Multivalent forms of the ribonuclease H1 hybrid binding domain are high-affinity binders of RNA-DNA hybrids.

The hybrid binding domain (HBD) is a conserved fold present in ribonucleases H1 that selectively recognizes RNA-DNA hybrids, which are structures present in cellular R-loops and participate in diverse biological processes. We engineered multivalent HBD proteins to create high-affinity hybrid binders. Using EMSA- and SPR-based analyses, we showed that the triple-HBD protein exhibits a ~ 22 000-fold increase in hybrid affinity (KD 370 pm) relative to the single HBD (KD 8.29 µm), with the length and sequence of the linkers enabling optimal function. These findings provide a framework for testing models that correlate multivalency and affinity to understand how multivalent proteins function and also can serve to guide applications that exploit multivalency as a strategy to enhance binding affinity.

Global and local mechanical properties control endonuclease reactivity of a DNA origami nanostructure.

We used coarse-grained molecular dynamics simulations to characterize the global and local mechanical properties of a DNA origami triangle nanostructure. The structure presents two metastable conformations separated by a free energy barrier that is lowered upon omission of four specific DNA staples (defect). In contrast, only one stable conformation is present upon removing eight staples. The metastability is explained in terms of the intrinsic conformations of the three trapezoidal substructures. We computationally modeled the local accessibility to endonucleases, to predict the reactivity of twenty sites, and found good agreement with the experimental data. We showed that global fluctuations affect local reactivity: the removal of the DNA staples increased the computed accessibility to a restriction enzyme, at sites as distant as 40 nm, due to an increase in global fluctuation. These results raise the intriguing possibility of the rational engineering of allosterically modulated DNA origami.

Binary control of enzymatic cleavage of DNA origami by structural antideterminants.

Controlling DNA nanostructure interaction with protein is essential in developing nanodevices with programmable function, reactivity, and stability for biological and medical applications. Here, we show that the sequence-specific action of restriction endonucleases towards sharp triangular or rectangular DNA origami exhibits a novel, binary 'on/off' behaviour, as canonical recognition sites are either essentially fully reactive, or strongly resistant to enzymatic cutting. Moreover, introduction of structural defects in the sharp triangle can activate an otherwise unreactive site, with a site-to-defect distance of ∼50 nm. We argue that site reactivity is dependent upon programmable, mechanical coupling in the two-dimensional DNA origami, with specific structural elements, including DNA nicks and branches proximal to the sites that can function as negative(anti) determinants of reactivity. Empirically modelling the constraints to DNA degrees of freedom associated with each recognition site in the sharp triangle can rationalize the pattern of suppressed reactivity towards nine restriction endonucleases, in substantial agreement with the experimental results. These results provide a basis for a predictive understanding of structure-reactivity correlates of specific DNA nanostructures, which will allow a better understanding of the behaviour of nucleic acids under nanoscale confinement, as well as in the rational design of functional nanodevices based on self-assembling nucleic acids.

Polymorphic thymidylate synthase gene impacts on overall survival of patients with epithelial ovarian cancer after platinum-based chemotherapy.

AIM: High levels of TS have been associated with a worse clinical outcome in several cancers including epithelial ovarian cancer (EOC). The TS gene (TYMS) is highly polymorphic and has an effect on mRNA/protein expression. MATERIALS & METHODS: Six TYMS polymorphisms were investigated for overall survival (OS) in 216 EOC patients: TYMS 1494ins/del, TSER (variable number of tandem repeats of 28 bp), TSER G>C, TYMS 1053C>T, TYMS IVS6-68C>T and TYMS 1122A>G. RESULTS: In a multivariate analysis, TYMS 1494 del/del genotype was associated with a significant increased OS compared with the ins/ins genotype (hazard ratio: 0.36; 95% CI: 0.16-0.82, p = 0.01). Similar results were obtained for the mutant genotypes TYMS 1053TT and TYMS IVS6-68TT. The event-free survival was significantly higher in TYMS 1053TT patients compared with wild-type patients (p = 0.05). CONCLUSION: TYMS 1494ins/del, 1053C>T and IVS6-68C>T polymorphisms can be prognostic markers for OS in patients with EOC, independently from stage at diagnosis, median age and tumor histotype.