In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions
Summary
Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely applicable for studying protein-nucleic acid interactions.
Abstract
Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.
Introduction
Protein-nucleic acid interactions are involved in many essential cellular processes such as DNA repair, replication, transcription, RNA processing, and translation. Protein interactions with specific DNA sequences within the chromatin are required for the tight control of gene expression at the transcriptional level1. Precise posttranscriptional regulation of numerous coding and noncoding RNAs necessitates extensive and complicated interactions between any protein and RNA2. These layers of gene expression regulatory mechanism comprise a cascade of dynamic intermolecular events, which are coordinated by interactions of transcription/epigenetic factors or RNA-binding proteins with their nucleic acid targets, as well as protein-protein interactions. To dissect whether proteins in vivo are directly or indirectly associated with nucleic acids and how such associations occur and culminate, in vitro biochemical assays are conducted to examine the binding affinity or enzymatic activity of proteins of interest on designed substrates of DNA and/or RNA.
Many techniques have been developed to detect and characterize nucleic acid-protein complexes, including the electrophoretic mobility shift assay (EMSA), also termed gel retardation assay or band shift assay3,4,5. EMSA is a versatile and sensitive biochemical method that is widely used for studying the direct binding of proteins with nucleic acids. EMSA relies on gel electrophoretic shift in bands, which are routinely visualized using chemiluminescence to detect biotin labels6,7, the fluorescence of fluorophore labels8,9, or by autoradiography of radioactive 32P labels10,11. Other purposes of biochemical studies are the identification and characterization of nucleic acid-processing activity of proteins, such as nuclease-based reactions to assess the cleavage products from nucleic acid substrates12,13,14 and DNA/RNA structure-unwinding assays to evaluate helicase activities15,16,17.
In such enzymatic activity assays, the radioisotope-labeled or fluorophore-labeled nucleic acids are often used as substrates due to their high sensitivity. Analysis of radiographs of enzymatic reactions involving 32P labeled radiotracers has been found to be sensitive and reproducible18. Yet, in an increasing number of laboratories in the world, the usage of radioisotopes is restricted or even prohibited due to the health risks associated with potential exposure. In addition to biosafety concerns, other drawbacks are the required necessary equipment to conduct work with radioisotopes, required radioactivity license, short half-life of 32P (about 14 days), and gradual deterioration of the probe quality due to radiolysis. Thus, alternative non-isotopic methods have been developed (i.e., labeling the probe with fluorophores enables detection by fluorescent imaging19). However, a high-resolution imaging system is needed when using fluorescently labeled probes. Biotin, a commonly used label, readily binds to biological macromolecules such as proteins and nucleic acids. Biotin-streptavidin system operates efficiently and improves detection sensitivity without increasing non-specific background20,21. Besides EMSA, biotin is widely used for protein purification and RNA pull-down, among others22,23,24.
This protocol successfully uses biotin-labeled nucleic acids as substrates for in vitro biochemical assays that include EMSA, in addition to enzymatic reactions in which biotin has not been commonly used. The MEIOB OB domain is constructed and two mutants (truncation and point mutation) are expressed as GST fusion proteins25,26,27, as well as mouse MOV10 recombinant FLAG fusion protein16. This report highlights the effectiveness of this combined protocol for protein purification and biotin-labeled assays for miscellaneous experimental purposes.
Protocol
Representative Results
Discussion
Investigating protein-nucleic acid interactions is critical to our understanding of molecular mechanisms underlying diverse biological processes. For example, MEIOB is a testis-specific protein essential for meiosis and fertility in mammals25,26,27. MEIOB contains an OB domain that binds to single-stranded DNA and exhibits 3' to 5' exonuclease activity26, which directly relates to its ph…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank P. Jeremy Wang (University of Pennsylvania) for helpful edits and discussions. We also thank Sigrid Eckardt for language editing. K. Z. was supported by National Key R&D Program of China (2016YFA0500902, 2018YFC1003500) and National Natural Science Foundation of China (31771653). L. Y. was supported by National Natural Science Foundation of China (81471502, 31871503) and Innovative and Entrepreneurial Program of Jiangsu Province. J. N. was supported by Zhejiang Medical Science and Technology Project (2019KY499). M. L. was supported by grants of National Natural Science Foundation of China (31771588) and the 1000 Youth Talent Plan.
