Protein–Nucleic Acid Interactions

Since the development of DNA footprinting in 1978, researchers have been interrogating the composition of protein–nucleic acid complexes. Innovative techniques allow for genome-wide analyses as well as the study of interactions in vivo. Some of the newer methods are outlined below.

1. Electrophoretic mobility shift assays (EMSA)
   A DNA or RNA EMSA is used to determine the specificity of a protein-nucleic acid interaction in vitro. The target nucleic acid is labeled, incubated with potential protein binding partners, then run on a gel. Any protein–nucleic interactions will retard the mobility of the nucleic acid in the gel and result in a shift of the band.
1. Pull-down assays
   Pull-down assays can be performed using either a target nucleic acid sequence or a protein to identify the missing partner(s) in the complex. In this in vitro assay, either the known nucleic acid sequence or the binding protein is attached to an affinity tag, which is used to isolate the complexes from a lysate. If nucleic acid is used for the pull-down, then candidate interacting proteins can be confirmed using antibodies. Alternatively, unknown proteins can be identified using mass spectrometry. If a protein is used for the pull-down, then sequencing can be used to identify any nucleic acids bound to the protein.
2. Microplate capture and detection
   Pull-down assays can be combined with enzyme-linked immunosorbent assays (ELISAs) to perform a technique known as microplate capture and detection. This method is a high-throughput assay to confirm the identity and relative amount of protein partners using antibodies.
3. ChIP assays
   1. ChIP-seq and ChIP-chip (DNA)
      Chromatin immunoprecipitation (ChIP) is used to determine the sequence of DNA bound to a known protein in a living cell.
      
      In a ChiP assay, cells are treated with a crosslinking reagent that stabilizes protein–DNA interactions. An antibody specific for the known protein is then used to purify the protein–DNA complexes. The bound DNA can be identified in several different ways. In traditional ChIP, polymerase chain reaction (PCR) is used to identify the particular DNA sequences bound to the protein. Alternatively, the DNA sequences can be identified using high throughput sequencing in a technique known as ChIP-seq or using a microarray, referred to as ChIP-on-chip.
   2. CLIP-seq (RNA)
      Originally termed HITS-CLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation), cross-linking immunoprecipitation (CLIP)-seq uses UV light as a crosslinking agent to stabilize protein–RNA interactions. The complexes are then immunoprecipitated using an antibody to the target protein. After immunoprecipitation, the complexes are digested with Proteinase K, which removes the bound protein targets and leaves a peptide cross-linked to the protein-binding site. High-throughput sequencing is used to identify the RNA sequences bound by the protein.
      
      CLIP-seq has been used to generate genome-wide splicing factor maps and has been extraordinarily useful in studying microRNAs. Immunoprecipitation of Argonaute, a protein component of the miRNA silencing complex, has been instrumental in identifying miRNA targets, as well as in defining the requirements for miRNA binding.¹
   3. Antibody considerations
      Immunoprecipitation assays are powerful techniques for studying protein-nucleic acid interactions in vivo. The reliability of the assays is directly dependent on the quality of the antibodies used. Antibodies used for these assays need to be very specific with the high binding efficiency and low background. Only ChIP- or CLIP-verified antibodies should be used.
      
      Bethyl Laboratories sells high-quality antibodies for immunoprecipitations and have been used recently to:
      • identify the binding sites of a protein involved in transformation resistance.²
      • study the interactions of drugs with their target proteins in tumor cells.³
      • study gene expression regulation.⁴
      • investigate miRNA processing.⁵

View Bethyl antibodies validated for use in ChIP.