No abstract

A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence. Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media. These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose. The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription. Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC). Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively. These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues. The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs. Examples of several such heteronuclear NMR experiments are shown.

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.

Aptamers recognize their targets with extraordinary affinity and specificity. The aptamer-based therapeutic, Macugen, is derived from a modified 2 fluoro pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now being used to treat the wet form of age-related macular degeneration. This VEGF 165 aptamer binds specifically to the VEGF165 isoform, a dimeric protein with a receptor-binding domain and a heparin-binding domain (HBD). To understand the molecular recognition between VEGF and this aptamer, binding experiments were used to show that the HBD contributes the majority of binding energy in the VEGF 165-aptamer complex. A tissue culture-based competition assay demonstrated that the HBD effectively competes with VEGF165 for aptamer binding in vivo. Comparison of NMR spectra revealed that structural features of the smaller HBD-aptamer complex are present in the full-length VEGF 164-aptamer complex. These data show that the HBD provides the binding site for the aptamer and is the primary determinant for the affinity and specificity in the VEGF 165-aptamer complex.age-related macular degeneration ͉ Macugen ͉ RNA ͉ NMR

The solution structure of a uniformly 13C/15N-labeled CUUG RNA hairpin loop has been determined by multidimensional heteronuclear magnetic resonance spectroscopy in combination with distance geometry and restrained molecular dynamics calculations. The structure of this CUUG tetraloop represents a novel RNA loop motif where the first and last loop nucleotides form a standard Watson-Crick C-G base pair and the second loop nucleotide interacts directly with the closing base pair of the stem by folding into the minor groove. This structure helps explain why the closing base pair is phylogenetically conserved and indicates a six-nucleotide G(CUNG)C motif for the CUUG RNA tetraloop. Implications for the function of this CUUG tetraloop in ribosomal RNA and in RNA tertiary interactions are discussed.