Materials
| Equipment | |||
| Centrifuge | Eppendorf, Germany | 5242R | |
| Chemiluminescent Imaging System | Tanon, China | 5200 | |
| Digital sonifer | Branson, USA | BBV12081048A | 450 Watts; 50/60 HZ |
| Semi-dry electrophoretic blotter | Hoefer, USA | TE77XP | |
| Tube Revolver | Crystal, USA | 3406051 | |
| UV-light cross-linker | UVP, USA | CL-1000 | |
| Materials | |||
| Amicon Ultra-4 Centrifugal Filter | Milipore, USA | UFC801096 | 4 ml/10 K |
| Nylon membrane | Thermo Scientific, USA | TG263940A | |
| TC-treated Culture Dish | Corning, USA | 430167 | 100 mm |
| TC-treated Culture Dish | Corning, USA | 430597 | 150 mm |
| Microtubes tubes | AXYGEN, USA | MCT-150-C | 1.5 mL |
| Tubes | Corning, USA | 430791 | 15 mL |
| Reagents | |||
| Ampicillin | SunShine Bio, China | 8h288h28 | |
| Anti-FLAG M2 magnetic beads | Sigma, USA | M8823 | |
| ATP | Thermo Scientific, USA | 591136 | |
| BCIP/NBT Alkaline Phosphatase Color Development Kit | Beyotime, China | C3206 | |
| CelLyticTM M Cell Lysis Reagent | Sigma, USA | 107M4071V | |
| ClonExpress II one step cloning kit | Vazyme, China | C112 | |
| Chemiluminescent Nucleic Acid Detection Kit | Thermo Scientific, USA | T1269950 | |
| dNTP | Sigma-Aldrich, USA | DNTP100-1KT | |
| DMEM | Gibco, USA | 10569044 | |
| DPBS buffer | Gibco, USA | 14190-136 | |
| EDTA | Invitrogen, USA | AM9260G | 0.5 M |
| EDTA free protease inhibitor cocktail | Roche, USA | 04693132001 | |
| EndoFree Maxi Plasmid Kit | Vazyme, China | DC202 |
|
| FastPure Gel DNA Extraction Mini Kit | Vazyme, China | DC301-01 | |
| FBS | Gibco, USA | 10437028 | |
| FLAG peptide | Sigma, USA | F4799 | |
| Glycerol | Sigma, USA | SHBK3676 | |
| GST Bulk Kit | GE Healthcare, USA | 27-4570-01 | |
| HEPES buffer | Sigma, USA | SLBZ2837 | 1 M |
| IPTG | Thermo Scientific, USA | 34060 | |
| KoAc | Sangon Biotech, China | 127-08-02 | |
| Lipofectamin 3000 Transfection Reagent | Thermo Scientific, USA | L3000001 | |
| MgCl2 | Invitrogen, USA | AM9530G | 1 M |
| NaCl | Invitrogen, USA | AM9759 |
5 M |
| NP-40 | Amresco, USA | M158-500ML | |
| Opti-MEM medium | Gibco, USA | 31985062 | |
| PBS | Gibco, USA | 10010023 | PH 7.4 |
| RNase Inhibitor | Promega, USA | N251B | |
| Streptavidin alkaline phosphatase | Promega, USA | V5591 | |
| TBE | Invitrogen, USA | 15581044 | |
| Tris-HCI Buffer | Invitrogen, USA | 15567027 | 1 M, PH 7.4 |
| Tris-HCI Buffer | Invitrogen, USA | 15568025 | 1 M, PH 8.0 |
| Tween-20 | Sangon Biotech, China | A600560 |
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