Understanding the Contributions of Conformational Changes, Thermodynamics, and Kinetics of RNA-Small Molecule Interactions

Abstract

The implication of RNA in multiple cellular processes beyond protein coding has revitalized interest in the development of small molecules for therapeutically targeting RNA and for further probing its cellular biology. However, the process of rationally designing such small molecule probes is hampered by the paucity of information about fundamental molecular recognition principles of RNA. In this Review, we summarize two important and often underappreciated aspects of RNA-small molecule recognition: RNA conformational dynamics and the biophysical properties of interactions of small molecules with RNA, specifically thermodynamics and kinetics. While conformational flexibility is often said to impede RNA ligand development, the ability of small molecules to influence the RNA conformational landscape can have a significant effect on the cellular functions of RNA. An analysis of the conformational landscape of RNA and the interactions of individual conformations with ligands can thus guide the development of new small molecule probes, which needs to be investigated further. Additionally, while it is common practice to quantify the binding affinities ( K_a or K_d) of small molecules for biomacromolecules as a measure of their activity, further biophysical characterization of their interaction can provide a deeper understanding. Studies that focus on the thermodynamic and kinetic parameters for interaction between RNA and ligands are next discussed. Finally, this Review provides the reader with a perspective on how such in-depth analysis of biophysical characteristics of the interaction of RNA and small molecules can impact our understanding of these interactions and how they will benefit the future design of small molecule probes.

Figure 1

Ligand-induced conformational changes in HIV-1 TAR. (a) Wild type HIV-TAR^, and HIV-1 TAR with a modified loop. Differences are colored red. (b) Reduction of interhelical angles in TAR RNA upon argininamide binding. (c) Structure of argininamide. (d) Structure of the arginine-derived probe.

Figure 2

Conformational spaces of diverse RNAs are affected by ligand binding. (a) Structures of small molecules that induce conformational changes in c-di-GMP-I riboswitch (kanamycin B), tRNA^Asp (tobramycin), and poly(A) (jatrorrhizine and coptisine).^,, (b) Unfolding of tRNA^Asp upon removal of magnesium. Nucleotides in the D-loop and anticodon stem bulge are colored red. Figure adapted from ref .

Figure 3

Ligand-induced conformational changes in a ribozyme and a pseudoknot. (a) Melamine-functionalized derivative of tris(2-aminoethyl)amine (t4M). (b) Restoration of ribozyme secondary structure (left) and tertiary contacts (right) upon t4M binding. Figure adapted from ref . (c) Naphthyridine carbamate tetramer (Z-NCTS). (d) Stabilization of a pseudoknot by Z-NCTS.^,

Figure 4

Select compounds for which calorimetric studies were reported by Suresh Kumar and co-workers as well as Patino and co-workers.^,–

Figure 5

Schematic illustration of techniques discussed herein. (a) Comparison between ITC and DSC. The two methods yield complementary information allowing full thermodynamic characterization of an interaction. T_m is the melting temperature. (b) Comparison between propagated and localized SPR. Localized SPR uses metal nanoparticles instead of metal film (kaki). In this format, free small molecules (purple) bind to immobilized biomolecules (black).^,

Figure 6

Compounds for which calorimetric studies were reported by (a) Pilch and co-workers and (b) Hergenrother and co-workers.^, Functional groups that differ between neomycin and paromomycin are colored red.

Figure 7

Select RNA constructs used in kinetics studies. (a) Domain II of HIV-1 RRE. The Rev binding site is colored magenta. (b) Upper stem-loop of the HIV-1 frameshift stimulatory signal. (c) Bacterial 16S and human 18S rRNA. Differences are colored red. Both RNAs were capped with the stable UUCG loop. (d) miR-29a precursor.