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1. Advancing the study of protein-G4 interactions in DNA repair: Insights from biolayer interferometry

   https://doi.org/10.1016/bs.mie.2023.12.005  

   Lowran, Kaitlin; Salib, Vereena; Cismas, Emma; Wu, Colin G (January 2024, Elsevier)

   Biolayer interferometry (BLI) is a powerful tool that enables direct observations of protein-G4 interactions in real-time. In this article, we discuss the crucial aspects in conducting a BLI experiment by using the TAR DNA-binding protein (TDP43) and a G4 DNA formed by (GGGGCC)4 as a sample application. We also describe the necessary precautions in designing the DNA substrate and evaluating the signal contributions arising from nonspecific binding interactions. A comprehensive guide is included that details the necessary materials and reagents, experimental procedures, and data analysis methods for researchers who are interested in using BLI for similar studies. The insights provided in this article will allow researchers to harness the potential of BLI and unravel the complexities of protein-G4 interactions with precision and confidence. 

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2. Using Restriction Endonuclease, Protection, Selection, and Amplification to Identify Preferred DNA-Binding Sequences of Microbial Transcription Factors

   https://doi.org/10.1128/spectrum.04397-22  

   Barrows, John K.; Van Dyke, Michael W. (February 2023, Microbiology Spectrum)

   Polen, Tino (Ed.)

   ABSTRACT Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism’s genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro , iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms’ transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods. 

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3. Electrophoretic Mobility Shift Assays using Infrared-Fluorescent DNA Probes

   https://doi.org/10.17504/protocols.io.mbdc2i6  

   Van Dyke, Michael; Cox, James (December 2018, Protocols.io)

   Protein-DNA binding interactions are critical in several biological processes, especially the regulation of gene expression at the level of transcription initiation. An important technique for studying these interactions is the electrophoretic mobility shift assay (EMSA), whereby protein-DNA complexes are resolved on the basis of their mass:charge ratio using native polyacrylamide gel electrophoresis (nPAGE). Here we describe EMSA using PCR-generated, near infrared-fluorescent DNA probes, and IR fluorescence imaging to qualitatively and quantitatively study the interaction of transcriptional regulatory proteins from thermophilic organisms with different DNAs. Direct imaging of IR fluorophore-labeled DNA probes is advantageous because it provides high sensitivity (subnanomolar) without the need for intermediate staining steps or costly and problematic radiolabeled probes, thereby providing a more affordable and sensitive option to image protein-DNA on polyacrylamide gels by techniques such as EMSA. 

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4. Cholesterol Chip for the Study of Cholesterol–Protein Interactions Using SPR

   https://doi.org/10.3390/bios12100788  

   He, Peng; Faris, Shannon; Sagabala, Reddy Sudheer; Datta, Payel; Xu, Zihan; Callahan, Brian; Wang, Chunyu; Boivin, Benoit; Zhang, Fuming; Linhardt, Robert J (October 2022, Biosensors)

   Cholesterol, an important lipid in animal membranes, binds to hydrophobic pockets within many soluble proteins, transport proteins and membrane bound proteins. The study of cholesterol–protein interactions in aqueous solutions is complicated by cholesterol’s low solubility and often requires organic co-solvents or surfactant additives. We report the synthesis of a biotinylated cholesterol and immobilization of this derivative on a streptavidin chip. Surface plasmon resonance (SPR) was then used to measure the kinetics of cholesterol interaction with cholesterol-binding proteins, hedgehog protein and tyrosine phosphatase 1B. 

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5. Interactions of HMGB Proteins with the Genome and the Impact on Disease

   https://doi.org/10.3390/biom11101451  

   Voong, Calvin K.; Goodrich, James A.; Kugel, Jennifer F. (October 2021, Biomolecules)

   High Mobility Group Box (HMGB) proteins are small architectural DNA binding proteins that regulate multiple genomic processes such as DNA damage repair, nucleosome sliding, telomere homeostasis, and transcription. In doing so they control both normal cellular functions and impact a myriad of disease states, including cancers and autoimmune diseases. HMGB proteins bind to DNA and nucleosomes to modulate the local chromatin environment, which facilitates the binding of regulatory protein factors to the genome and modulates higher order chromosomal organization. Numerous studies over the years have characterized the structure and function of interactions between HMGB proteins and DNA, both biochemically and inside cells, providing valuable mechanistic insight as well as evidence these interactions influence pathological processes. This review highlights recent studies supporting the roles of HMGB1 and HMGB2 in global organization of the genome, as well as roles in transcriptional regulation and telomere maintenance via interactions with G-quadruplex structures. Moreover, emerging models for how HMGB proteins function as RNA binding proteins are presented. Nuclear HMGB proteins have broad regulatory potential to impact numerous aspects of cellular metabolism in normal and disease states. 

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