Latest Innovations in Studying DNA-Protein Interactions
Interactions between DNA and proteins are of great biological significance as they govern numerous physiological
processes, including DNA replication and recombination, gene transcription, chromosome segregation, signal
transduction, and epigenetic silencing. For studying DNA-protein interactions, the analytical method of choice
should ideally ensure that:
True target protein-enriched regions are reliably identified, particularly from limited cell samples.
Enriched DNA contains minimal background.
Mapping of DNA-protein binding regions with minimal bias and at high resolution.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, or ChIP, is one of the most popular methods employed to study protein-DNA
interactions. ChIP can be used to identify the specific genomic DNA region that binds a protein of interest, such
as a modified histone or a transcription factor, in living cells.
A typical ChIP protocol involves the following sequential steps: in vivo cross-linking of cells; chromatin
extraction; DNA shearing, typically by sonication; protein-DNA immunoprecipitation with an antibody against a
protein of interest; cross-link reversal; and DNA purification. The enriched DNA fragments can then be analyzed
downstream by techniques like PCR or NGS. The main limitations of this method are that it requires a large amount
of input material, cells, or tissue to produce a strong enough signal over background noise, as well as the use of
cross-linking during an initial fixation step. Additionally, the majority of ChIP methods are considerably
time-consuming, are labor-intensive, or have low throughput.
ChIP Advancements
More advanced methods like ChIP-exo and ChIPmentation allow for reduced cell numbers and increased resolution.
ChIP-exo provides high-resolution mapping, but it is time-consuming and necessitates an ample supply of input
cells. ChIPmentation uses transposase and sequencing-compatible adaptors to enable the integration of ligation
during the ChIP process, but it follows a traditionally slow (2-day) ChIP procedure and cannot achieve
high-resolution mapping.
Cleavage Under Target & Release Using Nuclease (CUT&RUN) and Cleavage Under Target & Tagmenation (CUT&TAG) were
recently developed for mapping protein-DNA interaction with limited biological materials and have significantly
improved mapping resolution. However, they are both offered at a high cost. CUT&RUN needs expensive PA/Mnase
fusion protein that has significant A/T sequence bias, causing the target protein-interacted DNA region profiles to
be seriously affected by the level of MNase digestion. CUT&TAG displays the same digestion bias and is less
specific due to off-target accessibility of chromatin caused by the Tn5 transposase enzyme.
What’s (Epi)Next?
To address these issues, EpigenTek has developed two improved techniques, EpiNext CUT&RUN
Fast and EpiNext
CUT&Tag In-Place Sequencing, for enriching protein-bound DNA and mapping genome-wide
protein-DNA interactions. These innovative approaches combine the advantages of ChIP-exo, ChiPmentation, and
CUT&RUN with the fastest procedures in convenient and affordable all-in-one kits for reliably identifying true
target protein-enriched regions and achieving high-resolution mapping. Both techniques utilize a unique nucleic
acid cleavage enzyme mix, which has low sequence bias, to simultaneously fragment chromatin and cleave/remove
unbound DNA sequences in both ends of the target protein/DNA complex in situ without affecting DNA occupied by the
target protein, thereby minimizing immunocapture/sequencing background. With these techniques, library DNA
generation can be achieved in just a few hours, starting from as little as 500 of cells or 50 ng of chromatin.
These kits have the following advantages and features:
Fast and streamlined procedure, taking only 3 hours to complete for nucleic acid enrichment and only 5-6 hours
for enrichment plus library prep.
No sonication, which eliminates the optimization and reproducibility issues associated with this step.
No fixation step for the ChIP-related kits, which can otherwise cause issues with epitope recognition by the
antibody.
Fragmentation and immunocapture are both processed in the same single tube, which minimizes sample loss and
allows for a lower input amount.
Cleavage and removal of unbound nucleic acid occur in situ, minimizing the number of steps in the procedure, and
the nuclease mix used exhibits low sequence bias.
Nucleic acid is cut very close to the antibody binding site. This allows for enrichment of shorter nucleic acid
fragments, resulting in lower background levels and higher resolution mapping of target protein-DNA interaction
regions.