Development of nanobodies as tools to investigate G-quadruplex DNA secondary structures
Guanine-rich sequences in DNA can fold into four-stranded alternative secondary structures called G-quadruplexes (G4). G4s are suggested to be involved in key genome functions such as transcription, genome stability and epigenetic regulation, together with numerous connections to cancer biology. The detection G4s in cells has been facilitated by the use of structure-specific small molecules or antibody probes and much work has relied on G4 recognition by a single-chain variable fragment antibody, BG4. Other G4 probes have been reported but generally their usage has been limited to certain applications and in many cases the probes have limited selectivity for G4s.
The aim of this thesis was to develop an alternative probe with high affinity and good selectivity for G4s for use in applications for which BG4 in not suitable and to provide an important orthogonal validation of results previously obtained with BG4. A phage-display screen was used to select nanobodies, antibody fragments derived from of heavy-chain only antibodies of Camelid family, that have high affinity and selectivity for the G4 structure from the human MYC oncogene promoter (MycG4). Biophysical characterisation confirmed that one nanobody (B11) binds with high affinity to a wide range of DNA G4 conformations in vitro. B11 successfully confirmed the folding of G4s in chromatin isolated from cancer cells. Furthermore, mutational analysis coupled with molecular dynamics simulation identified specific arginines contributing to the interaction of B11 with MycG4. Lastly, I demonstrated that B11 can be expressed in human cells for use in experiments to map G4 genomic locations in situ.
The results from these experiments are significant to the field as they have demonstrated the validation of earlier data describing the location of folded G4 structures in the human genome. I have also extended previous studies by gaining molecular insights in the mode of action of an antibody-G4 interaction. Finally, I have provided initial proof-of-principle that a G4 antibody can be deployed in a cellular context to probe G4s in living cells. In sum, I have successfully generated a novel tool for the future study of G4s in key cellular processes, such as transcription, and in diseases such as cancer.