In this paper, we review recent DNA-binding agents that are expected to influence the field of DNA-targeting. We restrict ourselves to binders for which the three-dimensional structure in complex with DNA or RNA has been determined by X-ray crystallography or NMR. Furthermore, we primarily focus on unprecedented ways of targeting peculiar DNA structures, such as junctions, quadruplexes, and duplex DNAs different from the B-form. Classical binding modes of small molecular weight compounds to DNA, i.e. groove binding, intercalation and covalent addition are discussed in those cases where the structures represent a novelty. In addition, we review 3D structures of triple-stranded DNA, of the so-called Peptide Nucleic Acids (PNAs), which are oligonucleotide bases linked by a polypeptide backbone, and of aptamers, which are DNA or RNA receptors that are designed combinatorially. A discussion on perspectives in the field of DNA-targeting and on sequence recognition is also provided.

Significance: The plant hormone auxin controls many aspects of growth and development. It does so by changing the activity of AUXIN RESPONSE FACTOR (ARF) proteins that recognize specific DNA elements in plant genes and switch gene expression on or off. A major question in plant biology is how these ARF proteins bind unique DNA sequences and thus select which genes are regulated by auxin. Here, the authors systematically study the DNA architecture required for high-affinity binding of ARF proteins, and for auxin-dependent gene regulation in the plant Arabidopsis thaliana . This work shows that repeats of ARF recognition sites are critical for function, and reveals the molecular basis for the distinct activities of different repeat structures.

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.