Jan Ferner

Mapping the Landscape of RNA Dynamics with NMR Spectroscopy

Among the three major classes of biomacromolecules (DNA, RNA, and proteins) RNAs pronounced dynamics are the most explicitly linked to its wide variety of functions, which include catalysis and the regulation of transcription, translation, and splicing. These functions are mediated by a range of RNA biomachinery, including such varied examples as macromolecular noncoding RNAs, microRNAs, small interfering RNAs, riboswitch RNAs, and RNA thermometers. In each case, the functional dynamics of an interconversion is characterized by an associated rate constant. In this Account, we provide an introduction to NMR spectroscopic characterization of the landscape of RNA dynamics. We introduce strategies for measuring NMR parameters at various time scales as well as the underlying models for describing the corresponding rate constants. RNA exhibits significant dynamic motion, which can be modulated by (i) intermolecular interactions, including specific and nonspecific binding of ions (such as Mg(2+) and tertiary amines), (ii) metabolites in riboswitches or RNA aptamers, and (iii) macromolecular interactions within ribonucleic protein particles, including the ribosome and the spliceosome. Our understanding of the nature of these dynamic changes in RNA targets is now being incorporated into RNA-specific approaches in the design of RNA inhibitors. Interactions of RNA with proteins, other RNAs, or small molecules often occur through binding mechanisms that follow an induced fit mechanism or a conformational selection mechanism, in which one of several populated RNA conformations is selected through ligand binding. The extent of functional dynamics, including the kinetic formation of a specific RNA tertiary fold, is dependent on the messenger RNA (mRNA) chain length. Thus, during de novo synthesis of mRNA, both in prokaryotes and eukaryotes, nascent mRNA of various lengths will adopt different secondary and tertiary structures. The speed of transcription has a critical influence on the functional dynamics of the RNA being synthesized. In addition to modulating the local dynamics of a conformational RNA ensemble, a given RNA sequence may adopt more than one global, three-dimensional structure. RNA modification is one way to select among these alternative structures, which are often characterized by nearly equal stability, but with high energy barriers for conformational interconversion. The refolding of different secondary and tertiary structures has been found to be a major regulatory mechanism for transcription and translation. These conformational transitions can be characterized with NMR spectroscopy, for any given RNA sequence, in response to external stimuli.

NMR and MD studies of the temperature-dependent dynamics of RNA YNMG-tetraloops

In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.

Inhibition of HIV-1 by a Peptide Ligand of the Genomic RNA Packaging Signal Ψ

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV‐1) Gag polyprotein with the RNA packaging signal Ψ ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV‐1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV‐1 Ψ‐RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Ψ‐RNA binding. The optimized peptide, psi‐pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Ψ packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi‐pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV‐1.

Fragment based search for small molecule inhibitors of HIV-1 Tat-TAR.

Basic molecular building blocks such as benzene rings, amidines, guanidines, and amino groups have been combined in a systematic way to generate ligand candidates for HIV-1 TAR RNA. Ranking of the resulting compounds was achieved in a fluorimetric Tat-TAR competition assay. Although simple molecules such as phenylguanidine are inactive, few iteration steps led to a set of ligands with IC50 values ranging from 40 to 150 μM. 1,7-Diaminoisoquinoline 17 and 2,4,6-triaminoquinazoline 22 have been further characterized by NMR titrations with TAR RNA. Compound 22 is bound to TAR at two high affinity sites and shows slow exchange between the free ligand and the RNA complex. These results encourage investigations of dimeric ligands built from two copies of compound 22 or related heterocycles.

Tripeptides from Synthetic Amino Acids Block the Tat–TAR Association and Slow Down HIV Spread in Cell Cultures

Non‐natural amino acids with aromatic or heteroaromatic side chains were incorporated into tripeptides of the general structure Arg‐X‐Arg and tested as ligands of the HIV RNA element TAR. Some of these compounds could compete efficiently with the association of TAR and Tat and downregulated a TAR‐controlled reporter gene in HeLa cells. Peptide 7, which contains a 2‐pyrimidinyl‐alkyl chain, also inhibited the spread of HIV‐1 in cell cultures. NMR studies of 7 bound to HIV‐2‐TAR gave evidence for contacts in the bulge region.

Structures of HIV TAR RNA–Ligand Complexes Reveal Higher Binding Stoichiometries

Target TAR by NMR: Tripeptides containing arginines as terminal residues and non‐natural amino acids as central residues are good leads for drug design to target the HIV trans‐activation response element (TAR). The structural characterization of the RNA–ligand complex by NMR spectroscopy reveals two specific binding sites that are located at bulge residue U23 and around the pyrimidine‐stretch U40‐C41‐U42 directly adjacent to the bulge.