Structure, biology and medical potential of blood plasma-derived ultra-short cell-free DNA

• Applicant: Robert Hänsel-Hertsch, Ph.D.
• Subject Area: Hematology, Oncology ; General Genetics and Functional Genome Biology ; Immunology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504972506

The characteristics and diagnostic potential of single-stranded cell-free DNA (cfDNA) in healthy individuals and cancer patients remain unclear. We discovered that a large proportion of single-stranded cfDNA fragments are centered at ~50 nt and are strikingly depleted in cancer patients relative to healthy donors. Our data underline the diagnostic potential of ultrashort (US) cfDNA through classification for cancer patients but raise also questions why cancer-patients have so much less US cfDNA and whether less US cfDNA in blood would present an advantage for cancer development.We hypothesize that the pool of US cfDNAs, primarily consisting of single-stranded guanine-rich or cytosine-rich DANN, form tetraplex-based secondary structures (G-quadruplex, i-motif). We hypothesize that the tetraplex forming capabilities of US cfDNA are important for blood biology and can be rationally exploited for cancer diagnostics and therapy. The project follows four mutually related Aims:Aim 1: Uncovering structural characteristics of US cfDNA. Whether US cfDNAs are structured in blood plasma remain unknown. We will use a combination of high-resolution spectroscopic tools to characterize secondary structures of US cfDNA fragments found in blood plasma of healthy donors.Aim 2: Exploration of US cfDNA biogenesis. We postulate that US cfDNA’s ability to form secondary structure plays an important role in protection of the respective regions from degradation during programmed cell death. We will use state-of-the-art bioreactor assisted real-time in-cell NMR spectroscopy to characterize structural equilibria of precursors of US cfDNA in the course of cell death.Aim 3: Assessment of functional consequences of US cfDNA for blood biology and breast cancer development. We have established the links of US cfDNA in blood plasma to cancer but relationships to blood biology remain to be established. We will specifically search for factors responsible for the depletion of US cfDNA in blood plasma from breast cancer patients. We aim to reveal blood-related cells that alter their function by responding to US cfDNA and consequently lead to enhanced immune-surveillance of breast tumor development.Aim 4: Development and application of cell-free G4 DANN immunoprecipitation and sequencing. To reveal precise G4 landscapes from US cfDNA, we will develop cell-free G4 DANN immunoprecipitation and sequencing (cfG4DIP-seq) of blood plasma. We anticipate to find critical US cfDNA fragments that adopt G4 secondary structure in blood of healthy donors and cancer patients and infer insights on gene regulation.

• DFG Programme: Research Grants
• International Connection: Czech Republic
• Partner Organisation: Czech Science Foundation
• Cooperation Partner: Professor Lukas Trantirek, Ph.D.