Abstract
We introduce designed ankyrin repeat binding proteins (DARPins) as a novel class of highly specific and structure-selective DNA-binding proteins, which can be functionally expressed within all cells. Human telomere quadruplex was used as target to select specific binders with ribosome display. The selected DARPins discriminate the human telomere quadruplex against the telomeric duplex and other quadruplexes. Affinities of the selected binders range from 3 to 100 nM. CD studies confirm that the quadruplex fold is maintained upon binding. The DARPins show different specificity profiles: some discriminate human telomere quadruplexes from other quadruplex-forming sequences like ILPR, c-MYC and c-KIT, while others recognize two of the sequences tested or even all quadruplexes. None of them recognizes dsDNA. Quadruplex-binding DARPins constitute valuable tools for specific detection at very small scales and for the in vivo investigation of quadruplex DNA.
Highlights
The DNA double helix undoubtedly is one of the most important macromolecular structures
Folding of quadruplex structures was tested with CD spectroscopy for both sequences
The selection was performed in three rounds of ribosome display from two libraries (N2C and N3C) of designed ankyrin repeat binding proteins (DARPins) [39]
Summary
The DNA double helix undoubtedly is one of the most important macromolecular structures. It folds independently of its sequence, provided two complementary DNA strands are available. This seeming generality made it a dogma that all cellular DNA exists in this conformation. Depending on its sequence, DNA can adopt different conformations, such as triple helices and quadruplexes, where three or four strands come together to form a common helix. G-quadruplex DNA (G4) consists of stacks of planar G-quartets, where the four strands are connected via Hoogsteen base pairing and each guanine is donor and acceptor for two H-bonds [1,2]. Different arrangements of G-quartets are possible, which differ in the orientation of the four DNA strands and their thermodynamic stability [3]
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