NMR structure of the let-7 miRNA interacting with the site LCS1 of lin-41 mRNA from Caenorhabditis elegans

We have determined the 3D structure of a 34-nt RNA construct, herein named LCS1co, which mimics the interaction of let-7 microRNA (miRNA) to one of its complementary binding sites, LCS1, in the 3′-untranslated region of lin-41 mRNA by solution-state NMR spectroscopy. let-7 miRNAs control the timing of development of the nematode Caenorhabditis elegans and are highly conserved in mammals. The sequence and structure of the two conserved let-7 complementary sites, LCS1 and LCS2, in the 3′-untranslated region of lin-41 mRNA are important for a proper downregulation of lin-41. The high-resolution NMR structure reveals details of the binding of let-7 miRNA to lin-41 mRNA which involves formation of a complex with non-canonical structural elements within the seed region. LCS1co exhibits a stem-loop structure with two stems, an asymmetric internal loop and an adenine bulge. Comparison with the NMR solution-state structure of the let-7:lin-41 complex involving the LCS2-binding site shows that conformational freedom of the asymmetric internal loop of LCS1co correlates with a smaller bend between the upper and lower stems in comparison to the well-defined asymmetric loop of LCS2co

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA

Role of Loop Residues and Cations on the Formation and Stability of Dimeric DNA G-Quadruplexes^†

Formation of guanine-quadruplexes by four DNA oligonucleotides with common sequence dG4-loop-dG4 has been studied by a combination of NMR and UV spectroscopy. The loops consisted of 1‘,2‘-dideoxyribose, propanediol, hexaethylene glycol, and thymine residues. The comparison of data on modified and parent oligonucleotides gave insight into the role of loop residues on formation and stability of dimeric G-quadruplexes. All modified oligonucleotides fold into dimeric fold-back G-quadruplexes in the presence of sodium ions. Multiple structures form in the presence of potassium and ammonium ions, which is in contrast to the parent oligonucleotide with dT4 loop. 15N-filtered 1H NMR spectra demonstrate that all studied G-quadruplexes exhibit three 15NH4+ ion binding sites. Topology of intermolecular G-quadruplexes was evaluated by NMR measurements and diffusion experiments. The spherical, prolate-ellipsoid and symmetric cylinder models were used to interpret experimental translational diffusion constants in terms of diameters and lengths of unfolded oligonucleotides and their respective G-quadruplexes. UV melting and annealing curves show that oligonucleotides with non-nucleosidic loop residues fold faster, exhibit no hysteresis, and are less stable than dimeric d(G4T4G4)2 which can be attributed to the absence of H-bonds, stacking between loop residues and the outer G-quartets as well as cation−π interactions. Oligonucleotide consisting of hexaethylene glycol linkage with only two phosphate groups in the loop exhibits higher melting temperature and more negative ΔH° and ΔG° values than oligonucleotides with four 1‘,2‘-dideoxyribose or propanediol residues