FLIX-NANOPORE for analyzing transcription factor-DNA interactions 

FLIX-NANOPORE has been developed to provide the first rapid and genome-wide determination of transcription factor binding sites and transcription factor binding sequences. Transcription factors are cross-linked to DNA at the DNA binding sequence using a femtosecond laser. This is followed by proteolysis, which leaves only individual amino acids covalently bound to their DNA-binding sequence. In the nanopore, the complexes of covalently bound amino acids and nucleobases are sequenced simultaneously, allowing us to identify the transcription factor binding sites and the corresponding sequence.

Challenge

Identifying transcription factor-DNA interactions is time-consuming and costly. Chromatin immunoprecipitation (ChIP) is a well-known approach. An alternative method, called the FLIX method (femtosecond laser-induced transcription factor DNA cross-linking), converts the hydrogen bond into a covalent bond without the use of an additional chemical agent such as formaldehyde. The FLIX method allows rapid regulatory processes and binding kinetics to be analyzed efficiently and with minimal DNA damage. The FLIX method can be followed by mass spectrometry (FLIX-MS), which maps protein-DNA contacts with a resolution of one nucleotide down to a single amino acid. However, this type of downstream analysis focuses on protein information and is not only costly but also time-consuming. The newly developed FLIX-NANOPORE technology facilitates more specific, more sensitive and faster identification of genome-wide transcription factor binding sites.

Our Solution

The new FLIX NANOPORE technology enables the genome-wide identification of transcription factor binding sites and DNA-binding protein binding sites in general. First, the FLIX method enables the rapid and efficient analysis of regulatory processes and binding kinetics without the need for additional chemicals. A femtosecond laser is used to covalently bind the transcription factors to the transcription factor binding sites (Figure 1). This quickly “freezes” a natural molecular state. Zero-length cross-links are created without additional additive chemicals, as is usually the case with formaldehyde. Following cross-linking, the samples are processed, the bound transcription factors are digested and the DNA is purified with the covalently bound amino acids. This is followed by nanopore sequencing and subsequent determination of the nucleobase with the bound amino acids. In the analysis and data evaluation, the transcription factor binding sites including the corresponding sequence can be identified.

Figure 1) Workflow of the FLIX-NANOPORE technology.

Advantages

• FLIX procedure:
  ◦ high cross-linking efficiency and low DNA damage
  ◦ Fast "freezing" of the native molecular state
  ◦ zero-length cross-linking in the conversion of hydrogen bonds into a covalent bond without additional chemical agents for cross-linking
• NANOPORE sequencing:
  ◦ Small quantities of sample material are required
  ◦ DNA sequencing in real time with high accuracy, read length and high throughput
  ◦ cost-effective, fast and can be easily scaled up
  ◦ Nucleobases and covalently bound amino acid are identified simultaneously
• proof of concept shows availability of applicable sequences

Applications

This technology is used in all molecular biological approaches in which transcription factor binding sites, epigenetic markers, potential DNA damage or nucleobases to which amino acids are covalently bound are to be identified.

Development Status

Successful proof of concept for the availability of applicable sequences.

Patent Status

IP rights (DE102024104997.9) have been filed in the name of the HAWK University of Applied Sciences and Arts Hildesheim / Holzminden / Göttingen and a licensing partner is sought.

References

Reim A. et al Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry. Nat Commun. 2020 Jun 15;11(1):3019. doi: 10.1038/s41467-020-16837-x.

Contact

Dr. Vanessa Jensen
Patent Manager Life Sciences
E-Mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
Tel.: +49 551 30724 149
Reference: BioV-2494-HAWK

Tags: Nanopore Sequencing, Sequencing, Research tools, Life science