Binding to an RNA Aptamer Changes the Charge Distribution and Conformation of Malachite Green

Abstract

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. Isotopically labeled ligand in complex with RNA was analyzed by NMR spectroscopy in solution. The surprisingly asymmetric changes in the (13)C chemical shift of the ligand methyl groups indicate that the dye undergoes 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 that 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.

Structural Determinants of Conformationally Selective, Prion-binding Aptamers

“…1 (A and B). It must be noted that RNA structures are often dynamic (29). Aptamers, which have been selected for their ability to bind to a target protein with good affinity, may not always be fully structured in the absence of protein.…”

Recognition of Planar and Nonplanar Ligands in the Malachite Green–RNA Aptamer Complex

“…The reason for the observed differences are the different shapes of the two dyes in combination with the conformational changes detected in MG upon binding to the RNA. [11] The A and B rings in TMR are perfectly planar; however, the two rings are rotated relative to each other by approximately 498 in free MG and by 578 in the RNA-bound form. The different ligand structures make it impossible to accommodate favorable stacking interactions for both dyes in the same structural framework.…”

“…The stacking arrangement in the MG ± RNA complex is also in agreement with the observation that the B ring of MG rotates approximately 608 out of plane relative to the A and C rings due to the conformational changes caused by the RNA electrostatic field. [11] Position of A9 and A30…”

“…In addition to the structural changes in the RNA, it has recently been shown that the ligand can also undergo significant changes with respect to its conformation and charge distribution upon binding to RNA. [11] Aptamers are RNA or DNA molecules which are selected in vitro to bind ligand molecules with high specificity and affinity. [12,13] A great variety of these sequences have been identified to date (see refs.…”

Function and dynamics of aptamers: A case study on the malachite green aptamer

“…The modified aptamer is called a thioaptamer and is nuclease-resistant. Ds-DNA thioaptamers made from dATPαS, dTTP, dCTP and dGTP have been selected for NF-IL6 [38], NFκB(p50, p60, RelA) [39,40] and RNase H domain of HIV RT [41] with Kds in the low nM range. The half life of the thioaptamer against NF-IL6 was increased from about 5 min to about 20 min at the treatment of 0.12 pg/ml DNase I [38].…”