NEW YORK – Swedish life science firm Atlas Antibodies has developed a new kit for in situ measurements of protein-protein interactions, offering advancements on proximity ligation assay (PLA) technology.

纽约-瑞典生命科学公司Atlas Antibodies开发了一种用于原位测量蛋白质-蛋白质相互作用的新试剂盒，为邻近连接测定（PLA）技术提供了进步。

Called MolBoolean, the product allows researchers to distinguish between target proteins present in a sample bound together in a complex and those same proteins present as free, unbound molecules.

该产品被称为MolBoolean，研究人员可以区分复合物中结合在一起的样品中的目标蛋白质和作为游离未结合分子存在的那些蛋白质。

According to the company, the method is seeing uptake in areas like drug discovery and development and for research into conditions including Parkinson's disease.

据该公司称，该方法在药物发现和开发等领域以及帕金森病等疾病的研究中得到了应用。

Mikael Malmqvist, principal scientist at Atlas Antibodies, said that the MolBoolean assay emerged from a strategy the company embarked on in recent years to develop and offer immunoassay products leveraging its antibody catalog. Stockholm-based Atlas was founded in 2006 to commercialize antibodies used in the Human Protein Atlas project.

。斯德哥尔摩Atlas成立于2006年，旨在将人类蛋白质图谱项目中使用的抗体商业化。

The company currently offers more than 22,000 antibodies for research applications..

该公司目前提供22000多种用于研究应用的抗体。。

Several years ago, Atlas was approached by Ola Söderberg, the inventor of the MolBoolean technology and a professor in the department of pharmaceutical biosciences at Uppsala University. 'Professor Söderberg asked if we wanted to reserve the rights to patent the technology and develop it into a kit product, and we thought it was a great add-on to our existing catalog,' Malmqvist said.

几年前，MolBoolean技术的发明者、乌普萨拉大学制药生物科学系教授OlaSöderberg接触了Atlas马尔姆奎斯特（Malmqvist）说，Söderberg教授问我们是否想保留这项技术的专利权，并将其开发成套件产品，我们认为这是对现有目录的一个很好的补充。

Atlas secured the rights to the technology in 2019..

Atlas于2019年获得了该技术的权利。。

MolBoolean is based on the PLA technology developed almost 20 years ago by Uppsala scientists including Söderberg. PLA uses pairs of antibodies attached to unique DNA sequences to detect proteins of interest. When the antibodies bind to their targets, the attached DNA strands are brought into proximity and ligated, forming a new template that can then be amplified by rolling circle amplification.

MolBoolean基于近20年前乌普萨拉（Uppsala）科学家（包括Söderberg）开发的PLA技术。PLA使用附着在独特DNA序列上的成对抗体来检测感兴趣的蛋白质。当抗体与其靶标结合时，附着的DNA链被接近并连接，形成新的模板，然后可以通过滚环扩增进行扩增。

Readout of the new template can be done via PCR or next-generation sequencing or, in the case of in situ assays like MolBoolean, by hybridizing fluorescence-tagged oligos..

新模板的读数可以通过PCR或下一代测序完成，或者在原位检测（如MolBoolean）的情况下，通过杂交荧光标记的寡核苷酸来完成。。

In situ PLA assays have long been used to observe protein-protein interactions in cells and tissue samples, with Swedish life science firm Navinci offering a commercial version of the technology for spatial proteomics research. MolBoolean advances on traditional in situ PLA in that it allows researchers to simultaneously measure the amount of two target proteins bound together in a complex and the amount of these target proteins existing as free, unbound protein in the sample..

长期以来，原位PLA分析一直用于观察细胞和组织样品中的蛋白质-蛋白质相互作用，瑞典生命科学公司Navinci为空间蛋白质组学研究提供了该技术的商业版本。MolBoolean对传统原位PLA的改进在于，它允许研究人员同时测量复合物中结合在一起的两种靶蛋白的量以及样品中作为游离未结合蛋白存在的这些靶蛋白的量。。

In the MolBoolean assay, when two target proteins are bound together in a complex, the antibodies targeting them will be brought into close proximity, enabling the production of a specific oligo sequence via rolling circle amplification. In cases where the proteins are detected separately, the rolling circle amplification will produce a different oligo product, allowing researchers to distinguish between levels of bound and free protein.

在摩尔布尔分析中，当两个靶蛋白在复合物中结合在一起时，靶向它们的抗体将被紧密接近，从而能够通过滚环扩增产生特定的寡核苷酸序列。在分别检测蛋白质的情况下，滚环扩增将产生不同的寡核苷酸产物，使研究人员能够区分结合蛋白和游离蛋白的水平。

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Researchers use their own primary antibodies to their protein targets, with Atlas supplying the secondary antibodies conjugated to oligos that bind those primary antibodies to produce the MolBoolean signal. Malmqvist said that to effectively distinguish between free and bound proteins, users need a fluorescence microscopy system capable of spatial resolution of at least 100 nanometers..

研究人员使用他们自己的一抗来靶向蛋白质，Atlas提供与寡核苷酸结合的二抗，寡核苷酸结合这些一抗产生摩尔布尔信号。Malmqvist说，为了有效区分游离蛋白和结合蛋白，用户需要一种空间分辨率至少为100纳米的荧光显微镜系统。。

The ability to measure both bound and free protein allows researchers to normalize the number of protein-protein interactions to the total amounts of the target proteins in a sample. This can help prevent misinterpretation of protein interaction data in situations where, for instance, an increase in interaction signal stems from higher expression of the target proteins as opposed to increased interaction rates, Malmqvist said..

