Homogenous NAD assay for ARDT activity measurement

Sirtuins和ADP核糖基转移酶（ARDTs）是两个具有重要医学相关功能的相关酶家族。例如，SIRT3是一种线粒体sirtuin，控制线粒体功能，因此对代谢、肥胖、2型糖尿病和代谢综合征有很强的影响。目前主要需要一种简单的检测方法来测量SIRT3的活性，以及其他sirtuins和它们的近亲——ardt。这些酶都使用烟酰胺腺嘌呤二核苷酸（NAD），并产生烟酰胺作为这些反应的副产物。不幸的是，目前用于分析这些酶活性的方法并不是最佳的，因为它们不是均匀的，不是连续的，缺乏灵敏度，并且通常不能推广到筛选所有产生烟酰胺的不同酶。一种更直接的测定方法是直接测量这些酶的均相产物。因此，直接检测烟酰胺外观的分析将有助于测量这些酶的活性，而不考虑特定的底物。利用Lucerna开发的专利传感器技术，我们将开发一种通用的sirtuin/ARDT检测方法，该方法基于一种简单、均匀的荧光传感器，对烟酰胺具有高度特异性。该I期SBIR申请描述了在SIRT3活性分析中开发、表征和优化烟酰胺传感器的概念验证实验。对于II期，我们的检测将进行优化，以符合确保该检测可以包装并用于各种sirtuins和相关酶的试剂盒格式所需的标准。

DESCRIPTION: Sirtuins and ADP ribosyltransferases (ARDTs) are two families of related enzymes that have important medically relevant functions. For example, SIRT3, a mitochondrial sirtuin, controls mitochondrial function, and thereby exerts a strong effect on metabolism, obesity, type 2 diabetes, and metabolic syndrome. There is a major need to have simple assays to measure the activity of SIRT3, as well as other sirtuins and their close relatives, the ARDTs. Each of these enzymes uses nicotinamide adenine dinucleotide (NAD), and generates nicotinamide as a byproduct of these reactions. Unfortunately, current methods for assaying the activity of these enzymes are not optimal because they are not homogeneous, are not continuous, lack sensitivity, and often not generalizable to screening all of the diverse enzymes that generate nicotinamide. A more straightforward assay would be to directly measure the products of these enzymes in a homogeneous format. Therefore, assays that directly detect the appearance of nicotinamide would be useful for measuring the activity these enzymes, regardless of the specific substrate. Using a patented sensor technology developed by Lucerna, we will develop a universal sirtuin/ARDT assay based on a simple, homogeneous fluorescence sensor that is highly specific for nicotinamide. This Phase I SBIR application describes proof-of-concept experiments for developing, characterizing, and optimizing our nicotinamide sensors in an assay for SIRT3 activity. For Phase II, our assay will be optimized to comply with standards required for ensuring that this assay can be packaged and used in a kit format for a variety of sirtuins and related enzymes.