G-quadruplex DNA (G4-DNA) structures are four-stranded helical DNA (or RNA) structures, comprising stacks of G-tetrads, which are the outcome of planar association of four guanines in a cyclic Hoogsteen hydrogen-bonding arrangement. In the last decade the number of publications where CD spectroscopy has been used to study G4-DNAs, is extremely high. However, with very few exceptions, these investigations use an empirical interpretation of CD spectra. In this interpretation two basic types of CD spectra have been associated to a single specific difference in the features of the strand folding, i.e. the relative orientation of the strands, "parallel" (all strands have the same 5' to 3' orientation) or "antiparallel". Different examples taken from the literature where the empirical interpretation is not followed or is meaningless are presented and discussed. Furthermore, the case of quadruplexes formed by monomeric guanosine derivatives, where there is no strand connecting the adjacent quartets and the definition parallel/antiparallel strands cannot apply, will be discussed. The different spectral features observed for different G-quadruplexes is rationalised in terms of chromophores responsible for the electronic transitions. A simplified exciton coupling approach or more refined QM calculations allow to interpret the different CD features in terms of different stacking orientation (head-to-tail, head-to-head, tail-to-tail) between adjacent G-quartets irrespectively of the relative orientation of the stands (parallel/antiparallel).

Triplex with a twist: Through metadynamics calculations, the thrombin binding aptamer (TBA) has been shown to adopt a stable G‐triplex structural motif, in addition to the usual G‐quadruplex (see scheme). An 11‐mer oligonucleotide was also shown to form a stable G‐triplex, whose structural and thermodynamic properties have been characterized.

The study of DNA G-quadruplex stabilizers has enjoyed a great momentum in the late years due to their application as anticancer agents. The recognition of the grooves of these structural motifs is expected to result in a higher degree of selectivity over other DNA structures. Therefore, to achieve an enhanced knowledge on the structural and conformational requisites for quadruplex groove recognition, distamycin A, the only compound for which a pure groove binding has been proven, has been chemically modified. Surprisingly, structural and thermodynamic studies revealed that the absence of Coulombic interactions results in an unprecedented binding position in which both the groove and the 3' end of the DNA are occupied. This further contribution adds another piece to the so far elusive puzzle of the recognition between ligands and DNA quadruplexes and will serve as a platform for a rational design of new groove binders.

Triplex als Alternative: Metadynamikrechnungen deuten darauf hin, dass das Thrombin bindende Aptamer (TBA) neben dem üblichen G‐Quadruplex auch eine stabile G‐Triplex‐Struktur einnehmen kann (siehe Schema; rote Kugel: K+‐Ion). Ein 11‐mer‐Oligonucleotid bildet ebenfalls einen stabilen G‐Triplex, dessen Struktur und thermodynamische Eigenschaften charakterisiert wurden.

No abstract

Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is the subject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, it has been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promoting levels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. So far, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and to characterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasis on computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopies.

Autophagy is traditionally depicted as a signaling cascade that culminates in the formation of an autophagosome that degrades cellular cargo. However, recent studies have identified myriad pathways and cellular organelles underlying the autophagy process, be it as signaling platforms or through the contribution of proteins and lipids. The Golgi complex is recognized as being a central transport hub in the cell, with a critical role in endocytic trafficking and endoplasmic reticulum (ER) to plasma membrane (PM) transport. However, the Golgi is also an important site of key autophagy regulators, including the protein autophagy-related (ATG)-9A and the lipid, phosphatidylinositol-4-phosphate [PI(4)P]. In this review, we highlight the central function of this organelle in autophagy as a transport hub supplying various components of autophagosome formation. Basic Mechanisms of AutophagyAutophagy is a process whereby cellular material is degraded to procure nutrients or to remove organelles and proteins [1]. This highly conserved, essential process is mediated by a cohort of proteins called the ATG proteins, which are conserved from yeast to humans. There are many different cues that initiate autophagy, but perhaps the best known is amino acid starvation, which induces autophagy to offset the lack of nutrients. Autophagy can be seen as a complex pathway of membrane formation and reformation, centered on the de novo creation of a double-membraned autophagosome, which will fuse with the lysosome so as to degrade its cargo [1].Many aspects of autophagosome formation are by now well understood and, in simplified form, can be viewed as a cascade starting from the mammalian target of rapamycin (mTOR), which activates the Unc-51-like kinase 1 (ULK1) complex, followed by phosphatidylinositol 3-phosphate [PI(3)P] generation at the ER by the phosphatidylinositol 3-kinase catalytic subunit type 3 [PI(3) KC3] complex I. The generation of PI(3)P at the ER leads to the recruitment of autophagy effectors to form the omegasome, the earliest autophagic structure, which grows into the phagophore. One of the effectors that bind PI(3)P is WIPI2B, which has an important role in the lipidation and membrane association of LC3/GABARAPs (e.g., LC3-II; see Glossary), which are essential for autophagy and are the most widely used markers of autophagosomes. Once the phagophore has grown and enclosed its cargo, it closes to form an autophagosome [1,2].An important player in each step of this process is ATG9A, a transmembrane protein that cycles between the trans-Golgi network (TGN) and the ATG9 compartment [3]. Curiously, although essential at all stages for autophagosome formation, ATG9A does not have a defined function as far as we know [2]. Thus, many questions remain about autophagosomal membrane formation and the role of ATG9A. In particular, because it is the only transmembrane core autophagy protein, could ATG9A contribute lipids to help form the autophagosome? In addition, which proteins or lipid species could be trafficked by ATG9A to the...

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A–oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein–protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.