Protein-Nucleic Acid Interaction

Proteins interact with DNA and RNA through similar physical forces, which include electrostatic interactions (salt bridges), dipolar interactions (hydrogen bonding, H-bonds), entropic effects (hydrophobic interactions) and dispersion forces (base stacking). These forces contribute in varying degrees to proteins binding in a sequence-specific (tight) or non–sequence-specific (loose) manner. Nucleic acid-protein interactions are more common than nucleic acid-nucleic acid interactions. Gene replication, transcription, translation, modification and other processes are inseparable from the interaction between nucleic acid and protein.

Types

• Protein-DNA interactions
  The common DNA-binding domains, helix-turn-helix and zinc finger domains, are incorporated within numerous DNA-binding proteins expressed in the cell. Specificity is derived from higher order interactions involving nucleoprotein complexes. These DNA-binding protein complexes find their target by "sliding" along the genomic DNA until their specific DNA-docking site is discovered. The binding of protein to DNA controls the structure of genomic DNA (chromatin), RNA transcription, and DNA repair mechanisms.
• Protein-RNA interactions
  Proteins interact with RNA in order to splice, protect, translate or degrade the message. The first interaction occurs just after transcriptional initiation, when the complement to the promoter sequence is cleaved out of the mRNA and the capping machinery incorporates a "GpppN" cap at the 5' end of the mRNA. Elongation is followed by 3'-end processing and splicing, resulting in a mature RNA transcript that is exported to the cytoplasm for translation. All of these processes require significant protein–RNA interactions and are highly regulated and complex.

Methods

• Chromatin immunoprecipitation (ChIP) assay
  The chromatin immunoprecipitation (ChIP) method can be used to monitor transcriptional regulation through histone modification (epigenetics) or transcription factor–DNA binding interactions. The ChIP assay method allows analysis of DNA–protein interactions in living cells by treating the cells with formaldehyde or other crosslinking reagents in order to stabilize the interactions for downstream purification and detection.

Fig. 1 The basic flowchart of the chromatin immunoprecipitation assay technique.

• DNA pull-down assay
  The DNA pull down experiment is to design and prepare specific probes for the regulatory region of the target gene to be studied. At the same time, nuclear extracts were prepared, and the probes and nuclear extracts were co-incubated. The DNA-binding protein then specifically binds to the target sequence. Then, the protein-DNA complexes were purified by avidin magnetic beads. Finally, for the obtained protein, use WB verification or mass spectrometry to identify the protein type.
• RNA pull-down assay
  In the RNA pull down experiment, the in vitro transcription method was used to label the biotin RNA probe and incubate with the cytoplasmic protein extract to form the RNA-protein complex. This complex can be bound to streptavidin magnetic beads and then separated from other components of the incubation solution. After the complex was eluted, whether the specific RNA-binding protein interacted with RNA was detected by WB experiment.

Fig. 2 The basic flowchart of the RNA pull-down assay technique.

• Electrophoretic Mobility Shift Assay
  Electrophoretic Mobility Shift Assay (EMSA) is a technique for studying the interaction between DNA-binding proteins and their associated DNA-binding sequences, which can be used for qualitative and quantitative analysis. At present, it has been used to study the interaction between RNA binding proteins and specific RNA sequences, and is a classic method for transcription factor research.

Fig. 3 The basic flowchart of the electrophoretic mobility shift assay technique.

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STEMart provides you with a variety of protein-nucleic acid interaction technology equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for molecular interactions technology, please feel free to contact us.