in the cell as they happen at specific regulatory regions of DNA, during processes such as cell division and differentiation, stem cell renewal and disease onset and progression.

识别与细胞内基因表达结合并调节基因表达的蛋白质是一个重要的、持续的但耗时的过程。目前的方法，如染色质免疫沉淀（ChIP），需要预先知道一个特定的蛋白质可能会结合到细胞中的DNA的特定区域。然后，这种蛋白质被用作“把手”，以分离细胞中可能与该蛋白质结合的所有DNA。(This通常通过使用对特定蛋白质具有特异性的抗体来实现）。然后对该DNA进行评估，以确定感兴趣的原始DNA相对于其他序列是否富集。该方法可以通过使用蛋白质“手柄”作为分离可能与靶蛋白相互作用的其他额外蛋白质的手段而有所扩展，因此可以费力地组装关于给定启动子的一般蛋白质占有率的某种图片。然而，该方法存在几个缺陷：1）需要鉴定特定的蛋白质作为起点，2）需要获得对靶蛋白质特异的高质量抗体，3）这些抗体在单个批次之间和蛋白质靶标之间的亲和力/特异性不同，导致实验之间结果的潜在大变化，4）在整个细胞中被靶蛋白质结合的所有DNA区域将被分离，沿着与其在这些其他DNA基因座处相互作用的大多数其他蛋白质。因此，通过后续实验如质谱法来鉴定DNA区域特异性结合蛋白是不可能的。一种巧妙的方法来规避这些问题，即使用与感兴趣的DNA区域互补的小DNA分子（寡核苷酸）。这些具有连接的单个化学手柄（脱硫生物素），因此当这些寡核苷酸与其靶DNA序列结合时，它们可以从细胞中取回，沿着那些仍然与靶DNA结合的蛋白质。到目前为止，这种方法仅用于检查与重复DNA结合的蛋白质，从而人为地提高了该方法的灵敏度。此外，该方法的关键步骤之一是使细胞的DNA变性（熔化）以允许寡核苷酸结合它。这代表了一个可能的缺点，因为我们希望捕获的一些DNA：蛋白质相互作用可能在该过程中丢失。我们建议开发一种方法来规避这些问题，提供一种试剂，将有应用程序的科学家在世界各地，在一个去识别，所有的蛋白质与特定的，个别的基因。我们的方法是使用锌指蛋白（或类似作用的TALE蛋白），其可以被设计为结合几乎任何DNA序列，连接到在体内被修饰的多个小标签（生物素受体肽），并且将为DNA分离提供难以置信的强大“手柄”。我们认为，这种方法，它不需要任何DNA变性，可以有多个“手柄”，以增加灵敏度，将提供一个通用的和高度敏感的路线，分离整个基因组中的单个DNA区域和结合它们的蛋白质。另外的途径将涉及通过增加这些酶在体内的修饰程度来增强它们在细胞内的“处理”功效。最终目标是使科学家能够快速而自信地建立细胞中事件的图像，因为它们发生在DNA的特定调控区域，在细胞分裂和分化，干细胞更新以及疾病发作和进展等过程中。

项目成果

Triggering of Toll-like Receptors in Old Individuals. Relevance for Vaccination.

老年人中 Toll 样受体的触发。

• DOI: 10.2174/1381612825666191111155800
• 发表时间: 2019
• 期刊: Current pharmaceutical design
• 影响因子: 3.1
• 作者: Zareian N

Analysis of T and NK cell subsets in the Sicilian population from young to supercentenarian: The role of age and gender.

• DOI: 10.1111/cei.13606
• 发表时间: 2021-08
• 期刊: Clinical and experimental immunology
• 影响因子: 4.6
• 作者: Ligotti ME;Aiello A;Accardi G;Aprile S;Bonura F;Bulati M;Gervasi F;Giammanco GM;Pojero F;Zareian N;Caruso C;Farzaneh F;Candore G
• 通讯作者: Candore G

Glyco-engineered anti-EGFR mAb elicits ADCC by NK cells from colorectal cancer patients irrespective of chemotherapy.

• DOI: 10.1038/bjc.2014.35
• 发表时间: 2014-03-04
• 期刊: BRITISH JOURNAL OF CANCER
• 影响因子: 8.8
• 作者: Oppenheim, D. E.;Spreafico, R.;Etuk, A.;Malone, D.;Amofah, E.;Pena-Murillo, C.;Murray, T.;McLaughlin, L.;Choi, B. S.;Allan, S.;Belousov, A.;Passioukov, A.;Gerdes, C.;Umana, P.;Farzaneh, F.;Ross, P.
• 通讯作者: Ross, P.

Regeneration linked miRNA modify tumor phenotype and can enforce multi-lineage growth arrest in vivo.

• DOI: 10.1038/s41598-021-90009-9
• 发表时间: 2021-05-18
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Salehi S;Tavabie OD;Villanueva A;Watson J;Darling D;Quaglia A;Farzaneh F;Aluvihare VR
• 通讯作者: Aluvihare VR

Efficient Ex Vivo Expansion of ?d T-Cells from AML Patients Requires Elimination of Circulating Leukemic Blasts

AML 患者体内 ?d T 细胞的高效体外扩增需要消除循环白血病母细胞

• DOI: 10.36959/486/327
• 发表时间: 2020
• 期刊: Advances in Leukemia Research and Treatment
• 作者: Ana C P