"Probing the limits of the malachite green RNA aptamer’s adaptive binding"
Jason B. DaCosta, Pierre-Nicholas Roy, Thorsten Dieckmann
The multiple roles of RNA in living cells are today well established. Its functions include the catalysis of protein synthesis in the ribosome, post-t cellular function. Aptamers have been developed for a wide variety of targets and are excellent model systems for RNA-ligand interactions. In addition, they have found roles in pharmaceuticals, and analytical instrumentation. The mode of binding in different aptamers varies greatly depending on their targets and selection procedure. The malachite green aptamer (MGA) was originally engineered for binding specificity to the tri-phenyl dye malachite green (Figure 1 A and B). The structure of the aptamer was initially solved by X-ray crystallography in complex with the MG derivative tetramethylrosamine (TMR) and later by NMR spectroscopy in complex with the original selection target MG (Figure 1C). The binding pocket of the MGA (Figure 1A) consists of a base quadruple (C7:G24:A31:G29) and a Watson-Crick base pair (G8:C28) which serve as stacking platforms for malachite green (MG, Figure 1B). In addition, the nucleotides A9 and A30 are positioned in such a way that they almost completely close the pocket on one side (Figure 1C).The other residues in the internal loop region of the aptamer act as linkers and anchors for the nucleotides that are in contact with the ligand. The major difference between the structures of the MGA-TMR and the MGA-MG complexes is that in order to accommodate the non-planar MG, the aptamer undergoes a small rearrangement that can be best described as a rotation of the upper part of the binding pocket relative to the lower part. This provides enough space to accommodate the rings of MG in the binding pocket, but also leads to a loss of some stacking interactions.
RNA 2012
Annual Meeting of the RNA Society
Ann Arbor, Michigan, 5/29-6/2, 2012
"Expanding the Catalytic Repertoire of a Ribozyme by Ligand Engineering"
Jason Da Costa and Thorsten Dieckmann
The malachite green aptamer (MG RNA) was originally engineered for binding specificity to the malachite green (MG) dye This aptamer has since shown to be capable of more then selectively binding MG. MG, a cationic triphenyl methane dye, displays a significant change in the charge distribution in the presence of an in vitro selected aptamer. These changes within the ligand molecule were first discovered with the aid of 1H-13C HMQC spectra of the 13C labeled dye in complex with the RNA. Ab initiocalculations on the dye:aptamer system predict significant changes of the charge distribution within the dye upon binding to the RNA. The changes are consistent with the patterns of chemical shift changes observed in the heteronuclear NMR experiments.
The catalytic potential of the aptamer’s electronegative binding pocket was harnessed to catalyze an ester hydrolysis via the stabilization of positively charged intermediates. This novel catalytic activity demonstrated the significance of electrostatic forces in RNA enzymes. In order to further investigate the catalytic ability of the aptamer another MG derivative was designed, the MG-thiocyanate. This derivative also displays an increased hydrolysis in the presence of the aptamer compared to background hydrolysis. The accelerated hydrolysis is a result of structure based ligand design, demonstrating the ability of the aptamer to catalyze the hydrolysis of a C-N bond.
RNA 2007
Annual Meeting of the RNA Society
Madison, Wisconsin, 5/29-6/3, 2007
"A pH Controlled Conformational Switch In the leavage Site of the VS Ribozyme Substrate RNA"
Jeremy Flinders and Thorsten Dieckmann
The VS ribozyme is a 154 nucleotide sequence found in certain natural strains of Neurospora. The RNA can be divided into a substrate and a catalytic domain. Here we present the solution structure of the substrate RNA that is cleaved in a trans reaction by the catalytic domain in the presence of Mg2+. The 30 nucleotide substrate RNA forms a compact helix capped by a flexible loop. The cleavage site bulge contains three non-canonical base pairs, including an A+·C pair with a protonated adenine. This adenine (A622) is a pH controlled conformational switch that opens up the internal loop at higher pH. The possible significance of this switch for substrate recognition and cleavage is discussed.
RNA 2001
Annual Meeting of the RNA Society
Banff, Canada, 5/29-6/3, 2001
"Adaptability and Dynamics in the Malachite Green - RNA Aptamer Complex: Combined Use of Crystal Structure and NMR Spectroscopy for the Rapid Determination of Closely Related Structures?"
RNA plays a central role in many biological processes and is therefore an important target for drug development. In recent years an increasing wealth of structural and functional information about RNA – ligand complexes has been obtained using in vitro selected RNAs (aptamers). However, all those studies focused on structure and changes of the nucleic acid and mostly considered the ligand as a rigid target. In order to develop a detailed picture of ligand structure and dynamics in RNA – small molecule complexes the Malachite Green binding aptamer was studied. We determined the solution structure of the MG – RNA complex and used isotopically labeled ligand to probe the ligand structure in complex with RNA. The surprisingly asymmetric changes in the 13C chemical shift of the ligand methyl groups indicate that the dye undergoes significant changes in its conformation and charge distribution upon binding. The role of the RNA electrostatic field in this interaction was explored using ab initio calculations of the ligand structure and charge distribution. The results indicate that the uneven charge distribution in the RNA binding pocket provides a major contribution to the driving force of the ligand structural changes. The observation that not only the RNA adapts to the ligand in what is called adaptive binding, but the ligand itself also undergoes conformational changes (“induced fit”), is crucial for the rational design of RNA ligands and for understanding the properties of RNA – ligand complexes?
ICMRBS 2002
International Conference for Magnetic Resonance in Biological Systems
Toronto, Canada, August 25-30, 2002
"Computational and Experimental Studies of Ligand Binding in the Malachite Green RNA Aptamer"
Jeremy C. Flinders, Steven C. DeFina, and Thorsten Dieckmann
RNA plays a central role in many biological processes and is therefore an important target for drug development. In recent years an increasing wealth of structural and functional information about RNA-ligand complexes has been obtained using in vitro selected RNAs (aptamers). However, all those studies focused on structure and changes of the nucleic acid and mostly considered the ligand as a rigid target. To develop a detailed picture of ligand structure and dynamics in RNA-small molecule complexes, the malachite green binding aptamer was studied.
We present the solution structure of an RNA aptamer that binds triphenyl dyes in complex with malachite green and compare it with a previously determined crystal structure of a complex formed with tetramethylrosamine. The structures illustrate how the same RNA binding pocket can adapt to accommodate both planar and nonplanar ligands. The two RNA-ligand complex structures allow a discussion of structural changes that have been observed in the ligand in the context of the overall complex structure.
During the structural characterization of the RNA aptamer, isotopically labeled ligand in complex with RNA was analyzed by NMR spectroscopy in solution. The surprisingly asymmetric changes in the 13C chemical shift of the ligand methyl groups indicate that the dye undergoes changes in its conformation and charge distribution upon binding. First, the role of the RNA electrostatic field in this interaction was explored using ab initio calculations of the ligand structure and charge distribution.
Next, base-stacking interactions and the dynamics of a malachite green (MG)-RNA aptamer complex were examined to help explain the observed 14 nm red-shift in the maximum absorption frequency of MG. In particular, we examined the conformational dynamics of the MG molecule, in terms of the interplanar dihedrals of its aromatic rings. A correlation between the degree of planarity of the ligand and binding strength was found in terms of the extension of p-system base-stacking forces.
Gordon Research Conference on Computational Aspects - Biomolecular NMR
Ventura, California, January 18-23, 2004