Direct structural analysis of modified RNA by fluorescent in-line probing

Chemical probing is a common method for the structural characterization of RNA. Typically, RNA is radioactively end-labelled, subjected to probing conditions, and the cleavage fragment pattern is analysed by gel electrophoresis. In recent years, many chemical modifications, like fluorophores, were introduced into RNA, but methods are lacking that detect the influence of the modification on the RNA structure with single-nucleotide resolution. Here, we first demonstrate that a 5′-terminal 32P label can be replaced by a dye label for in-line probing of riboswitch RNAs. Next, we show that small, highly structured FRET-labelled Diels–Alderase ribozymes can be directly probed, using the internal or terminal FRET dyes as reporters. The probing patterns indeed reveal whether or not the attachment of the dyes influences the structure. The existence of two dye labels in typical FRET constructs is found to be beneficial, as ‘duplexing’ allows observation of the complete RNA on a single gel. Structural information can be derived from the probing gels by deconvolution of the superimposed band patterns. Finally, we use fluorescent in-line probing to experimentally validate the structural consequences of photocaging, unambiguously demonstrating the intentional destruction of selected elements of secondary or tertiary structure

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups

Mg concentration dependence of the average inter-dye distance (filled squares) and the width of the distance distribution (open squares) of the state

<p><b>Copyright information:</b></p><p>Taken from "Mg-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis"</p><p></p><p>Nucleic Acids Research 2007;35(6):2047-2059.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874616.</p><p>© 2007 The Author(s)</p> Solid lines represent a global fit according to the Hill equation, yielding a midpoint Mg concentration of (3.8 ± 0.5) mM and a cooperativity parameter = 4.2 ± 0.8

Thermodynamic scheme describing the Mg-dependent folding of DAse ribozyme

<p><b>Copyright information:</b></p><p>Taken from "Mg-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis"</p><p></p><p>Nucleic Acids Research 2007;35(6):2047-2059.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874616.</p><p>© 2007 The Author(s)</p> () Scheme involving three Mg-free states, denoted as , and , and three Mg-bound states, denoted as , and . Folding is induced by Mg-dependent equilibrium coefficients and () Free energy diagram of the observed populations of ( + ), ( + ) and ( + ) is shown for 0 and 40 mM Mg

Histograms of FRET efficiency values, , taken from single molecules exposed to buffer solutions with 0, 0

<p><b>Copyright information:</b></p><p>Taken from "Mg-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis"</p><p></p><p>Nucleic Acids Research 2007;35(6):2047-2059.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874616.</p><p>© 2007 The Author(s)</p>625, 1.25, 2.5, 5, 10, 20, 40 and 100 mM Mg concentration. The lowest panel shows the histogram for molecules exposed to 0 mM Mg immediately after exposure to 40 mM Mg. Three subpopulations, denoted as (〈〉 ∼ 0), (〈〉 ∼ 0.7) and (〈〉 ∼ 0.9), can be distinguished. Dotted, dashed and solid thick lines represent fit results using model distributions for the and states, respectively. The solid line represents the sum over the three distributions