In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

A PilZ domain protein interacts with the transcriptional regulator HinK to regulate type VI secretion system in Pseudomonas aeruginosa.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.

Experimental Validation of RNA-RNA Interactions by Electrophoretic Mobility Shift Assay.

Regulatory RNAs in bacteria are known to act by base pairing with other RNAs. Interactions between two partner RNAs can be investigated by electrophoretic mobility shift assays. The regions predicted to be engaged in base pairing are analyzed by introducing mutations in one RNA that prevent RNA-RNA complex formation. Next, base pairing is restored by introducing complementary mutations in its partner RNA. Here, we describe the mutational strategy and experimental methods used to validate specific base pairing between two RNA species.

Complexes and Supramolecular Associates of Dodecyl-Containing Oligonucleotides with Serum Albumin.

Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that the interaction of human serum albumin with the duplex of complementary dodecyl-containing oligonucleotides resulted in the formation of circular associates with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and interaction with polydeoxyadenylic acid and dodecyl-containing oligothymidylate resulted in formation of supramolecular associates with the size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm, respectively. The obtained data allow considering the dodecyl-containing oligonucleotides and albumin as potential components of the designed self-assembling systems for solving problems of molecular biology, biomedicine, and development of unique theranostics with targeted action.

Identification of tau-tubulin kinase 1 inhibitors by microfluidics-based mobility shift assay from a kinase inhibitor library.

Tau tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that phosphorylates multiple residues in tau protein. Hyperphosphorylated tau is the main cause of tauopathy, such as Alzheimer's disease (AD). Therefore, preventing tau phosphorylation by inhibiting TTBK1 has been proposed as a therapeutic strategy for AD. However, few substrates of TTBK1 are reported for a biochemical assay and few inhibitors targeting TTBK1 have been reported so far. In this study, we identified a fluorescein amidite (FAM)-labeled peptide 15 from a small peptide library as the optimal peptide substrate for human TTBK1 (hTTBK1). We then developed and validated a microfluidics-based mobility shift assay (MMSA) with peptide 15. We further confirmed that peptide 15 could also be used in the ADP-Glo kinase assay. The established MMSA was applied for screening of a 427-compound kinase inhibitor library, yielding five compounds with IC50s of several micro molars against hTTBK1. Among them, three compounds, AZD5363, A-674,563 and GSK690693 inhibited hTTBK1 in an ATP competitive manner and molecular docking simulations revealed that they enter the ATP pocket and form one or two hydrogen bonds to the hinge region with hTTBK1. Another hit compound, piceatannol, showed non-ATP competitive inhibitory effect on hTTBK1 and may serve as a starting point to develop highly selective hTTBK1 inhibitors. Altogether, this study provided a new in vitro platform for the development of novel hTTBK1 inhibitors that might have potential applications in AD prevention.

The Interactome of Protein, DNA, and RNA.

Proteins participate in many processes of the organism and are very important for maintaining the health of the organism. However, proteins cannot function independently in the body. They must interact with proteins, DNA, RNA, and other substances to perform biological functions and maintain the body's health. At present, there are many experimental methods and software tools that can detect and predict the interaction between proteins and other substances. There are also many databases that record the interaction between proteins and other substances. This article mainly describes protein-protein, protein-DNA, and protein-RNA interactions in detail by introducing some commonly used experimental methods, the software tools produced with the accumulation of experimental data and the rapid development of machine learning, and the related databases that record the relationship between proteins and some substances. By this review, we hope that through the analysis and summary of various aspects, it will be convenient for researchers to conduct further research on protein interactions.

Uridylation of the histone mRNA stem-loop weakens binding interactions with SLBP while maintaining interactions with 3'hExo.

