Probing of Metal-Binding Domains of RNA Hairpin Loops by Laser-Induced Lanthanide(III) Luminescence

Abstract

Direct laser excitation of aqueous Eu(III) bound to specific RNA fragments was used to probe the metal-binding sites of the anticodon loop of tRNA(Phe) from E. coli and of a tetraloop containing a GNRA consensus sequence. Binding of Mg(II) or Eu(III) to either RNA fragment resulted in a higher melting transition, but no global change in structure was observed. Aqueous Eu(III) exhibits a single weak excitation peak at 17273 cm(-1), the intensity of which increased upon addition of the tRNA loop fragment. Analysis of incremental increases in the luminescence intensity upon complexation with the tRNA loop indicated a stoichiometry of one high-affinity Eu(III)-binding site per loop fragment, with a Kd of 1.3 +/- 0.2 microM. Competition experiments between Eu(III) and Mg(II) were consistent with the two metal ions binding to a common site and with an approximately 30-fold lesser affinity of the tRNA loop for Mg(II) than for Eu(III). The rate of luminescence decay following excitation of Eu(III) bound to the tRNA loop corresponded to displacement of up to 4-5 (of a possible 9) waters of hydration on binding to the tRNA loop. By comparison, Eu(III) binds to the DNA analogue of the tRNA loop with an 8-fold lesser affinity and one fewer direct coordination site than to the RNA sequence, suggesting that a 2'OH of RNA is one of the direct ligands. In contrast with the absence of a shift in the excitation peak of aqueous Eu(III) upon formation of the tRNA loop complex, direct excitation of Eu(III) bound to a GNRA tetraloop fragment resulted in a substantially blue-shifted excitation peak (17290 cm(-1)). The tetraloop fragment also has a single Eu(III)-binding site, with a Kd of 12 +/- 3 microM. The bound Eu(III) was competed by Mg(II), although the relative affinity for Mg(II) was approximately 150-450-fold less than that for Eu(III). The Eu(III)-binding site of the tetraloop site is highly dehydrated, with approximately 7 water molecules displaced upon binding by RNA ligands, suggesting that the blue-shift of the excitation peak is the result of Eu(III) specifically bound in a nonpolar site within the GNRA loop structure.

Probing Metal Binding in the 8–17 DNAzyme by TbIII Luminescence Spectroscopy

“…[40] Lanthanide ions were used as effective cofactors for a Pb II -dependent DNAzyme to probe metal-binding sites, [50] and luminescence lifetime experiments were used to find the number of coordinated waters on the lanthanide ions binding to the hammerhead ribozyme [39] and RNA hairpin loops. [51] In this study, we report that Tb III Figure 1A. Previous studies had shown that a single base change-T2.1C-producing a GC base pair instead of the GT wobble pair adjacent to the cleavage site abolished the metal-dependent activity.…”

7-Amino actinomycin D bound to single-stranded hairpin DNA enhanced by loop sequence-dependent luminescent Eu3+ and Tb3+ binding

“…Clearly, Eu 3+ or Tb 3+ can destabilize It should be noted that the destabilization effect on GTT 3 -7AACTD complex is stronger than GAA 3 -7AACTD complex at the same binding ratio (Table 1), and Eu 3+ has even stronger effect than Tb 3+ . This is different from a previous report [9] that Eu 3+ can stabilize DNA hairpin structure [9]. The difference can be caused by the different route for hairpin formation.…”

“…The DNA used in Ref. [9] is a naturally formed hairpin DNA. However, the DNA we used cannot form hairpin structure by itself under our experimental conditions, the hairpin structure is induced upon 7AACTD binding.…”

“…Their binding is essential to maintain RNA and DNA structure or conformation [8,9]. Metal ions can also modulate DNA stability and topology [10,11].…”

Use of a novel Forster resonance energy transfer method to identify locations of site-bound metal ions in the U2-U6 snRNA complex

“…Concentration was measured by titration with EDTA as described previously (19). The excitation wavelength was 280 nm.…”