Regulating the dynamic folding of a DNA hairpin at the expense of a small, molecular fuel

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nan- otechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a fast, chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in fast and precise DNA nanodevices.

Computational design of single-stranded DNA hairpin aptamers immobilized on a biosensor substrate

Aptamer interactions with a surface of attachment are central to the design and performance of aptamer-based biosensors. We have developed a computational modeling approach to study different system designs—including different aptamer-attachment ends, aptamer surface densities, aptamer orientations, and solvent solutions—and applied it to an anti MUC1 aptamer tethered to a silica biosensor substrate. Amongst all the system designs explored, we found that attaching the anti MUC1 aptamer through the 5′ terminal end, in a high surface density configuration, and solvated in a 0.8 M NaCl solution provided the best exposure of the aptamer MUC1 binding regions and resulted in the least amount of aptamer backbone fluctuations. Many of the other designs led to non-functional systems, with the aptamer collapsing onto the surface. The computational approach we have developed and the resulting analysis techniques can be employed for the rational design of aptamer-based biosensors and provide a valuable tool for improving biosensor performance and repeatability.

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin, to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

An extended APOBEC3A mutation signature in cancer

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition

Artemis (DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in DNA double-strand break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Consistently, Artemis deficiency leads to radiosensitive congenital severe immune deficiency (RS-SCID). Artemis belongs to a structural superfamily of nucleases that contain conserved metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. Here, we present crystal structures of the catalytic domain of wild type and variant forms of Artemis that cause RS-SCID Omenn syndrome. The truncated catalytic domain of the Artemis is a constitutively active enzyme that with similar activity to a phosphorylated full-length protein. Our structures help explain the basis of the predominantly endonucleolytic activity of Artemis, which contrast with the predominantly exonuclease activity of the closely related SNM1A and SNM1B nucleases. The structures also reveal a second metal binding site in its β-CASP domain that is unique to Artemis. By combining our structural data that from a recently reported structure we were able model the interaction of Artemis with DNA substrates. Moreover, co-crystal structures with inhibitors indicate the potential for structure-guided development of inhibitors.