Histone mRNA degradation is controlled by the unique 3' stem-loop of histone mRNA and the stem-loop binding protein (SLBP). As part of this process, the 3' stem-loop is trimmed by the histone-specific 3' exonuclease (3'hExo) and uridylated by the terminal uridylyl transferase 7 (TUT7), creating partially degraded intermediates with short uridylations. The role of these uridylations in degradation is not fully understood. Our work examines changes in the stability of the ternary complex created by trimming and uridylation of the stem-loop to better understand the role of this process in the histone mRNA life cycle. In this study, we used fluorescence polarization and electrophoretic mobility shift assays to demonstrate that both SLBP and 3'hExo can bind to uridylated and partially degraded stem-loop intermediates, although with lower affinity. We further characterized this complex by performing 1-µs molecular dynamics simulations using the AMBER force field and Nanoscale Molecular Dynamics (NAMD). These simulations show that while uridylation helps maintain the overall shape of the stem-loop, the combination of uridylation and dephosphorylation of the TPNK motif in SLBP disrupts key RNA-protein interactions. They also demonstrate that uridylation allows 3'hExo to maintain contact with the stem-loop after partial degradation and plays a role in disrupting key base pairs in partially degraded histone mRNA intermediates. Together, these experiments and simulations suggest that trimming by 3'hExo, uridylation, and SLBP dephosphorylation weakens both RNA-protein interactions and the stem-loop itself. Our results further elucidate the role of uridylation and SLBP dephosphorylation in the early stages of histone mRNA degradation.

Electrophoretic Mobility Shift Assay (EMSA) and Microscale Thermophoresis (MST) Methods to Measure Interactions Between tRNAs and Their Modifying Enzymes.

The Elongator complex is a unique tRNA acetyltransferase; it was initially annotated as a protein acetyltransferase, but in-depth biochemical analyses revealed its genuine function as a tRNA modifier. The substrate recognition and binding of the Elongator is mainly mediated by its catalytic Elp3 subunit. In this chapter, we describe protocols to generate fluorescently labeled RNAs and outline the principles underlying electrophoretic mobility shift assays (EMSA) and microscale thermophoresis (MST). These two methods allow qualitative and quantitative examinations of the binding affinity of various tRNAs toward the homologs of Elp3 from various organisms. The rather qualitative results from EMSA analyses can be nicely complemented by MST measurements allowing precise determination of the dissociation constant (KD). We also provide detailed notes for users to mitigate potential ambiguities and technical pitfalls during the procedures.

Quantitative analyses of interactions between SpoVG and RNA/DNA.

The Borrelia burgdorferi SpoVG protein has previously been found to be a DNA- and RNA-binding protein. To aid in the elucidation of ligand motifs, affinities for numerous RNAs, ssDNAs, and dsDNAs were measured and compared. The loci used in the study were spoVG, glpFKD, erpAB, bb0242, flaB, and ospAB, with particular focus on the untranslated 5' portion of the mRNAs. Performing binding and competition assays yielded that the 5' end of spoVG mRNA had the highest affinity while the lowest observed affinity was to the 5' end of flaB mRNA. Mutagenesis studies of spoVG RNA and ssDNA sequences suggested that the formation of SpoVG-nucleic acid complexes are not entirely dependent on either sequence or structure. Additionally, exchanging uracil for thymine in ssDNAs did not affect protein-nucleic acid complex formation.

Evaluating two steps in transcription using a fluorescence-based electrophoretic mobility shift assay.

Transcription is the critical first step in expressing a gene, during which an RNA polymerase (RNAP) synthesizes an RNA copy of one strand of the DNA that encodes a gene. Here we describe a laboratory experiment that uses a single assay to probe two important steps in transcription: (1) RNAP binding to DNA, and (2) the transcriptional activity of the polymerase. Students probe both these steps in a single experiment using a fluorescence-based electrophoretic mobility shift assay (EMSA) and commercially available Escherichia coli RNAP. As an inquiry-driven component, students add the transcriptional inhibitor rifampicin to reactions and draw conclusions about its mechanism of inhibition by determining whether it blocks polymerase binding to DNA or transcriptional activity. Depending on the curriculum and learning goals of individual courses, this experimental module could be easily expanded to include additional experimentation that mimics a research environment more closely. After completing the experiment students understand basic principles of transcription, mechanisms of inhibition, and the use of EMSAs to probe protein/DNA interactions.

A DNA Pull-Down Assay with Diversity Forms of Competitor for Detecting or Evaluating Protein-DNA Interactions.

