Investigation of Mg2+- and temperature-dependent folding of the hairpin ribozyme by photo-crosslinking: effects of photo-crosslinker tether length and chemistry

Abstract

We have used photo-crosslinking to investigate the structure and dynamics of four-way junction hairpin ribozyme constructs. Four phenylazide photo-crosslinkers were coupled to 2′-NH2-modified U+2 in the substrate and irradiated at different Mg2+ concentrations and temperatures. Consistent with the role of divalent metal ions in hairpin ribozyme folding, we observed more interdomain crosslinks in the presence of Mg2+ than in its absence. In general, we observed intradomain crosslinks to nucleotides 2–11 and interdomain crosslinks to the U1A binding loop. Crosslinks to A26 and G36 in domain B were also observed when crosslinking was carried out at −78°C. In contrast to crosslinking results at higher temperatures (0, 25 and 37°C), similar crosslinks were obtained in the presence and absence of Mg2+ at −78°C, suggesting Mg2+ stabilizes a low-energy hairpin ribozyme conformation. We also evaluated the effects of photo-crosslinker structure and mechanism on crosslinks. First, most crosslinks were to unpaired nucleotides. Second, shorter and longer photo-crosslinkers formed crosslinks to intradomain locations nearer to and farther from photo-crosslinker modification, respectively. Finally, fluorine substitutions on the phenylazide ring did not change the locations of crosslinks, but rather decreased crosslinking efficiency. These findings have implications for the use of phenylazide photo-crosslinkers in structural studies of RNA.

Design and Synthesis of (+)-Discodermolide–Paclitaxel Hybrids Leading to Enhanced Biological Activity

“…The amine coupling partners were envisioned to arise via condensation reactions of the photoaffinity tags possessing carboxylic acid functionality with the diamine tethers of suitable length. The photoaffinity tags for this study were prepared following known synthetic routes, 35,36,37 wherein acids 8 and 13 were activated as hydroxysuccinamide esters, 38 followed by condensation with the alkyl diamine linkers (Scheme 2). For the aryl-(trifluoromethyl)diazirine component 18 , the mono-Boc protected amine linker was employed with N,N′-Carbonyl-diimidazole (CDI) 39 to reduce possible side reactions; the Boc group was subsequently removed under acidic conditions.…”

Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat

“…In general, ribozymes require magnesium for correct folding and for catalysis per se 23 , 43 but, although magnesium is essential for the hammerhead ribozyme to catalyze cleavage, it is not required for catalysis by the hairpin ribozyme. 44 Thus, the intracellular concentration of free magnesium ion in rat hepatoma cells in culture may be too low for the RzGUC20 hammerhead ribozyme, which showed best in vitro cleavage at 10 mmol/l; the value reported for rat liver is ~1 mmol/l. 45…”

Investigation and Conformational Analysis of Fluorinated Nucleoside Antibiotics Targeting Siderophore Biosynthesis

“…HPLC purifications were performed on instruments equipped with a reversed-phase Phenomenex Gemini 10 μm C18 110 Å (250 × 21.2 mm) column. All NMR spectra were recorded on a 400 MHz spectrometer at 400 MHz for 1 H, 100 MHz for 13 C, and 376 MHz for 19 F. 1 H NMR spectra were referenced to residual CDCl 3 (7.27 ppm), DMSO- d 6 (2.50 ppm), or CD 3 OD (3.31 ppm); 13 C NMR spectra were referenced to CDCl 3 (77.23 ppm) DMSO–D 6 (39.51 ppm), or CD 3 OD (49.15 ppm); and 19 F NMR spectra were referenced to hexafluorobenzene (−162.9 ppm) or trifluoroacetic acid (−76.5 ppm). NMR chemical shift data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, ABq = AB quartet, dm = doublet of multiplets), coupling constant, integration.…”