20 research outputs found

    Discovering ligands for a microRNA precursor with peptoid microarrays

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    We have screened peptoid microarrays to identify specific ligands for the RNA hairpin precursor of miR-21, a microRNA involved in cancer and heart disease. Microarrays were printed by spotting a library of 7680 N-substituted oligoglycines (peptoids) onto glass slides. Two compounds on the array specifically bind RNA having the sequence and predicted secondary structure of the miR-21 precursor hairpin and have specific affinity for the target in solution. Their binding induces a conformational change around the hairpin loop, and the most specific compound recognizes the loop sequence and a bulged uridine in the proximal duplex. Functional groups contributing affinity and specificity were identified, and by varying a critical methylpyridine group, a compound with a dissociation constant of 1.9 μM for the miR-21 precursor hairpin and a 20-fold discrimination against a closely-related hairpin was created. This work describes a systematic approach to discovery of ligands for specific pre-defined novel RNA structures. It demonstrates discovery of new ligands for an RNA for which no specific lead compounds were previously known by screening a microarray of small molecules

    NMR characterization of an oligonucleotide model of the miR-21 pre-element.

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    We have used NMR spectroscopy to characterize an oligonucleotide stem loop structure based on the pre-element of an oncogenic microRNA, miR-21. This predicted stem-loop structure is cleaved from the precursor of miR-21 (pre-miR-21) by the nuclease Dicer. It is also a critical feature recognized by the protein complex that converts the primary transcript (pri-miR-21) into the pre-miRNA. The secondary structure of the native sequence is poorly defined by NMR due to rapid exchange of imino protons with solvent; however, replacement of two adjacent putative G•U base pairs with G•C base pairs retains the conformation of the hairpin observed by chemical probing and stabilizes it sufficiently to observe most of the imino proton resonances of the molecule. The observed resonances are consistent with the predicted secondary structure. In addition, a peak due to a loop uridine suggests an interaction between it and a bulged uridine in the stem. Assignment of non-exchangeable proton resonances and characterization of NOEs and coupling constants allows inference of the following features of the structure: extrahelicity of a bulged adenosine, deviation from A-form geometry in a base-paired stem, and consecutive stacking of the adenosines in the 5' side of the loop, the guanosine of the closing base pair, and a cross-strand adenosine. Modeling of the structure by restrained molecular dynamics suggests a basis for the interaction between the loop uridine, the bulged uridine in the stem, and an A•U base pair in the stem

    Stereoview of a representative structure for nucleotides 8–23 of RNA 2 (PDB ID 2MNC).

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    <p>Stereoview of a representative structure for nucleotides 8–23 of RNA 2 (PDB ID 2MNC).</p

    A manganese(II)-Based Responsive Contrast Agent Detects Glucose- Stimulated Zinc Secretion from the Mouse Pancreas and Prostate by MRI.

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    A Mn(II)-based zinc-sensitive MRI contrast agent, Mn(PyC3A)-BPEN, was prepared and characterized and the agent was used in imaging experiments to detect glucose-stimulated zinc secretion (GSZS) from the mouse pancreas and prostate in vivo. Thermodynamic and kinetic stability tests showed that Mn(PyC3A-BPEN) has superior kinetic inertness compared to Gd(DTPA), is less susceptible to transmetallation in the presence of excess Zn2+ ions, and less susceptible to transchelation by albumin. In comparison with other gadolinium-based zinc sensors bearing a single zinc binding moiety, Mn(PyC3A-BPEN) appears to be a reliable alternative for imaging b-cell function in the pancreas and glucose-stimulated zinc secretion from prostate cells

    Mg<sup>2+</sup>-stimulated (in-line) cleavage of RNAs 1 and 2.

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    <p>A. Electrophoretic analysis of cleavage. Mg<sup>2+</sup>: RNA treated with 5 mM MgCl<sub>2</sub>. OH: hydrolysis ladder. T1: Ribonuclease T1 digestion. Untreated RNA in lanes labeled “-“. Bands due to guanosines identified from RNAse T1 digestion are indicated. B. Mapping of cleavage onto predicted secondary structures of <b>1</b> and <b>2</b>. Cleavage after each nucleotide is indicated by a line with length proportional to band intensity.</p

    Stereoviews of structural features apparent in the NMR-based model of 2.

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    <p>A. Stacking of consecutive purines G13–A15 and cross-strand stacking of A20. B. Orientation of U12 with respect to U18. The fourteen lowest energy structures were aligned on U18 and the pyrimidine rings of U18 and U12 are shown.</p

    NMR spectra of imino protons for RNAs 1–7 in H<sub>2</sub>O.

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    <p>Peaks for <b>2</b> are labeled according to assignments described in the text. Conditions were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108231#s4" target="_blank">Materials and Methods</a>.</p

    Portion of the NOESY spectrum showing NOEs between H8/H6/H2 (7.4–8.0 ppm) and H1′/H5 (5.2–6.0 ppm) protons.

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    <p>Mixing time was 400 ms and temperature was 25°C. Cross-peaks due to NOEs from a nucleotide aromatic proton to the H1′ proton of its own sugar are labeled. A. Sequential NOEs from C9 to U12. B. Sequential NOEs from G13 to U16. Crosspeaks due to H2 of A14 and A15 and the U16 H5 to U16 H6 are labeled. C. Sequential NOEs from U16 to C21.</p
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