DNA-protein interactions (DPIs) are critical to all living organisms, particularly in the regulation of gene expression, replication, packing, recombination, and DNA repair, as well as RNA transport and translation. Many laboratory techniques have been developed to study the complex interactions of proteins with DNA, such as chromatin immunoprecipitation (ChIP) assays, DNA electrophoretic mobility shift assay (EMSA), and oligonucleotide pull-down assays. Here we describe an effective approach to identify potential DNA-binding proteins: a pull-down assay using DNA-conjugated beads with a customized competition strategy, which conferred a more effective and efficient approach to determine the interaction between DNA and protein(s), therefore dramatically improving the progress to investigate novel DNA-binding proteins.

Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay.

Electrophoretic mobility shift assay (EMSA) is a classical and popular approach for DNA/RNA protein-binding affinity detection in vitro. This protocol describes a competitive EMSA assay using digoxigenin (DIG)-labeled probe, which solves the safety issues and limitations attributed to the short lifespan of the 32P-radiolabeled DNA probe. We detail steps for DNA probe preparation, protein-DNA mixture coincubation, EMSA, and competitive EMSA process. We optimize the standard DIG-ddUTP-labeling EMSA protocol to high sensitivity with reproducible results. For complete details on the use and execution of this protocol, please refer to Feng et al. (2022).

Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme.

The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR-Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min-1 at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9-DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR-Cas9 technology.

Separation and Characterization of Protein-DNA Complexes by EMSA and In-Gel Footprinting.

In-gel footprinting enables the precise identification of protein binding sites on the DNA after separation of free and protein-bound DNA molecules by gel electrophoresis in native conditions and subsequent digestion by the nuclease activity of the 1,10-phenanthroline-copper ion [(OP)2-Cu+] within the gel matrix. Hence, the technique combines the resolving power of protein-DNA complexes in the electrophoretic mobility shift assay (EMSA) with the precision of target site identification by chemical footprinting. This approach is particularly well suited to characterize distinct molecular assemblies in a mixture of protein-DNA complexes and to identify individual binding sites within composite operators, when the concentration-dependent occupation of binding sites, with a different affinity, results in the generation of complexes with a distinct stoichiometry and migration velocity in gel electrophoresis.

Oncogenic Mutations in the DNA-Binding Domain of FOXO1 that Disrupt Folding: Quantitative Insights from Experiments and Molecular Simulations.

FOXO1, a member of the family of winged-helix motif Forkhead box (FOX) transcription factors, is the most abundantly expressed FOXO member in mature B cells. Sequencing of diffuse large B-cell lymphoma (DLBCL) tumors and cell lines identified specific mutations in the forkhead domain linked to loss of function. Differential scanning calorimetry and thermal shift assays were used to characterize how eight of these mutations affect the stability of the FOX domain. Mutations L183P and L183R were found to be particularly destabilizing. Electrophoresis mobility shift assays show these same mutations also disrupt FOXO1 binding to their canonical DNA sequences, suggesting that the loss of function is due to destabilization of the folded structure. Computational modeling of the effect of mutations on FOXO1 folding was performed using alchemical free energy perturbation (FEP), and a Markov model of the entire folding reaction was constructed from massively parallel molecular simulations, which predicts folding pathways involving the late folding of helix α3. Although FEP can qualitatively predict the destabilization from L183 mutations, we find that a simple hydrophobic transfer model, combined with estimates of unfolded-state solvent-accessible surface areas from molecular simulations, is able to more accurately predict changes in folding free energies due to mutations. These results suggest that the atomic detail provided by simulations is important for the accurate prediction of mutational effects on folding stability. Corresponding disease-associated mutations in other FOX family members support further experimental and computational studies of the folding mechanism of FOX domains.

Regulation of the Human IL-10RB Gene Expression by Sp8 and Sp9.