测量结合蛋白和游离蛋白的能力使研究人员能够将蛋白质-蛋白质相互作用的数量标准化为样品中目标蛋白的总量。Malmqvist说，这有助于防止对蛋白质相互作用数据的误解，例如，相互作用信号的增加源于目标蛋白质的高表达，而不是相互作用率的增加。。

He said that Atlas has received substantial interest from pharma companies in the targeted protein degradation space, in which small molecule agents are used to connect E3 ubiquitin ligases involved in the degradation and elimination of nonnative misfolded proteins to protein targets involved in disease.

他说，Atlas在靶向蛋白质降解领域引起了制药公司的极大兴趣，在该领域中，小分子试剂用于将参与降解和消除非天然错误折叠蛋白质的E3泛素连接酶连接到与疾病有关的蛋白质靶标。

In this case, MolBoolean can be used to measure levels of interaction between E3 ligases and protein targets following drug treatment while normalizing against the amount of free E3 ligase and target protein present..

在这种情况下，MolBoolean可用于测量药物治疗后E3连接酶与蛋白质靶标之间的相互作用水平，同时针对存在的游离E3连接酶和靶蛋白的量进行标准化。。

Thomas Sakmar, a professor at the Rockefeller University, has been using MolBoolean for his research into G protein coupled receptors (GPCRs). In a recent study in Science Advances, he and his colleagues used the approach to confirm in vivo interaction of GPCRs and receptor activity-modify proteins (RAMPs) — which can impact the function of GPCRs — that were found to bind in a large-scale in vitro screening assay..

洛克菲勒大学（RockefellerUniversity）教授托马斯·萨马尔（ThomasSakmar）一直在使用摩尔布尔（MolBoolean）研究G蛋白偶联受体（GPCR）。在《科学进步》最近的一项研究中，他和他的同事使用这种方法来确认GPCR和受体活性修饰蛋白（RAMPs）的体内相互作用-这可能会影响GPCR的功能-这些蛋白在大规模体外筛选试验中被发现结合。。

Sakmar noted that while the in vitro screen provided evidence that the observed GPCR-RAMP pairs could bind, its didn't conclusively demonstrate that these complexes actually formed in cells.

Sakmar指出，虽然体外筛选提供了观察到的GPCR-RAMP对可以结合的证据，但它并没有最终证明这些复合物实际上在细胞中形成。

'We looked for a long time for methods that would show that the complexes actually existed in the cell membrane,' he said. Upon coming across the paper describing the MolBoolean technique, which Söderberg and his colleagues published in Nature Communications in 2022, Sakmar reached out to Atlas, with which his lab had a preexisting relationship, and began working with the method..

他说：“我们花了很长时间寻找能够证明复合物实际上存在于细胞膜中的方法。”。在看到Söderberg及其同事于2022年在《自然通讯》（Nature Communications）上发表的描述MolBoolean技术的论文后，Sakmar联系了Atlas，他的实验室与Atlas有着预先存在的关系，并开始使用该方法。。

The MolBoolean work was 'extremely important' to the Science Advances work as it allowed the researchers to validate the in vitro screening approach, Sakmar said.

萨马尔说，摩尔布尔工作对科学进步工作“极其重要”，因为它使研究人员能够验证体外筛选方法。

'The cool thing about MolBoolean is that it is basically a colocalization assay where you have very good quantitative readouts for each component, and you can use that information to calculate an apparent affinity constant for complexes,' he said. 'That's something you can't do with [older] strategies where you just say 'yes' or 'no' about a complex.'.

他说：“MolBoolean的酷之处在于，它基本上是一种共定位测定法，你可以对每种成分都有很好的定量读数，你可以利用这些信息来计算复合物的表观亲和力常数。”这是[旧的]策略无法做到的，你只能对一个复杂的事物说“是”或“否”。

Sakmar said the spatial information provided by the technique also offers insights, citing his work on GPCR-RAMP interactions.

Sakmar说，该技术提供的空间信息也提供了见解，并引用了他在GPCR-RAMP相互作用方面的工作。

'Say the [GPCR] needs a RAMP to get to the cell surface, but if the RAMP is not there or is defective, then the [GPCR] is still going to get synthesized, but it will get stuck in the endoplasmic reticulum, for example,' he said. 'So, knowing the localization of the [GPCR] is interesting.'

他说，“（GPCR）需要一个斜坡才能到达细胞表面，但如果斜坡不存在或有缺陷，那么（GPCR）仍然会被合成，但它会卡在内质网中。”因此，了解（GPCR）的本地化很有趣。”

He suggested the approach could be useful across a range of applications including, for instance, observing changes in protein interactions in response to cell stress or other perturbations.

他建议该方法在一系列应用中都很有用，例如，观察蛋白质相互作用对细胞应激或其他扰动的反应。

Sakmar said his lab is also interested in devising ways to scale up the MolBoolean approach, perhaps by converting it to a flow cytometry-based assay.

萨马尔说，他的实验室也有兴趣设计扩大摩尔布尔方法的方法，也许可以将其转换为基于流式细胞术的检测方法。