Recent studies indicate that i-DNA, afour-stranded cytosine-richD NA also knowna st he i-motif,i sa ctually formed in vivo;however,asystematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences ( 271) bearing four runs of 3-6 cytosines with different spacer lengths has been tested. While i-DNAstability is nearly independent on total spacer length, the central spacer plays as pecial role on stability.S tability also depends on the length of the C-tracts at both acidic and neutral pHs.T his study provides ag lobal picture on i-DNAs tability thanks to the large size of the introduced data set;i tr eveals unexpected features and allows to conclude that determinants of i-DNAstability do not mirror those of G-quadruplexes.Our results illustrate the structural roles of loops and C-tracts on i-DNAs tability,c onfirm its formation in cells,a nd allow establishing rules to predict its stability.

The i-motif DNA, also knowna si -DNA, is an oncanonical DNAs econdary structure formed by cytosine-rich sequences,c onsisting of two intercalated parallel-stranded duplexes held together by hemi-protonated cytosine-cytosine + (C:C + )base pairs.The growing interest in the i-DNAstructure as atarget in anticancer therapyincreases the need for tools for arapid and meaningful interpretation of the spectroscopic data of i-DNAsamples.Herein, we analyzed the circular dichroism (CD) and thermal difference UV-absorbance spectra (TDS) of 255 DNAs equences by means of multivariate data analysis, aiming at unveiling peculiar spectral regions that could be used as diagnostic features during the analysis of i-DNA-forming sequences.

A high catalytic efficiency associated to a robust chemical structure are among the ultimate goals when developing new biocatalytic systems for biosensing applications. To get ever closer to these goals, we report here on a combination of metal-organic framework (MOF)-based nanozymes and G-quadruplex (G4)-based catalytic system known as G4-DNAzyme. This approach aims at combining the advantages of both partners (chiefly, the robustness of the former, the modularity of the latter). To this end, we used MIL-53(Fe) MOF and linked it covalently to a G4-forming sequence (F3TC), itself covalently linked to its cofactor hemin. The resulting complex (referred to as MIL-53(Fe)/G4-hemin) exhibited exquisite peroxidase-mimicking oxidation activity and an excellent robustness (being stored in water for weeks). These properties were exploited to devise a new biosensing system, based on a cascade of reactions catalyzed by the nanozyme (ABTS oxidation) and an enzyme, the alkaline phosphatase (or ALP, ascorbic acid 2-phosphate dephosphorylation). The product of the latter poisoning the former, we thus designed a biosensor for ALP (a marker of bone diseases and cancers), with a very low limit of detection (LOD, 0.02 U L -1 ) which is operative in human plasma samples.

G-quadruplex/hemin (G4/hemin) DNAzymes are biosensing systems, but their application remains limited by an overall low activity and a rather high level of unwarranted background reactions. Here, these issues were addressed through the rational design of F3T-azaC-hemin, a G4-based construct in which the hemin is covalently linked to the G4 core and its binding site flanked with a nucleotide activator, here d(T-azaC). This design led to a G4-DNAzyme whose performances have been ca. 150-fold increased compared to the parent G4-based system. The utility of F3T-azaChemin was demonstrated here through the ultrasensitive chemiluminescent detection of miRNA-221. The limit of detection (LOD) has been decreased to the femtomolar range, making it a new and highly efficient molecular tool in the biosensing technology field.

Massive efforts are currently being invested to improve the performance, versatility, and scope of applications of nucleic acid catalysts. G-quadruplex (G4)/hemin DNAzymes are of particular interest owing to their structural programmability and chemical robustness. However, optimized catalytic efficiency is still bottleneck and the activation mechanism is unclear. Herein, we have designed a series of parallel G4s with different proximal cytosine (dC) derivatives to fine-tune the hemin-binding pocket for G4-DNAzymes. Combining theoretical and experimental methods, we have assessed the dependence of catalytic enhancement on the electronic properties of proximal dCs and demonstrated how proximal dCs activate catalytic proficiency. These results provide interesting clues in recapitulating the push-pull mechanism as the basis of peroxidase activity and help to devise a new strategy to design highly competent DNA catalysts whose performances are of the same order as protease.