BACKGROUND: Interleukin-10 (IL-10) is a classic anti-inflammatory cytokine that exerts its effects via the receptor complexes IL-10RA and IL-10RB. Loss of IL-10RB results in many diseases. Moreover, IL-10RB is closely associated with neuronal survival and synaptic formation. However, the regulation of IL-10RB gene expression remains elusive. OBJECTIVE: To investigate whether the expression of IL-10RB gene is increased in brain of Alzheimer's disease (AD) and its transcriptional regulation. METHODS: We examined the gene expression of AD patient brain from public database and detected the protein expression of AD model mouse brain by western blot. We constructed a variety of reporter gene plasmids with different lengths or mutation sites, tested the promoter activity and defined the functional region of the promoter with the luciferase reporter assay. The protein-DNA binding between transcription factors and the promoter was analyzed using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). RESULTS: We found that the IL-10RB is elevated in the brain of AD patient and AD model mice. The minimal promoter of the IL-10RB gene is located in the -90 to +51 bp region (relative to the transcriptional start site) and is sufficient for high-level expression of the IL-10RB gene. Transcription factors Sp8 and Sp9 bind to the IL-10RB promoter in vitro. The overexpression or knockdown of Sp8 and Sp9 affected the IL-10RB promoter activity and its gene expression. CONCLUSION: Our study functionally characterized the promoter of the IL-10RB gene and demonstrated that Sp8 and Sp9 regulated its expression.

Studying R-Loop Recognizing Proteins Using Single-Molecule DNA Curtain Technique and Electrophoretic Mobility Shift Assay.

R-loops are nucleic acid structures containing a DNA-RNA hybrid and the associated non-template single-stranded DNA. R-loops are not only involved in many biological processes but also cause genomic instability when they are abnormally regulated. The R-loop regulation pathway consists of multiple steps associated with diverse proteins. The initial and essential step of the pathway is to recognize R-loops in long DNA of human genome. To elucidate the molecular mechanism underlying R-loop recognition by proteins, we utilize a novel high-throughput single-molecule approach called "DNA curtain" as well as electrophoretic mobility shift assays. Here, we describe the detailed protocols for these techniques that both can be used for studying the R-loop recognition mechanisms.

Fluorescent annulated imidazo[4,5-c]isoquinolines via a GBB-3CR/imidoylation sequence - DNA-interactions in pUC-19 gel electrophoresis mobility shift assay.

Herein we report the development of a sequential synthesis route towards annulated imidazo[4,5-c]isoquinolines comprising a GBB-3CR, followed by an intramolecular imidoylative cyclisation. X-Ray crystallography revealed a flat 3D structure of the obtained polyheterocycles. Thus, we evaluated their interactions with double-stranded DNA by establishing a pUC-19 plasmid-based gel electrophoresis mobility shift assay, revealing a stabilising effect on ds-DNA against strand-break inducing conditions.

Determination of Optimum Ratio of Cationic Polymers and Small Interfering RNA with Agarose Gel Retardation Assay.

Nanomaterials have aroused attention in the recent years for their high potential for gene delivery applications. Most of the nanoformulations used in gene delivery are positively charged to carry negatively charged oligonucleotides. However, excessive positively charged carriers are cytotoxic. Therefore, the complexed oligonucleotide/nanoparticles should be well-examined before the application. In that manner, agarose gel electrophoresis, which is a basic method utilized for separation, identification, and purification of nucleic acid molecules because of its poriferous nature, is one of the strategies to determine the most efficient complexation rate. When the electric field is applied, RNA fragments can migrate through anode due to the negatively charged phosphate backbone. Because RNA has a uniform mass/charge ratio, RNA molecules run in agarose gel proportional according to their size and molecular weight. In this chapter, the determination of complexation efficiency between cationic polymer carriers and small interfering RNA (siRNA) cargos by using agarose gel electrophoresis is described. siRNA/cationic polymer carrier complexes are placed in an electric field and the charged molecules move through the counter-charged electrodes due to the phenomenon of electrostatic attraction. Nucleic acid cargos are loaded to cationic carriers via the electrostatic interaction between positively charged amine groups (N) of the carrier and negatively charged phosphate groups (P) of RNA. The N/P ratio determines the loading efficiency of the cationic polymer carrier. In here, the determination of N/P ratio, where the most efficient complexation occurs, by exposure to the electric field with a gel retardation assay is explained.