Programmable and Chemoselective Protein-DNA Crosslinking for Sensitive Detection of 5-Formylcytosine

5-Formylcytosine (fC) is the seventh nucleobase of the mammalian genome. fC has recently been found to be present at high levels in genes associated with transcription and differentiation, and crystal structures have shown that its presence in DNA can strongly distort the duplex structure. These findings are suggestive of roles in gene expression regulation. However, the underlying control mechanism of such potential functions is the introduction and removal of fC in DNA, i.e. its dynamics. Information on this aspect is crucial for a full understanding of fC´s function, but has not been provided by previous studies that focused only on static aspects. Measuring fC dynamics requires simple and sensitive assays for the time-resolved quantification of fC at relevant genomic loci. However, current detection methods for fC do not provide a direct sequence-selectivity, resulting in disadvantages in respect to simplicity and/or resolution. We have recently introduced the concept of an expanded programmability of DNA recognition based on TALE proteins that consist of multiple concatenated repeats with individual selectivities for the recognition of both canonical and epigenetic nucleobases in DNA.

With this project, to meet the specific challenges of fC detection, we will advance our concept from a solely recognition-based approach to a chemoselective fC crosslinking-based approach. We will achieve this by 1.) cotranslational incorporation of para-acetyl-L-phenylalanine (pAcF) into appropriate sites of TALEs by genetic encoding in vivo, 2.) binding of the modified TALE to the fC-containing DNA target site, and 3.) chemoselective crosslinking between pAcF and fC by catalytic oxime formation with a bifunctional aminooxy-linker under conditions that are compatible with selective TALE-DNA complex formation. This will significantly increase the selectivity and sensitivity of our approach, and for the first time enable the direct and highly resolved quantification of fC at user-defined genomic loci. We will establish a bead-based assay for genomic affinity enrichment coupled to qPCR quantification and employ it to study the kinetics of post-replicative fC formation in the mouse genome.This will lead to the first insights into the dynamics of fC as a basis of its biological function, and represents a starting point for follow-up studies aiming at a better understanding of the dynamic consequences of fC formation in respect to further oxidation/repair processes, as well as the recruitment/release of key repair and epigenetic reader proteins.

Dr. Daniel Summerer
Universität Konstanz

Tel.: +49 7531 88 5669

Email Dr. Summerer

Publications within the SPP 1623 project

G. Kubik, D. Summerer
ACS Chem. Biol., 2015, accepted
Deciphering epigenetic cytosine modifications by direct molecular recognition
Link to the article

G. Kubik, D. Summerer
ChemBioChem 2015, 16(2), 228-231
Achieving single nucleotide resolution of 5-methylcytosine detection with TALEs
Link to the article

G. Kubik, S. Batke, D. Summerer
J. Am. Chem. Soc. 2015, 137, 2-5
Programmable sensors of 5-hydroxymethylcytosine
Link to the article

M. Pott, M.J. Schmidt, D. Summerer
ACS Chem. Biol. 2014, 9(12), 2815-2822
Evolved sequence contexts for highly efficient amber suppression with noncanonical amino acids
Link to the article

G. Kubik, M.J. Schmidt, J.E. Penner, D. Summerer
Angew. Chem. Int. Ed., 2014, 53(23), 6002-6006.
Programmable and highly resolved detection of 5-methylcytosine by TALE-controlled DNA replication
Link to the article

M.J. Schmidt, A. Weber, M. Pott, W. Welte, D. Summerer
ChemBioChem, 2014, in press
Structural basis of furan-amino acid recognition by a polyspecific aminoacyl-tRNA-synthetase for its genetic encoding in human cells
Link to the article

M.J. Schmidt, D. Summerer
Front. Chem.2014, 2(7), 1-11
Genetic code expansion as a tool to study regualatory processes of trascription
Link to the article

M.J. Schmidt, J. Borbas, M. Drescher, D. Summerer
J. Am. Chem. Soc.2014, 136, 1238-1241
A genetically encoded spin label for electron paramagnetic resonance distance measurements
Link to the article

M.J. Schmidt, D. Summerer
Angew. Chem. Int. Ed. 2013, 52, 4690-4693
Red-light-controlled protein-RNA crosslinking with a genetically encoded furan
Link to the article