That's right, conducting single-molecule research does not require protein purification!

In a recent paper published in Nuclear Acids Research, Professor Bennett Van Houten from the University of Pittsburgh School of Medicine in the United States developed a revolutionary new method that combines C-Trap dynamic single-molecule technology with nuclear protein extraction technology, without the need for complex protein purification and fluorescence labeling, to directly study the dynamic interaction process between proteins and nucleic acids in nuclear extracts.

 

 

Single molecule technology, relying on its powerful manipulation and imaging performance, has helped scientists achieve success in multiple research fields. The research data obtained has been repeatedly published in high-level journals; Especially in the field of protein-DNA interaction research, it plays a decisive role in multiple dynamic biological processes such as DNA damage repair, DNA replication, DNA transcription and chromatin processing, and epigenetics in multiple cellular metabolic processes. Single molecule technology can directly obtain quantitative data related to protein DNA binding, including binding duration (Koff), number of binding times per unit time (K on), so as to calculate binding strength Kd value, specific localization of binding, direction and speed of movement on DNA, and other biological function related data.

 

However, when scientists use single-molecule technology for research, the methods commonly used are mainly divided into two categories: relying on purified proteins under limited conditions and single-molecule technology under live cells. Single molecule techniques that rely on protein purification and fluorescence labeling are often affected by low yields and impaired activity, while single molecule techniques in living cells are mostly affected by temporal spatial resolution. Therefore, there is an urgent need for a single molecule technique to address the shortcomings of these two aspects, obtain more information on protein DNA interactions under physiological conditions, and uncover the molecular mechanisms under the most realistic conditions.

 

The method SMADNE (Single Molecular Analysis of DNA binding proteins from Nuclear Extracts) developed by Professor Bennett Van Houten's research group can directly detect the interactions between several types of proteins in the nucleus and DNA, reducing the threshold of single-molecule technology. Scientists studying DNA binding proteins can also easily obtain the functions and mechanisms of these protein molecules.

 

 

Figure 1. SMADNE experimental process and output data (A) SMADNE experimental process: (B) Dynamic single-molecule experimental protocol and 2D and 1D experimental graphs, showing the binding of YFP-PARP1 to single chain damage protein; (C) Obtain four quantifiable data through SMADNE.

In this paper, the author presents a detailed workflow of SMADNE, demonstrating that binding quantification information can be obtained through the construction of DNA tethered structures, mechanical regulation, 2D imaging, and 1D scanning, as shown in Figure 1. Specifically, scientists constructed three different substrate models for DNA damage: UV damage, oxidative damage, and single strand break damage. Using commercial nuclear protein extraction kits, nuclear proteins were extracted from transfected cells, including the fluorescent labeled DNA damage protein eGFP-DDB1, mNeonGreen-DDB2，eGFP-XPC，YFP-PARP1，tGFP-Polb，tGFP-APE1。 These proteins specifically bind to DNA of different types of damage, and information on protein DNA interactions was obtained through 2D fluorescence imaging and 1D Kymograph data.

 

 

Figure 2. Binding behavior of dual color labeled DDB protein to UV damaged DNA in the SMADNE method.

 

In addition, the multi-color fluorescence imaging function in the C-Trap dynamic single-molecule system helps scientists simultaneously detect the binding of multiple protein molecules at the same site, and through co localization analysis, obtain the binding and dissociation behavior of multiple protein molecules that interact to form complexes at the same site. In the article, the author compared the binding behavior of dimers formed by DDB1 and DDB2 with UV damaged DNA, as shown in Figure 2. The co localization binding behavior of protein molecule complexes was divided into 11 types. Finally, through statistical analysis of a large amount of data, it was confirmed that DDB1 and DDB2 simultaneously reach the damage site and dissociate from DNA at the same time is the most common binding behavior.

 

 

Figure 3. Real time binding behavior between proteins and damaged DNA under mechanical regulation in the SMADNE method.

 

The unique mechanical manipulation and measurement characteristics of the C-Trap system regulate the application of force to individual DNA molecules, detecting the binding behavior of protein molecules with damaged DNA under different DNA tensions. The author used single stranded DNA as a model in the article to study the binding behavior of PARP1 protein with YFP tag and its mechanical regulation, as shown in Figure 3. The author found that when the force applied to DNA increased to 30pN, the binding sites of PARP1 protein on DNA significantly increased, indicating that the regulation of force increased the damage sites on DNA and facilitated the binding of PARP1 protein.

 

The single-molecule method provided in the article helps scientists study the molecular mechanisms of DNA damage, DNA replication, and DNA transcription by extracting DNA binding proteins from cell nuclei. These DNA metabolic behaviors are crucial for maintaining genomic stability, which is a crucial discovery in the field of molecular biology and can lead to new discoveries and innovative treatment plans for a range of human diseases, including cancer.

 

As the author stated, combining the LUMICKS C-Trap system for single-molecule analysis of DNA binding proteins in nuclear extracts is a novel, scalable, and relatively high-throughput method that can obtain key protein-DNA interaction single-molecule mechanisms in environments similar to mammalian nuclei.